Natl Toxicol Program Tech Rep Ser, 2003 Oct, (509), 1 - 290 NTP toxicology and carcinogensis Studies of 2,4-hexadienal (89% trans,trans isomer, CAS No . 142-83-6; 11% cis,trans isomer) (Gavage Studies); National Toxicology Program; 2,4-Hexadienal, a colorless to yellow liquid with a pungent "green" or citrus odor, is used as a food additive for flavor enhancement, as a fragrance agent, as a starting material or intermediate in synthetic reactions in the chemical and pharmaceutical industries, as a fumigant, and as a corrosion inhibitor for steel . 2,4-Hexadienal was nominated for study by the National Cancer Institute because of the potential for carcinogenicity based on its alpha,beta-unsaturated aldehyde structure and the potential link between exposure to lipid peroxidation products in the diet and human malignancies . The commercial product is a mixture containing chiefly trans,trans-2,4-hexadienal in equilibrium with cis,trans-2,4-hexadienal . Male and female F344/N rats and B6C3F1 mice received 2,4-hexadienal (89% trans,trans; 11% cis,trans) in corn oil by gavage for 16 days, 14 weeks, or 2 years . Tissues and plasma from dosed rats were examined for malondialdehyde and glutathione concentrations, and DNA adducts were characterized in liver and forestomach samples from dosed rats and mice . Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes . 16-DAY STUDY IN RATS: Groups of five male and five female rats were administered 0, 3, 9, 27, 80, or 240 mg 2,4-hexadienal/kg body weight in corn oil by gavage, 5 days per week, for 16 days . Three male and three female 240 mg/kg rats died before the end of the study . Mean body weight gains of 240 mg/kg rats were significantly less than those of the vehicle controls . Clinical findings included diarrhea, ataxia, lethargy, and nasal/eye discharge in males, and lethargy, paleness, and abnormal breathing in females in the 240 mg/kg groups . Liver weights of 240 mg/kg females were significantly greater than those of the vehicle controls . Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in most 240 mg/kg rats, and forestomach epithelial hyperplasia was microscopically evident in most 80 mg/kg rats . 16-DAY STUDY IN MICE: Groups of five male and five female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 3, 9, 27, 80, or 240 mg/kg, 5 days per week, for 16 days . Chemical-related deaths occurred in one male and one female in the 240 mg/kg groups . Female mice in the 240 mg/kg group lost weight during the study . Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in all 240 mg/kg mice, and forestomach epithelial hyperplasia and hyperkeratosis were microscopically evident in 80 mg/kg mice . 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks . All rats survived to the end of the study . Mean body weights of 30, 60, and 120 mg/kg males were significantly less than those of the vehicle controls . The only clinical finding attributed to 2,4-hexadienal administration was hypersalivation in 30 and 120 mg/kg males and females . The incidences of forestomach hyperplasia and nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg rats . Nasal lesions occurred in most 120 mg/kg male rats . 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks . No deaths were attributed to administration of 2,4-hexadienal . Mean body weights of males and females were similar to those of the vehicle controls throughout the study . Clinical findings included salivation and anal wetness in males and females . Kidney weights of 60 and 120 mg/kg males and liver weights of 60 mg/kg males and females were significantly greater than those of the vehicle controls . The incidences of forestomach hyperplasia and/or nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg mice . 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 2,4-hexministered 2,4-hexadienal in corn oil by gavage at doses of 0, 22.5, 45, or 90 mg/kg, 5 days per week, for up to 105 weeks . Survival of all dosed groups of rats was similar to that of the vehicle control groups . The mean body weights of 90 mg/kg males were generally less than those of the vehicle controls throughout the study . The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female rats . This neoplasm was found in 58% of males and 34% of females in the 90 mg/kg groups . In the forestomach of male rats, papilloma multiplicity was increased in the 90 mg/kg group, and squamous cell carcinomas were found in one 45 mg/kg male and two 90 mg/kg males . Epithelial hyperplasia of the forestomach occurred in most 45 and 90 mg/kg rats . 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 30, 60, or 120 mg/kg, 5 days per week, for up to 105 weeks . Survival of dosed mice was similar to that of the vehicle controls . The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study . The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female mice; squamous cell carcinomas were present in 120 mg/kg males and females . Epithelial hyperplasia of the forestomach occurred in many 120 mg/kg mice . Two 120 mg/kg males had uncommon squamous cell carcinoma of the oral cavity (tongue) . GENETIC TOXICOLOGY: 2,4-Hexadienal was mutagenic in S . typhimurium strain TA100 with and without induced hamster or rat liver enzymes; no mutagenic activity was detected with strains TA1535 or TA98, with or without S9 . Results of bone marrow tests in male rats and male mice given intraperitoneal injections of 2,4-hexadienal showed a small increase in the induction of micronucleated erythrocytes . However, neither test was repeated, and the test results were judged to be inconclusive . Results of peripheral blood micronucleus tests in male and female mice treated with 2,4-hexadienal by gavage for 14 weeks were negative . CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity* of 2,4-hexadienal in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of squamous cell neoplasms of the forestomach . The occurrence of squamous cell carcinoma of the oral cavity (tongue) in male B6C3F1 mice may have been related to the administration of 2,4-hexadienal . Hyperplasia of the forestomach in male and female rats and mice was associated with administration of 2,4-hexadienal . Synonyms: Hexa-2,4-dienal; 2,4-hexadienal; 2,4-hexadien-1-al; 2,4-Hx; 1,3-pentadiene-1-carboxaldehyde; 2-propylene acrolein; sorbaldehyde; sorbic aldehyde Toxicol Appl Pharmacol, 2004 Mar 1, 195(2), 166 - 81 In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1; Ross MK et al.; The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1) . Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2) . First, rodent hepatic cytosol incubations containing {(14)C}CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA . The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2) . Second, exposure of GSTT1-1-expressing S . typhimurium to {(14)C}CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity . Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis . A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed . To circumvent problems of adduct instability, reactions of {(14)C}CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine . Hydroxyapatite chromatography of {(14)C}-labeled DNA or HPLC chromatography of {(14)C}-labeled deoxyguanosine derivatives demonstrated the covalent binding of {(14)C}CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of {(14)C}CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts) . Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue) . Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared . In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues . The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats). Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3398 - 403 Epub 2004 Mar 01. Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome; Deng S et al.; Transcription and replication both influence and are influenced by superhelical changes in DNA . Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years . Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion . Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins . Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA . Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains . High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module . Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome . In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval . A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span . These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes. Mol Cell, 2004 Feb 27, 13(4), 453 - 4 Actin lessons from pathogens; Le Clainche C et al.; Salmonella typhimurium secretes proteins that co-opt the host actin cytoskeleton to induce membrane ruffling, leading to the uptake of the bacterium . New information about the biochemical activities of the Salmonella protein SipA suggests that this protein might inhibit host cell actin dynamics by competing with ADF/cofilin and gelsolin, two key proteins that promote the turnover of actin filaments. Environ Mol Mutagen, 2004, 43(2), 128 - 33 Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method; Araki A et al.; The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays . In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method . Tests were also conducted with a glutathione-supplemented S9 mix . Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537 . Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix . Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix . The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds . Nat Biotechnol, 2004 Mar, 22(3), 313 - 20 Epub 2004 Feb 08. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins; Yu YA et al.; We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases . The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases . Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated . Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging . These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication . We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors . Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer. Int Arch Allergy Immunol, 2004 Mar, 133(3), 295 - 304 Epub 2004 Feb 25. DNA vaccines designed to inhibit tumor growth by suppression of angiogenesis; Reisfeld RA et al.; The development of new blood vessels, i.e . angiogenesis, is a rate-limiting step in the development of tumors since tumor growth is generally limited to 1-2 mm3 in the absence of a blood supply . Thus, the inhibition of tumor growth by attacking the tumor's vascular supply offers a primary target for antiangiogenic intervention by DNA-based vaccines . Here, we describe two novel orally delivered DNA vaccines which suppress tumor angiogenesis and induce a robust cell-mediated immune response that provides for long-lived protection against melanoma, colon, breast and non-small-cell lung carcinoma in mouse model systems . These vaccines, which are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs, are directed against such targets as vascular endothelial growth factor receptor 2 (FLK-1) and transcription factor Fos-related antigen 1 (Fra-1) . Both vaccines break peripheral T cell tolerance against these self-antigens and induce a robust T cell-mediated immune response leading to suppression of tumor angiogenesis and resulting in effective suppression of tumor growth and metastases . Such research efforts may open up new possibilities for the rational design of future DNA vaccines effective for the prevention and treatment of cancer . Nat Prod Res, 2004 Apr, 18(2), 189 - 95 Analysis of essential oil of Satureja thymbra by hydrodistillation, thermal desorber, and headspace GC/MS techniques and its antimicrobial activity; Goren AC et al.; The essential oil composition of Satureja thymbra was analyzed by direct thermal desorber and Headspace GC/MS analysis methods . Its constituents were determined to be mainly carvacrol (40.15%), gamma-terpinene (26.56%), p-cymene (16.39%), and thymol (13.16%) . The other techniques, thermal desorber and Headspace GC/MS, were used for the plant leaves at three different temperature, which showed similar results . The thermal desorber GC/MS gave better and more sensitive results than Headspace GC/MS . The essential oil was found to be active against the bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Staphylococcus aureus and the yeast Candida albicans. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2422 - 7 Interplay between antibacterial effectors: a macrophage antimicrobial peptide impairs intracellular Salmonella replication; Rosenberger CM et al.; Antimicrobial peptides have established an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated . Macrophage expression of the murine cathelicidin-related antimicrobial peptide (CRAMP) was increased after infection by the intracellular pathogen Salmonella typhimurium, and this increase required reactive oxygen intermediates . By using CRAMP-deficient mice or synthetic CRAMP peptide, we found that CRAMP impaired Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria . This impaired bacterial cell division also depended on intracellular elastase-like serine protease activity, which can proteolytically activate cathelicidins . Macrophage serine protease activity induced filamentation and enhanced the activity of CRAMP in vitro . A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment . Although cationic peptides have been hypothesized to have activity against pathogens within macrophages, this work provides experimental evidence that the antimicrobial arsenal of macrophages includes cathelicidins . These results show that intracellular reactive oxygen intermediates and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen, and they highlight the cooperativity between macrophage antibacterial effectors. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2404 - 9 Evolutionary comparisons suggest many novel cAMP response protein binding sites in Escherichia coli; Brown CT et al.; The cAMP response protein (CRP) is a transcription factor known to regulate many genes in Escherichia coli . Computational studies of transcription factor binding to DNA are usually based on a simple matrix model of sequence-dependent binding energy . For CRP, this model predicts many binding sites that are not known to be functional . If they are indeed spurious, the underlying binding model is called into question . We use a species comparison method to assess the functionality of a population of such predicted CRP sites in E . coli . We compare them with orthologous sites in Salmonella typhimurium identified independently by CLUSTALW alignment, and find a dependence of mutation probability on position in the site . This dependence increases with predicted site binding energy . The positions where mutation is most strongly suppressed are those where mutation would have the biggest effect on predicted binding energy . This finding suggests that many of the novel sites are functional, that the matrix model correctly estimates their binding strength, and that calculated CRP binding strength is the quantity that is conserved between species . The analysis also identifies many new E . coli binding sites and genes likely to be functional for CRP. Mutagenesis, 2004 Mar, 19(2), 149 - 56 Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells; Arlt VM et al.; 3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter . 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites . In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5 . DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis . DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time) . At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA . Median CTLs in control incubations were in the range 7.7-13.1 micro m . Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea . Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay . 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05) . Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites . DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively . DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA . All major 3-NBA-derived DNA adducts produced in MCL-5 cells were found to be formed from reductive metabolites bound to purine bases and lacked an N-acetyl group . These results demonstrate that 3-NBA and its metabolites are effectively activated to DNA-damaging species in human MCL-5 cells, which may reflect the genotoxic potential of 3-NBA in humans . Environmental exposure to 3-NBA may be a health hazard for large sections of the population and the risks associated with such exposure require further assessment. Bioorg Med Chem, 2004 Mar 1, 12(5), 853 - 7 Synthesis and antimicrobial properties of imidazolium and pyrrolidinonium salts; Demberelnyamba D et al.; For the purpose of developing new disinfectants and antiseptics, we searched for compounds having high bactericidal activity against gram-positive bacteria, gram-negative bacteria, and fungi . Three different series of quaternary imidazolium and pyrrolidinonium salts were synthesized: series A (1-alkyl-3-methylimidazolium chlorides and bromides); series B (1-alkyl-2-methyl-3-hydroxyethylimidazolium chlorides); and series C (N-alkyl-N-hydroxyethylpyrrolidinonium) . Series B and C were newly designed . These three series were tested to evaluate their antibacterial and antifungal properties for the first time . Seven microbial strains were used in the study: Escherichia coli KCTC1924, Salmonella typhimurium KCTC1926, Staphylococcus aureus 209 KCTC1916, Staphylococcus aureus R209 KCTC1928, Bacillus subtilis KCTC1914, Candida albicans KCTC1940, and Chlorella regularis . The antimicrobial efficiency was measured by bacterial and fungal growth inhibition expressed as minimal inhibitory concentration (MIC) values . Series A and B imidazolium salts had very good antimicrobial activity against the examined Gram-negative bacteria, Gram-positive bacteria, and fungi . Also the pyrrolidinonium salt was found to have low MIC for some of tested microorganisms . The antibacterial and antifungal active properties of the salts depend upon the structure of functional groups and the alkyl chain length in the imidazolium and pyrrolidinonium ring . Among the synthesized quaternary imidazolium and pyrrolidinonium salts, the imidazolium salts containing a long alkyl chain and the introduction of a hydroxyethyl chain and methyl group into the imidazolium ring structure leads to broad spectrum active antimicrobial agents which not only have bacteriostatic properties but could be powerful bactericides. J Agric Food Chem, 2004 Feb 25, 52(4), 781 - 7 Volatile constituents from the leaves of Callicarpa japonica Thunb . and their antibacterial activities; Kim YS et al.; Volatile substances of Callicarpa japonica Thunb . were examined for their antibacterial activities against six foodborne microorganisms using the optical densitometer Bioscreen C . Extracts of C . japonica were obtained by simultaneous steam distillation and solvent extraction (SDE), and those extracted for 1.5 and 2.0 h at pH 6.0 strongly inhibited the growth of Bacillus cereus and Salmonella typhimurium; the content of the volatile substances of leaves at these pH levels were 543.1 and 706.7 mg/kg, respectively . All foodborne microorganisms tested were strongly inhibited by the addition of >8% (v/v) of the SDE extracts to broth medium . The major volatile components of the SDE extracts obtained at 1.5 h and pH 6.0 were gamma-caryophyllene, 1-octen-3-ol, 2-hexenal, germacrene B, and aromadendrene II, with corresponding peak areas of 44.14, 15.6, 9.86, 5.24, and 4.01%, respectively, and major antibacterial components were 1-octen-3-ol and 2-hexenal . Among the 32 materials identified as volatile flavor components, 2-hexenal, 2,4-hexadienal, 1-octen-3-ol, 2,4-heptadienal, and epiglobulol strongly inhibited microorganism growth . In particular, 2-hexenal (107.52 mg/L) and 1-octen-3-ol (678.64 mg/L) inhibited the growth of most microorganisms tested by >90%. J Food Prot, 2004 Feb, 67(2), 371 - 7 Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon; Yuste J et al.; Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C . Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E . coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days . The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells . The pathogens were gradually inactivated by the acidic pH of apple juice . Nisin and cinnamon greatly contributed to the inactivation . The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E . coli O157:H7 . Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature . The TAL method was as effective as TSA in recovering injured cells of the pathogens . The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E . coli O157:H7 in apple juice and so enhances the safety of the product. Int J Food Microbiol, 2004 Feb 15, 91(1), 91 - 8 Viability loss and morphology change of foodborne pathogens following exposure to hydrostatic pressures in the presence and absence of bacteriocins; Kalchayanand N et al.; Cell suspensions of three pathogens were exposed to hydrostatic pressure (HP), bacteriocin mixture (nisin and pediocin) or a combination of HP+bacteriocins and changes in colony forming units (cfu) and cell-morphology by scanning electron microscopy (SEM) were studied . Cell viability loss, as determined from the reduction in cfu before and after a treatment, occurred in Listeria monocytogenes by all three treatments and in Salmonella typhimurium and Escherichia coli O157:H7 by HP and HP+bacteriocin combination . Cell wall and cell membrane collapse and cell lysis was indicated in L . monocytogenes exposed to bacteriocin or HP+bacteriocin and in Salmonella and E . coli exposed to HP or HP+bacteriocin. Life Sci, 2004 Mar 5, 74(16), 2023 - 36 Role of histamine and acid back-diffusion in modulation of gastric microvascular permeability and hemorrhagic ulcers in Salmonella typhimurium-infected rats; Hung CR et al.; Documentation concerning the pathogenesis of gastric hemorrhagic ulcer in Salmonella typhimurium (Salmonella typhi)-infective disease is lacking . This research first proposed that alterations of mast cell histamine release, gastric acid back-diffusion and mucosal microvascular permeability are important in modulating gastric ulcer and hemorrhage in Salmonella typhi-infected rats . Additionally, effects of several histamine-related drugs on this ulcer model were evaluated . Male Wistar rats were deprived food for 36 h . Live cultures of Salmonella typhi (OU 5045, 1 x 10(10) CFU in 1.0 mL of sterilized phosphate buffer saline) were challenged, intrajejunally to rats just before withdrawal of food . Control rats received the same volume of sterilized vehicle only . Rat stomachs were irrigated for 3 h with either normal saline or simulated gastric juice . Gastric acid back-diffusion, mucosal histamine concentration, microvascular permeability as well as luminal hemoglobin content and ulcer areas were determined . Severe gastric hemorrhage and mucosal ulcerations, particularly in acidic stomachs, were observed in Salmonella typhi-infected rats . A positive correlation of histamine to gastric hemorrhage and ulcer was found in those rats with Salmonella typhi-infection . This hemorrhagic ulcer in Salmonella typhi-infected rats was effectively ameliorated by intraperitoneal ketotifen, diphenhydramine and ranitidine but was worsen by exogenous histamine or diamine oxidase . In conclusion, enhancement of acid back-diffusion, mast cell histamine release and microvascular permeability is important in modulating gastric hemorrhage and ulcer in Salmonella typhi-infected rats. Rev Esp Quimioter, 2003 Dec, 16(4), 398 - 402 {Different antibiotic resistance mechanisms associated with integrons in clinical isolates of Salmonella typhimurium}; Gallardo F et al.; Antibiotic resistance in clinical isolates of Salmonella typhimurium has steadily risen in recent years . Some of the resistance genes may be carried into integrons . In this study, integrons, both from 10 epidemiologically related and unrelated S . typhimurium clinical isolates, were characterized, showing that epidemiologically different strains can carry the same integron, and that epidemiologically related strain can carry different integrons . Among the resistance genes detected in this study were genes encoding b-lactamases (bla(oxa-30) in two strains, and bla(pse-1) in five strains, one of which was carrying this cassette in two different integrons); aminoglycoside-modifying enzymes (aadA2 in four strains, one of which was carrying this cassette in two different integrons, and aadA1 in six strains); as well dihydrofolate reductases (dfrAI in three strains). Autoimmun Rev, 2004 Jan, 3(1), 61 - 9 Is there a role for viruses in triggering autoimmune hepatitis? Vento S, Cainelli F. A role for viruses in autoimmune hepatitis (AH) has been repeatedly proposed but convincing evidence links only two viruses, hepatitis A and Epstein-Barr virus, to the type 1 form of the disease, and only in those rare cases where a genetic predisposition exists and the viral infection occurs at the right time, i.e . when other unknown factors are cooperating . In spite of an impressive amount of information conclusively showing molecular mimicry between cytochrome P450IID6 (the target autoantigen of autoantibodies characteristic of AH type 2) sequences and viral (hepatitis C virus, herpes simplex virus 1, cytomegalovirus, human T lymphotropic viruses 1 and 2) or bacterial (Salmonella typhimurium) antigens, no infectious agent is clearly able to induce this second form of the disease . In conclusion, the molecular mimicry theory has so far found little clinical evidence in its support and many more clinical observations are needed in order to unreveal possible links between viruses and AH. Planta Med, 2004 Jan, 70(1), 17 - 22 Suppression of infection-induced endotoxin shock in mice by a citrus flavanone naringin; Kawaguchi K et al.; The protective effect of the Citrus flavanone naringin was demonstrated in an endotoxin shock model based on Salmonella infection . Intraperitoneal ( i . p.) infection with 10 (8) CFU Salmonella typhimurium aroA caused lethal shock in lipopolysaccharide (LPS) -responder but not LPS-non-responder mice . Administration of 1 mg naringin 3 h before infection resulted in protection from lethal shock, similar to LPS-non-responder mice . The protective effect of naringin was time- and dose-dependent . Treatment with naringin resulted not only in a significant decrease in bacterial numbers in spleens and livers, but also in a decrease in plasma LPS levels . In addition, naringin markedly suppressed TNF-alpha and normalized the activated states of blood coagulation factors such as prothrombin time, fibrinogen concentration and platelet numbers caused by infection . Interestingly, treatment with naringin suppressed high levels of soluble CD14 and high mobility group-1 molecule caused by infection. Microb Ecol, 2004 Feb, 47(2), 175 - 85 Epub 2004 Feb 09. A portable array biosensor for detecting multiple analytes in complex samples; Taitt CR et al.; The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation . This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument . These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested . Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology . Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading . This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis . J Biol Chem, 2004 Apr 23, 279(17), 17054 - 62 Epub 2004 Feb 02. Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity; Leonardi R et al.; The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI . We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E . coli 83-1 cells . The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E . coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes . By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E . coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells . The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH. Mol Microbiol, 2004 Jan, 51(2), 483 - 95 Salmonella type III secretion-associated chaperones confer secretion-pathway specificity; Lee SH et al.; Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants . Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus . One of the domains is located within the amino terminal first approximately 20 amino acids and the second domain, located within the first approximately 140 amino acids, serves as a binding site for specific chaperones . It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment . We have found that in the absence of their chaperone-binding domains, the Salmonella typhimurium TTSS-secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway . These results indicate the existence of an 'ancestral' flagellar secretion signal within TTSS-exported proteins that is revealed in the absence of the chaperone-binding domain . Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone-binding domains . We conclude from these studies that a critical function for the TTSS-associated chaperones is to confer secretion-pathway specificity to their cognate secreted proteins. Genome Biol . 2004;5(2):R9 . Epub 2004 Jan 29. In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection; Marchal K et al.; BACKGROUND: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence . PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages . The PmrAB system also confers resistance to Fe3+-mediated killing . New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets . RESULTS: We performed an in silico analysis of possible targets of the PmrAB system . Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes . To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure . As well as the known targets, we detected additional targets with unknown functions . Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ) . Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements . CONCLUSIONS: We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally . We also pinpointed directions for further research that could help elucidate the S . typhimurium virulence pathway. Carcinogenesis, 2004 May, 25(5), 779 - 86 Epub 2004 Jan 30. Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of sulfotransferases and acetyltransferases in nitrofen mutagenicity; Glatt H et al.; Nitrofen had been used as a herbicide, until its carcinogenic and teratogenic activity in rodents was detected . A food contamination occurring in 2002 in Germany led to the initiation of new studies in order to better understand the potential risk for humans . Nitrofen is a nitroarene and as such might be activated to a mutagen via reduction to the corresponding hydroxylamine and subsequent formation of a reactive acetic or sulfuric acid ester . Therefore, we have investigated the mutagenicity of nitrofen in Salmonella typhimurium strains engineered for the expression of all human xenobiotic-metabolizing sulfotransferases (SULTs) and acetyltransferases (NATs) identified . Nitrofen was inactive in the parental strains TA1538, TA98 and TA100, but was mutagenic even at low doses when human sulfotransferase SULT1A1 (the major broad-spectrum phenol SULT) was expressed in these strains, but not when it was expressed in a TA1538-derived strain deficient in an endogenous nitroreductase . Several other human SULTs (in particular 1A3 and 1C1) as well as human NAT2 (unlike NAT1) also activated nitrofen, but were markedly less efficient than SULT1A1 . Likewise, expression of rat and mouse SULT1A1 led to weaker mutagenic activity of nitrofen than expression of the corresponding human enzyme . An endogenous acetyltransferase only activated nitrofen to a mutagen when it was strongly over-expressed in the TA98-derived strain YG1024 . Thus, humans might be more susceptible to the carcinogenic effects of nitrofen than mice and rats, which have been used in long-term studies . The fact that several SULTs show particular high expression in fetal tissues suggests that this activation pathway may also play a role in the teratogenic effects observed. Int J Food Microbiol, 2004 Feb 1, 90(3), 331 - 9 Studies to determine the critical control points in pork slaughter hazard analysis and critical control point systems; Pearce RA et al.; Aerobic mesophilic counts (AMC), coliform (CC) and coliform resuscitation counts (CRCs) were obtained by swabbing 50 cm(2) areas at three sites (ham, belly and neck) on pig carcasses, after each of seven stages of the slaughter/dressing process (bleeding, scalding, dehairing, singeing, polishing, evisceration and chilling) . In most cases, there were no statistical differences (P>0.05) among the counts derived by these three methods . Reductions in counts at individual sites were observed after scalding (3.5 log(10) cfu cm(-2)), and singeing (2.5 log(10) cfu cm(-2)) . Increases in counts at individual sites were observed after dehairing (2.0 log(10) cfu cm(-2)) and polishing (1.5 log(10) cfu cm(-2)) . The incidence of Salmonella on pig carcasses was also obtained by swabbing the outside surfaces of 100 half carcasses . Information on the incidence of Salmonella in scald tank water (108 samples) was also investigated . Carcass swabs and scald tank water were examined for the presence of Salmonella using standard enrichment methods . Salmonella were detected on 31% of carcasses immediately after bleeding, 7% of carcasses immediately after dehairing and evisceration, and 1% of carcasses immediately after scalding . Serovars included Salmonella Typhimurium, Salmonella Hadar, Salmonella Infantis and Salmonella Derby . No Salmonella were recovered from samples of scald tank water . The impact of pig slaughter/dressing processes on carcass microbiology and their potential use as critical control points (CCPs) during pork production are discussed. Nucleic Acids Res, 2004 Jan 26, 32(2), 522 - 34 Print 2004. Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives; Sibley MH et al.; A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species . We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli . This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there . To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C . Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2 . To discuss this variation, we adopt the term 'framework' to refer to genes that are stable components of genomes within related lineages, while 'migratory' genes are transient inhabitants of the genome . Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined . The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation . The patterns observed are consistent with the action of a site-specific recombinase . Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked. Biochemistry, 2004 Feb 3, 43(4), 837 - 42 Structural elements, mechanism, and evolutionary convergence of Rho protein-guanine nucleotide exchange factor complexes; Erickson JW et al.; Rho GTPases act as key regulators of cellular biochemistry by determining the timing, direction, and amplitude of signal transduction in a number of important pathways . The rate of activation of a GTPase-controlled reaction is limited by the rate of GTP binding to the Rho protein, and this, in turn, depends on the rate that GDP dissociates from the GTPase . The latter is controlled by the action of guanine nucleotide exchange factors (GEFs) that catalyze GDP-GTP exchange by increasing the rate of GDP dissociation . Here, the recently reported structural information for Rho GTPase-GEF complexes and the molecular basis for the specificity of their interactions are discussed . Underscoring the importance of regulating the Rho GTPase activation pathway, genetically unrelated proteins have evolved which complement or mimic the Dbl homology-Pleckstrin homology (DH-PH) domain-containing family of proteins in their ability to catalyze GDP-GTP exchange . In particular, the structure of the mammalian Cdc42 protein bound to the SopE protein from Salmonella typhimurium illustrates how two unrelated protein folds are able to carry out guanine nucleotide exchange by a remarkably similar mechanism . It will be interesting to see if this conservation of mechanism extends to a newly recognized class of GEFs related to the DOCK180 family. Acta Microbiol Pol, 2003, 52(3), 285 - 92 Amlodipine: a cardiovascular drug with powerful antimicrobial property; Kumar KA et al.; Ten cardiovascular drugs were procured in pure form from their manufacturers in India and screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive and gram-negative types . These bacteria were inhibited by the common antibiotics at 1-5 mg ml(-1) level through our earlier studies . Since most of the bacteria were moderate to highly responsive to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera of gram-positive and 15 genera of gram-negative bacteria . Most of these were inhibited by the drug at 50-200 microg ml(-1) level and few strains were sensitive even at lower concentrations (10 microg ml(-1)) . The bacteria could be arranged in the decreasing order of sensitivity towards amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella spp . and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the drug . Amlodipine was found to be bactericidal in nature when its mode of action was studied against S . aureus 6571, V . cholerae 14035 and Sh boydii 8 NCTC 254/66 . The antibacterial activity of amlodipine could also be confirmed in vivo . When it was given to Swiss strain of white mice at different dosages (30 and 60 microg/mouse), it could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . According to Chi square test the in vivo data were highly significant (p<0.001). Environ Mol Mutagen, 2004, 43(1), 10 - 9 Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter; Oda Y et al.; In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S . typhimurium strain NM2009 to detect genotoxic activity in small volume samples . The assay was used to test the genotoxicity of several standard mutagens and environmental samples . Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9 . The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity . The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain . Induction of umuC gene expression was detected more readily with CPRG than ONPG . The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan . The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons . Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples . J Mol Biol, 2004 Feb 6, 336(1), 81 - 92 Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily; Zaremba M et al.; The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence . Unlike other restriction enzymes, it functions without metal ions . The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily . Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically . BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer . On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer . The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second . Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus . It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily . BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence. J Mol Biol, 2004 Feb 6, 336(1), 25 - 42 Purine and pyrimidine-specific repression of the Escherichia coli carAB operon are functionally and structurally coupled; Devroede N et al.; Transcription of the carAB operon encoding the sole carbamoylphosphate synthetase of Escherichia coli proceeds from a tandem pair of promoters . P2, downstream, is repressed by arginine and the ArgR protein, whereas P1 is submitted to pyrimidine-specific regulation and as shown here to purine-specific control exerted by binding of the PurR protein to a PUR box sequence centered around nucleotide -128.5 with respect to the start of P1 transcription . In vivo analyses of the effects of trans and cis-acting mutations on the regulatory responses and single round in vitro transcription assays indicated that ligand-bound PurR is by itself unable to inhibit P1 promoter activity . To exert its effect PurR relies on the elaborated nucleoprotein complex that governs P1 activity in a pyrimidine-specific manner . Thus we reveal the existence of an unprecedented functional and structural coupling between the modulation of P1 activity by purine and pyrimidine residues that appears to result from the unique position of the PUR box in the carAB control region, far upstream of the promoter . Missing contact and premethylation binding interference studies revealed the importance of base-specific groups and of structural aspects of the PUR box sequence in complex formation . Permutation assays indicated that the overall PurR-induced bending of the carAB control region is slightly less pronounced than that of the purF operator . The PUR boxes of the carAB operon of E.coli and Salmonella typhimurium are unique in that they have a guanine residue at position eight . Interestingly, guanine at this position has been proposed to be extremely unfavorable on the basis of modeling and binding studies, as its exocyclic amino group would enter into a steric clash with the side-chain of lysine 55 . To analyze the effect of guanine at position eight in the upstream half-site of the carAB operator we constructed the adenine derivative and assayed in vivo repressibility of P1 promoter activity and in vitroPurR binding to the mutant operator, and constructed a molecular model for the unusual lysine 55-guanine 8 interaction. Am J Physiol Gastrointest Liver Physiol, 2004 Jun, 286(6), G1024 - 31 Epub 2004 Jan 22. Salmonella typhimurium SipA-induced neutrophil transepithelial migration: involvement of a PKC-alpha-dependent signal transduction pathway; Silva M et al.; Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen . We previously showed that S . typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN . Here we sought to determine the specific PKC isoforms that are induced by the S . typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration . In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S . typhimurium and recombinant SipA induced activation of PKC-alpha, -delta, and -epsilon . To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles . Inhibitors selective for PKC-alpha (Go-6976 and 2,2',3,3',4,4'-hexahydroxyl-1,1'-biphenyl-6,6'-dimethanoldimethyl ether) markedly reduced S . typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-delta (rottlerin) or PKC-epsilon (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN . These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy . Our results are the first to show that the S . typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-alpha is involved in the signal transduction cascade leading to PMN transepithelial migration. Vet Microbiol, 2004 Jan 14, 98(1), 37 - 43 Intestinal colonisation-inhibition and virulence of Salmonella phoP, rpoS and ompC deletion mutants in chickens; Methner U et al.; Administration of live Salmonella strains to day-old chicks provides profound protection against superinfection with a related strain within a matter of hours by a colonisation-inhibition mechanism, which is primarily a bacterial physiological process . Although currently available, commercial, live attenuated Salmonella vaccines induce protection by adaptive immunity, none of them is able to induce protection against Salmonella organisms by colonisation-inhibition and, therefore, they are unable to protect newly-hatched birds immediately after oral vaccination . In this study, mutants of Salmonella Typhimurium and Enteritidis with deletions in phoP and rpoS, either alone or in combination with ompC, were characterised and tested for their level of attenuation and their ability to inhibit the intestinal colonisation of the isogenic parent strains in chickens . Mutants with deletions only in rpoS demonstrated an unaffected potential to inhibit the intestinal colonisation of the challenge strain but were still fully virulent for the chickens . Mutants with deletions in phoP, either alone or in combination with rpoS, resulted in a high level of attenuation, unimpaired ability to colonise the gut and a nearly unaffected potential to inhibit the challenge strain from caecal colonisation . Mutants with an additional deletion in ompC revealed a reduced capacity of intestinal colonisation-inhibition when compared to the control strains and both the single rpoS and the phoP deletion mutants . Mutations in phoP- or phoP-regulated genes may therefore be used for the development of live attenuated Salmonella vaccines possessing these novel characteristics. J Endotoxin Res, 2003, 9(6), 381 - 4 The dual role of LBP and CD14 in response to Gram-negative bacteria or Gram-negative compounds; Heumann D et al.; Innate immunity initiates protection of the host organism against invasion of micro-organisms by specific recognition mechanisms . This article reviews the dual role of LBP/CD14 in innate immunity, focusing mostly on experiments performed in mice by the authors . LPS induces uncontrolled pro-inflammatory response that kills the host and is LBP- and CD14-dependent, as neutralization of LBP and CD14 prevents lethal shock . However, surprisingly, the synthetic Pam3CysSerLys4 bacterial lipoprotein from Escherichia coli (BLP), which is well tolerated in mice, kills the mice upon LBP or CD14 blockade . Furthermore, after blockade of LBP and CD14, the mice succumb to a challenge with virulent Klebsiella pneumoniae or Salmonella typhimurium . Therefore, host responses to Gram-negative bacteria are not identical to that of LPS or BLP . When the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted . Under these conditions, LBP, CD14, and likely Toll-like receptors (TLRs) are a prerequisite to trigger a pro-inflammatory response of macrophages, which is crucial for keeping an infection under control . These studies indicate that we are very far from understanding how the innate system works and more work needs to be done concerning LBP, CD14 or TLRs . Therefore, caution should be the rule about the use of therapeutic approaches to block the pro-inflammatory response in Gram-negative infections. Int J Antimicrob Agents, 2004 Jan, 23(1), 99 - 102 Studies on the antibacterial potentiality of isoflavones; Dastidar SG et al.; The isoflavonoid compounds 'YS11-YS21' were screened for possible antimicrobial property against 12 known Gram-positive and Gram-negative sensitive bacteria . YS11 and YS16 failed to show antimicrobial activity and YS12, 13, 14, 15, 17, 18 and 20 had moderate antimicrobial action . Compounds YS19 and YS21 showed pronounced antimicrobial property . YS19 and YS21 were then tested in vitro against 214 strains of bacteria from one Gram-positive and six Gram-negative genera . The minimum inhibitory concentration (MIC) of YS19 and YS21 was determined by agar dilution method and ranged from 25 to 200 mg/l in most strains . At concentrations of 30 and 60 microg/mouse these compounds offered significant protection to mice challenged with 50 median lethal dose (MLD) of a virulent strain of Salmonella Typhimurium. J Antimicrob Chemother, 2004 Feb, 53(2), 266 - 70 Epub 2004 Jan 16. Antimicrobial susceptibility of Salmonella isolated from cattle, swine and poultry (2001-2002): report from the Japanese Veterinary Antimicrobial Resistance Monitoring Program; Esaki H et al.; OBJECTIVES: The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) Program was established in 1999 to examine the susceptibility of bacteria from food-producing animals to antimicrobial agents . This study tested the susceptibility of Salmonella isolates collected during 2001-2002 to 20 antimicrobials.Materials and methods: MICs of antimicrobial agents were determined using the NCCLS agar dilution method, and interpreted according to breakpoints obtained from the bimodal MIC distributions . RESULTS: A total of 82 Salmonella were isolated from food-producing animals and tested for antimicrobial susceptibility . Isolates resistant to ampicillin, dihydrostreptomycin, kanamycin, oxytetracycline, chloramphenicol, bicozamycin, nalidixic acid, oxolinic acid and trimethoprim were obtained from healthy animals and diagnostic sample submissions . Salmonella Dublin was isolated only from cattle and showed resistance to older quinolones . Resistance to ampicillin, dihydrostreptomycin, kanamycin and oxytetracycline was common across all serotypes . Fluoroquinolone-resistant Salmonella Choleraesuis was isolated from swine and was the first Japanese report on this type of resistance in Salmonella from an animal origin . Most Salmonella Typhimurium isolates showed resistance to ampicillin, chloramphenicol, dihydrostreptomycin and oxytetracycline . S . Typhimurium DT104 accounted for 40.7% of S . Typhimurium isolates and was more often multi-drug resistant . Most Salmonella Infantis isolates from poultry showed resistance to dihydrostreptomycin, oxytetracycline, trimethoprim or kanamycin . In Salmonella Enteritidis, the major serotype isolated from food-poisoning in Japan, only resistance to dihydrostreptomycin was observed . CONCLUSIONS: This is the first JVARM report of Salmonella isolates, and continuous investigations at the national level on antimicrobial resistance in Salmonella isolated from food-producing animals will be important in the JVARM Program. J Biol Chem, 2004 Apr 2, 279(14), 13555 - 63 Epub 2004 Jan 16. Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on M-CSF/RANKL-induced in vitro osteoclastogenesis; Saito K et al.; Bacterial infection sometimes impairs bone metabolism . In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guerin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method . A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-alpha receptor family, was found to be one of the up-regulated genes . Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG . Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL) . Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-kappaB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells . Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation . A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling . r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-kappaB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway . r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2 . This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis. Carcinogenesis, 2004 May, 25(5), 801 - 7 Epub 2004 Jan 16. Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido {2,3-b}indole (MeAalphaC) in recombinant test systems expressing human xenobiotic-metabolizing enzymes; Glatt H et al.; 2-Amino-3-methyl-9H-pyrido{2,3-b}indole (MeAalphaC) and some metabolites were investigated for mutagenicity in mammalian cell lines and bacterial strains engineered for the expression of human enzymes . MeAalphaC induced gene mutations (studied at the hprt locus) in Chinese hamster V79-derived cells co-expressing cytochrome (CYP) 1A2 and sulphotransferase (SULT) 1A1 even at a concentration of 30 nM, but was inactive in cells co-expressing CYP1A2 and N-acetyltransferase (NAT) 1 or 2 . MeAalphaC, tested in the presence of rat liver post-mitochondrial fraction, showed strongly enhanced mutagenicity in a Salmonella typhimurium strain expressing human SULT1A1 compared with the control (recipient) strain TA1538/1,8-DNP (deficient in endogenous acetyltransferase) . Mutagenicity was also enhanced, although to a lesser extent, when NAT2 was expressed in the latter strain . The metabolite, 2-hydroxylamino-3-methyl-9H-pyrido{2,3-b}indole (N-OH-MeAalphaC) was a direct mutagen to strains TA1538 and TA1538/ 1,8-DNP . This mutagenicity was strongly enhanced in corresponding strains expressing SULT1A1 . A moderate enhancement was observed when SULT1A2, SULT1B1, SULT1C2 or NAT2 were expressed in strain TA1538 . The remaining enzymes studied (SULT1A3, 1C1, 1E1, 2A1, 2B1a, 2B1b, 4A1 and NAT1) did not indicate any activation of N-OH-MeAalphaC . Preliminary mutagenicity experiments in SULT-expressing S.typhimurium strains were conducted with other hydroxylated metabolites of MeAalphaC . The phenols, 6- and 7-hydroxy-MeAalphaC, were inactive under the conditions studied . The benzylic alcohol, 2-amino-3-hydroxymethyl-9H-pyrido{2,3-b}indole, was mutagenic in a strain expressing SULT1A1, but its activity was much weaker than that of N-OH-MeAalphaC . Thus, N-hydroxylation (e.g . mediated by CYP1A2) and sulpho conjugation (primarily mediated by SULT1A1) was the dominating activation pathway of MeAalphaC in model systems engineered for human enzymes . Some other SULT forms as well as NAT2 were also capable of activating N-OH-MeAalphaC, although with much lower efficiency than SULT1A1 . Another minor activation pathway involved benzylic hydroxylation followed by sulpho conjugation by SULT1A1. Mutat Res, 2004 Feb 14, 557(2), 137 - 49 Effects of paving asphalt fume exposure on genotoxic and mutagenic activities in the rat lung; Zhao HW et al.; Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs) . Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung . In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied . Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day . Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay . To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored . Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo{a}pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029 . This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3) . The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner . In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479+/-33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S . typhimurium YG1024 or YG1029 . At a higher total asphalt fume exposure level (1150+/-63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control . However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test . These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content. J Mol Biol, 2004 Jan 30, 335(5), 1151 - 71 Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup; Scholl D et al.; We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively . The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities . The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized . The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats . About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage . The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved . Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies. J Appl Microbiol, 2004, 96(2), 271 - 8 Oxidation-reduction potential regulates RpoS levels in Salmonella Typhimurium; Komitopoulou E et al.; AIMS: The aim of this work was to investigate the connection between oxidation-reduction (redox) potential and stationary phase induction of RpoS in Salmonella Typhimurium . METHODS AND RESULTS: A lux-based reporter was used to evaluate RpoS activity in S . Typhimurium pure cultures . During growth of S . Typhimurium, a drop in the redox potential of the growth medium occurred at the same time as RpoS induction and entry into stationary phase . An artificially induced decrease in redox potential earlier during growth reduced the time to RpoS induction and Salmonella entered the stationary phase prematurely . In contrast, under high redox conditions, Salmonella grew unaffected and entered the stationary growth phase as normal, although RpoS induction did not occur . As a consequence, stationary phase cells grown in the high redox environment were significantly more heat sensitive (P < 0.05) than those grown under normal conditions . CONCLUSIONS: This work suggests that redox potential can regulate RpoS levels in S . Typhimurium and can thus, control the expression of genes responsible for thermal resistance . SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to manipulate RpoS induction and control stationary phase gene expression can have important implications in food safety . Early RpoS induction under low redox potential conditions can lead to enhanced resistance in low cell concentrations to inimical processes such as heat stress . Inhibition of RpoS induction would abolish stationary phase protective properties making cells more sensitive to common food control measures. Indian J Med Res, 2003 Nov, 118, 192 - 6 Antibacterial potential of an antispasmodic drug dicyclomine hydrochloride; Karak P et al.; BACKGROUND & OBJECTIVES: Several compounds are known to possess antimicrobial activity in addition to their predesignated pharmacological actions . In the present study, dicyclomine hydrochloride, an antispasmodic drug, was tested for possible antimicrobial property in vitro and in vivo . METHODS: The minimum inhibitory concentration (MIC) of dicyclomine against the bacteria was determined by agar and broth dilution methods in vitro . The antibacterial activity of dicyclomine was confirmed by animal experiments . Toxicity and protective efficacy of the drug were tested in vivo . RESULTS: Dicyclomine inhibited most of the bacterial isolates tested at 25-100 microg/ml concentration, and a few were sensitive even at a lower concentration (10 microg/ml) . Dicyclomine was found to be bacteriostatic in nature against Shigella dysenteriae 7, and bactericidal against S . aureus NCTC 6571, 8530, and 8531 . When administered to Swiss white mice at doses of 30 and 60 microg/mouse, dicyclomine protected the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . INTERPRETATION & CONCLUSION: Dicyclomine showed inhibitory action against several pathogenic bacteria . It also offered significant protection to mice against the bacterial challange . As dicyclomine is in routine therapeutic use, it may be developed as a potent antimicrobial agent in many infections. Poult Sci, 2003 Dec, 82(12), 1886 - 97 Effect of intravenous endotoxin on blood cell profiles of broilers housed in cages and floor litter environments; Wang W et al.; Commercial broilers are constantly exposed to airborne microorganisms and endotoxin (lipopolysaccharide, LPS) . It has been shown that microbial contamination of the air was higher in broiler houses using floor litter than in broiler houses using netting-type floors . The current study evaluated the effect of housing conditions on blood leukocyte profiles and tested the hypothesis that, when compared to broilers reared in clean stainless steel cages (Cage group), broilers raised on floor litter (Floor group) should experience a higher environmental challenge and have a desensitized immune system that may exhibit better tolerance/resistance to subsequent intravenous LPS challenge . Hematological parameters were evaluated prior to and following i.v . administration of 1 mg/kg BW Salmonella typhimurium LPS (dissolved at 1 mg/0.25 mL in PBS) or i.v . injection of 0.25 mL/kg BW PBS alone . The results showed that prior to LPS/PBS injection, broilers in the cage group had higher heterophil and monocyte concentrations, a higher B cell percentage within the lymphocyte population, and a higher heterophil to lymphocyte (H:L) ratio in the blood . The i.v . LPS injection resulted in 25% mortality in the cage group and 42% mortality in the floor group within 8 h post-injection . LPS reduced the concentrations of total white blood cells (WBC) and all differential WBC except eosinophils and increased thrombocyte concentrations within 1 h post-injection in both groups . All of these values returned to their respective pre-injection levels within 48 h post-injection in the surviving birds . The two groups exhibited similar overall hematological changes after LPS injection except that the cage group showed a higher H:L ratio at 8 h post-injection and a lower B-cell percentage within the lymphocyte population at 48 h post-injection when compared with the floor group . We concluded that the immune systems of broilers reared on floor litter were desensitized and exhibited less pronounced leukocyte responses to i.v . LPS when compared with those of broilers reared in clean stainless steel cages . However, such desensitization of the immune system did not help broilers survive subsequent i.v . LPS challenge. J Food Prot, 2004 Jan, 67(1), 178 - 80 Salmonella in sesame seed products; Brockmann SO et al.; In the context of an international outbreak of multiresistant Salmonella Typhimurium DT 104 that was correlated to the consumption of halvah ("helva," an Asian candy made from sesame seed), we examined several sesame seed products for the occurrence of Salmonella . Of 117 ready-to-eat food items containing sesame, we isolated salmonellae from 11 (9.4%) samples . In addition to finding Salmonella Typhimurium DT 104 in the halvah involved in the outbreak, we also isolated different Salmonella Typhimurium strains out of halvah from other manufacturers and countries of origin, as well as Salmonella Offa, Salmonella Tennessee, and Salmonella Poona from sesame paste (tahini) and sesame seed, which is sold for raw consumption in cereals. J Food Prot, 2004 Jan, 67(1), 156 - 61 Antigenotoxic effects of water extract from Korean fermented soybean paste (doen-jang); Kim JG; Aflatoxin B1 is a major metabolite of the toxigenic molds Aspergillus flavus and Aspergillus parasiticus . In this study, a bacterial reverse mutation assay with Salmonella Typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 and an in vitro chromosome aberration test with Chinese hamster lung (CHL) cells were used to investigate the genotoxicity of water extract from Korean soybean paste (doen-jang {dwen-jahng}) and its antigenotoxic activity against aflatoxin B1 . The water extract itself did not exhibit cytotoxicity or mutagenicity . The extract significantly reduced the numbers of revertants when it was added to the assay system with Salmonella Typhimurium TA100 (P < 0.05) . The extract also exhibited significant inhibitory effects on chromosome aberration in CHL cells (P < 0.05) . Dose-response relationships were observed between the concentration of the water extract and both its antimutagenic effect and its suppression of chromosome aberration . The results of this work indicate that water extract from Korean soybean paste could have potential as an antigenotoxic substance. J Food Prot, 2004 Jan, 67(1), 148 - 55 Antimicrobial and antioxidant activities of natural extracts in vitro and in ground beef; Ahn J et al.; Inhibition of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes by grape seed extract (ActiVin) and pine bark extract (Pycnogenol) and the effect of these natural extracts on the oxidative stability of raw ground beef were studied . In an agar dilution test, the MICs of ActiVin and Pycnogenol were determined to be 4.0 mg/ml for 4.43 log CFU per plate of E . coli O157:H7 and 4.0 mg/ml for 4.38 log CFU per plate of L . monocytogenes . In an inhibition curve test, populations of E . coli O157:H7, Salmonella Typhimurium, and L . monocytogenes fell to below the detection limit (10 CFU/ml) after 16 h of incubation . The numbers of E . coli O157:H7, L . monocytogenes, and Salmonella Typhimurium declined by 1.08, 1.24, and 1.33 log CFU/g, respectively, in raw ground beef treated with 1% Pycnogenol after 9 days of refrigerated storage . ActiVin (1%) and oleoresin rosemary (1%) resulted in an approximately 1-log CFU/g reduction in the populations of all three pathogens after 9 days . The addition of 1% ActiVin and Pycnogenol contributed to the maintenance of an acidic pH of 5.80 and 5.58, respectively, in raw ground beef . Compared to the control, all treatments increased in L* (lightness), with the exception of ActiVin . ActiVin and oleoresin rosemary had the highest a* (redness) and b* (yellowness) values, respectively . ActiVin most effectively retarded lipid oxidation, followed by Pycnogenol . The results suggest that these natural extracts have potential to be used with other preservative methods to reduce pathogenic numbers, lipid oxidation, and color degradation in ground beef. J Food Prot, 2004 Jan, 67(1), 53 - 9 Modeling the boundaries of growth of Salmonella Typhimurium in broth as a function of temperature, water activity, and pH; Koutsoumanis KP et al.; The growth limits of a mixture of five strains of Salmonella Typhimurium in tryptic soy broth were examined at different environmental conditions . The response of the pathogen was monitored in a total of 350 combination treatments of temperature (10 to 35 degrees C), pH (3.76 to 6.44), and water activity (aw, 0.913 to 0.990) for 62 days . No growth/growth (turbidity) data were modeled by logistic polynomial regression . The concordance index of the logistic model was 99.8%, indicating a good fit to the observed data . The minimum pH and aw values that permitted growth were 3.94 and 0.942, respectively, and occurred in the temperature range of 25 to 35 degrees C . At temperatures below this range, the minimum pH and aw allowing growth increased as the temperature decreased . The results showed an abrupt change in the probability of growth close to the boundary with minor changes of the environmental factors . The probabilities predicted by the model were compared with published data on the actual response of Salmonella Typhimurium or other salmonellae serotypes in 50 cases of food products, including salad dressing, mayonnaise, meat, cheese, vegetables, and fruits . The model predicted successfully the response of the pathogen in 90% of the tested cases . The results of the study indicated that the developed model predicts satisfactorily the growth/no growth interface of Salmonella Typhimurium in foods and can provide useful quantitative data for the development of safer food products and processes. J Food Prot, 2004 Jan, 67(1), 46 - 52 A rapid and automated fiber optic-based biosensor assay for the detection of Salmonella in spent irrigation water used in the sprouting of sprout seeds; Kramer MF et al.; Recent outbreaks of foodborne illness have been linked to the consumption of contaminated sprouts . The spent irrigation water used to irrigate sprouts can carry many microorganisms, including pathogenic strains of Escherichia coli and Salmonella enterica . These pathogens are believed to originate from the seeds . The U.S . Food and Drug Administration recommends that sprout producers conduct microbiological testing of spent irrigation water from each production lot at least 48 h after seeds have germinated . Microbial analysis for the detection of Salmonella is labor-intensive and takes days to complete . A rapid and automated fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water was developed in this study . Alfalfa seeds contaminated with various concentrations of Salmonella Typhimurium were sprouted . The spent irrigation water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research International, Monroe, Wash.), an automated fiber optic-based detector . Salmonella Typhimurium could be positively identified in spent irrigation water when seeds were contaminated with 50 CFU/g . Viable Salmonella Typhimurium cells were also recovered from the waveguides after the assay . This biosensor assay system has the potential to be directly connected to water lines within the sprout-processing facility and to operate automatically, requiring manual labor only for preventative maintenance . Therefore, the presence of Salmonella Typhimurium in spent irrigation water could be continuously and rapidly detected 3 to 5 days before the completion of the sprouting process. J Food Prot, 2004 Jan, 67(1), 27 - 33 Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR; Li Y et al.; With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella . Under the optimized conditions, the assay yielded a 252-bp product from E . coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively . When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed . In the specificity test, 10 E . coli O157:H7 strains and one E . coli O157:NM strain showed the expected 252-bp amplicon . Seven other E . coli strains yielded no signal . Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species . No nonspecific amplification was observed with DNA from 48 other bacterial strains . Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts . The whole procedure can be easily completed within 30 h . The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E . coli O157:H7, Salmonella, and/or Shigella. J Food Prot, 2004 Jan, 67(1), 4 - 11 An assessment of the microbiological risks involved with egg washing under commercial conditions; Hutchison ML et al.; The potential benefits of washing eggs is offset by a historical perception in the European Union that wetted eggs are prone to spoilage and water loss . This study describes the effects of spray jet washing under various processing conditions to shell surface counts of Salmonella and the presence of bacteria in egg contents . Experiments used eggs that were contaminated with Salmonella Enteritidis PT4 or Salmonella Typhimurium DT104 before cuticle hardening . Washing of contaminated eggs under optimum conditions resulted in a more than 5-log reduction of Salmonella counts from the shell surface . Salmonella was not isolated from the yolk or albumen of any egg washed by the optimal protocol, suggesting that when properly controlled, egg washing did not cause Salmonella to enter the contents . However, contamination did arise if strict control was not maintained over the wash and rinse water temperatures . Both Salmonella Enteritidis and Salmonella Typhimurium were shown to enter the egg contents when water temperatures were lowered, indicating that strict temperature control must be maintained in order to prevent the ingress of Salmonella into egg contents . Other washing machine parameters that were investigated did not significantly affect Salmonella entry into the egg contents but influenced shell surface kill levels to varying degrees. Am J Physiol Gastrointest Liver Physiol, 2004 Jun, 286(6), G1050 - 8 Epub 2004 Jan 08. Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice; Clarke LL et al.; Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen . Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice . On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine . Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion . Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution . Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates . Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine . Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis . Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Avian Dis, 2003 Oct-Dec, 47(4), 1474 - 80 Infection models for Salmonella typhimurium DT110 in day-old and 14-day-old broiler chickens kept in isolators; Bjerrum L et al.; A series of experiments was undertaken to investigate the infection dynamics of various doses of S . typhimurium in day-old and 14-day-old broiler chickens kept in isolators . The infections were followed quantitatively in ceca and ileum by enumerating the colony forming units (cfu) of the challenge strain . It was found that the inoculation of 10(7) cfu of S . typhimurium to day-old chickens established stable cecal infection in all the animals for 35 days . For 14-day-old chickens, stable and lasting infections were seen with inoculation of 10(9) cfu . Lower doses yielded more variable results, and the bacteria were rapidly eliminated from most birds, especially in 14-day-old inoculated chickens . Salmonella was found in spleen and liver 2-3 days postinoculation . Salmonella was cleared from both organs or reduced to very low numbers within 3 weeks. Mutat Res, 2004 Jan 10, 557(1), 99 - 108 Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list; Yan J et al.; The photomutagenicity of 16 polycyclic aromatic hydrocarbons (PAHs), all on the United States Environmental Protection Agency (US EPA) priority pollutant list, was studied . Concomitant exposing the Salmonella typhimurium bacteria strain TA102 to one of the PAHs and light (1.1 J/cm2 UVA+2.1 J/cm2 visible) without the activation enzyme S9, strong photomutagenic response is observed for anthracene, benz{a}anthracene, benzo{ghi}perylene, benzo{a}pyrene, indeno{1,2,3-cd}pyrene, and pyrene . Under the same conditions, acenaphthene, acenaphthylene, benzo{k}fluoranthene, chrysene, and fluorene are weakly photomutagenic . Benzo{b}fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenz{a,h}anthracene are not photomutagenic . These results indicate that PAHs can be activated by light and become mutagenic in Salmonella TA102 bacteria . At the same time, the mutagenicity for all the 16 PAHs was examined with the standard mutagenicity test with 10% S9 as the activation system . Benzo{b}fluoranthene, benzo{k}fluoranthene, chrysene, acenaphthylene, and fluorene are weakly mutagenic, while the rest of the PAHs are not . In general, the photomutagenicity of PAHs in TA102 does not correlate with their S9-activated mutagenicity in either TA102 or TA98/TA100 since they involve different activation mechanisms. Mutat Res, 2004 Jan 10, 557(1), 85 - 97 Genotoxicity studies with pure trans-capsaicin; Chanda S et al.; Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin . However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles . Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances . These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays . In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range . trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range . Limited evidence for very weak activity was also obtained in the absence of S9 mix . trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen . Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes . Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear . Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations. Nucleic Acids Res, 2004 Jan 02, 32(1), 143 - 50 Print 2004. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes; Nahvi A et al.; Recent studies have begun to reveal that numerous fundamental metabolic pathways in bacteria are regulated by riboswitches residing within certain messenger RNAs . These riboswitches selectively bind metabolites and modulate gene expression in response to changing ligand concentrations . Previously, we provided evidence that the btuB mRNAs of Escherichia coli and Salmonella typhimurium each carry a coenzyme B12-dependent riboswitch that causes repressed translation of the encoded cobalamin-transport protein at elevated coenzyme concentrations . Herein, we use a phylogenetic analysis to define a consensus sequence and secondary structure model for the ligand- binding domain of this riboswitch class . RNA structures that conform to this model are widespread in both Gram-positive and Gram-negative organisms . In addition, we find that the 5'-untranslated region (5'-UTR) of the cobalamin biosynthesis (cob) operon of S.typhimurium carries an RNA motif that matches this consensus sequence . Biochemical and genetic characterization of this motif confirms that the RNA directly binds coenzyme B12, and that it likely serves as a genetic control element for regulating expression of the 25-gene operon for cobalamin production in this pathogen. J Am Soc Mass Spectrom, 2004 Jan, 15(1), 1 - 11 Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem mass spectrometry with electrospray ionization; Hsu FF et al.; Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization . The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)) . This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule . The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., {M - H - R'(2)CH=CO}(-) > {M - H - R(2)CO(2)H}(-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., {M - H - R(1)CO(2)H}(-) > {M - H - R'(1)CH=CO}(-); {M - H - R(3')CO(2)H}(-) > {M - H -R'(3')CH=CO}(-)) . The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS) . However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of {M - H - R'(2)CH=CO - R(3)CO(2)H - 74}(-) > {M - H - R'(2)CH=CO - R(1)CO(2)H - 74}(-) z.Gt; {M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74}(-) . This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents . Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated . The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined. Mutat Res, 2004 Jan 12, 545(1-2), 37 - 47 Biphasic effects of the flavonoids quercetin and naringenin on the metabolic activation of 2-amino-3,5-dimethylimidazo{4,5-f}quinoline by Salmonella typhimurium TA1538 co-expressing human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and cytochrome b5; Kang IH et al.; Heterocyclic amines (HCAs) produced by cooking meat products at high temperatures are promutagens that are activated by cytochrome P450 (CYP) lA2 . Using a newly developed Salmonella typhimurium TA1538/1A2bc-b5 strain, we tested the effect of quercetin and naringenin on the mutagenicity of 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) . TA1538/1A2bc-b5 bears two plasmids, one expressing human CYP1A2 and NADPH-P450 reductase (NPR), and the other plasmid which expresses human cytochrome b5 (cyp b5) . TA1538/1A2bc-b5 cells showed high activities of 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) associated with CYP1A2 and are very sensitive to mutagenesis induced by several HCAs . MeIQ was found to be the strongest mutagen among the HCAs tested in this system . Mutagenicity of MeIQ was enhanced 50 and 42% by quercetin at 0.1 and 1 microM, respectively, but suppressed 82 and 96% at 50 and 100 microM . Naringenin also increased the MeIQ-induced mutation about 37 and 22% at 0.1 and 1 microM, but suppressed it 32 and 63% at 50 and 100 microM concentrations, respectively, in TA 1538/1A2bc-b5 cells . Thus, they stimulated the MeIQ induced mutation at low concentrations, but strongly suppressed it at high concentrations . This biphasic effect of flavonoids was due to the stimulation or the inhibition of CYP1A2 activity in a dose-dependent manner judging by the activities of EROD or MROD in the Salmonella cells . These results indicate that quercetin and naringenin can exhibit inhibitory or stimulating effects on CYP1A2 mediated mutagenesis by MeIQ, depending on their concentrations. Mutat Res, 2004 Jan 12, 545(1-2), 11 - 21 Mutagenicity of benzo{b}phenanthro{2,3-d}thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT; Kumar S et al.; Benzo{b}phenanthro{2,3-d}thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S . typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006 . Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9 . In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9 . Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9 . BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9 . In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation . In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9 . Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide . The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S . typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products . Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer. Teratog Carcinog Mutagen, 2003, Suppl 2, 31 - 41 Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents; Harrington DJ et al.; Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells . However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity . For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102) . DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes . The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells . The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis . Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele . However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells . Lett Appl Microbiol, 2004, 38(1), 8 - 12 The effect of copper on the death rate of Salmonella typhimurium DT104:30 in food substrates acidified with organic acids; Beal JD et al.; AIMS: The objective of this study was to investigate the effect of copper ions on the survival of Salmonella typhimurium DT104:30 in acidified liquid food substrates . METHODS AND RESULTS: The decimal reduction time (Dvalue) of Salm . typhimurium DT104:30 was determined in acidified liquid pig feed (LPF) and skimmed milk (SM) containing a range of copper concentrations (0-50 ppm) . As copper concentration increased, the death rate of Salm . typhimurium DT104:30 increased . In LPF acidified with 150 mmol l-1 lactic acid the presence of 50 ppm copper resulted in a 10-fold increase in the death rate . CONCLUSIONS: The presence of copper salts in acidic liquid food substrates significantly increases the death rate of Salm . typhimurium DT104:30 . SIGNIFICANCE AND IMPACT OF THE STUDY: This finding could influence policy on levels of copper in pig feed. J Gastroenterol Hepatol, 2003 Dec, 18(12), 1384 - 91 Helicobacter pylori-induced enlarged-fold gastritis is associated with increased mutagenicity of gastric juice, increased oxidative DNA damage, and an increased risk of gastric carcinoma; Nishibayashi H et al.; BACKGROUND AND AIM: The severe inflammation, increased cell proliferation and marked acid inhibition observed in subjects with Helicobacter pylori-associated enlarged-fold gastritis suggest that enlarged-fold gastritis may be a risk factor for gastric carcinoma . The purpose of the present study was to determine whether a relationship exists between enlarged-fold gastritis and gastric carcinoma . METHODS: One hundred and thirty-five H . pylori-positive patients with early gastric carcinoma and 141 age- and sex-matched H . pylori-positive controls without gastric carcinoma were involved in the study . The widths of gastric body folds were measured by double-contrast radiographs . The mutagenicity of gastric juice was assayed using the Ames test and Salmonella typhimurium TA-98 or TA-100 with S9-mix . Levels of 8-hydroxydeoxyguanosine (8-OHdG) in gastric mucosa were examined using high-performance liquid chromatographic-electrochemical detection . RESULTS: An upward shift in the distribution of gastric fold widths in H . pylori-positive patients with early gastric carcinoma was found . Enlarged-fold gastritis (fold width >/=5 mm) was observed in 81% of the patients with gastric carcinoma, compared with 46% of H . pylori-positive controls . The odds ratio for gastric carcinoma increased with increasing fold width to a maximum of 35.5 in persons with fold width >/=7 mm . The prevalence of diffuse-type early gastric carcinoma in the body region increased with increasing fold width . The mutagenicity of gastric juice from the patients with enlarged-fold gastritis was significantly higher than that in H . pylori-negative controls and H . pylori-positive patients without enlarged folds . Mucosal 8-OHdG levels in the body region of patients with enlarged-fold gastritis were significantly higher than in H . pylori-negative controls and H . pylori-positive patients without enlarged-fold gastritis . Eradication of H . pylori significantly decreased the mutagenicity of gastric juice and 8-OHdG levels in the gastric mucosa from patients with enlarged-fold gastritis . CONCLUSION: A significant association is suggested between enlarged-fold gastritis and gastric carcinoma. Environ Mol Mutagen, 2003, 42(4), 233 - 42 Genetic toxicity of methamphetamine in vitro and in human abusers; Li JH et al.; Methamphetamine (METH) is a widely abused psychomotor stimulant . Although numerous studies have examined METH-induced neurotoxicity, its ability to produce genotoxic effects has not been evaluated . In this article, we report on the genotoxicity of METH in vitro and in human METH abusers . METH induced his(+) revertants in Salmonella typhimurium strains TA98 and TA100, and increased the frequency of hprt mutants, micronuclei, and sister chromatid exchange (SCE) in cultured Chinese hamster ovary K1 (CHO-K1) cells . These METH-induced genotoxic effects were eliminated if METH exposure was conducted in the presence of rat liver S9, indicating that the genotoxicity was caused by METH, and not by metabolites of METH . In addition, reactive oxygen species (ROS) scavengers inhibited the METH-induced micronuclei in CHO-K1 cells . Further investigation with 76 human long-term METH abusers and 98 unexposed controls demonstrated that total METH exposure correlated with micronucleus and SCE frequencies in cultured lymphocytes . The results of this study indicate that METH is a genotoxic agent and that ROS may play a role in METH-induced genotoxicity . BMC Mol Biol . 2003 Dec 13;4(1):11. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E . coli; Murphy KC et al.; BACKGROUND: The lambda Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E . coli such as EHEC and EPEC . Our early experiments with the use of lambda Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E . coli . RESULTS: In this report, we have identified conditions that optimize the use of lambda Red for recombineering in EHEC and EPEC . Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes . In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors . We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (approximately 1 kb of flanking homology); both work well and each have their own advantages . The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam . This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin . Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC . CONCLUSIONS: The lambda Red recombineering technology has been optimized for use in pathogenic species of E . coli, namely EHEC and EPEC . As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA . These results should encourage the use of lambda Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development. J Food Prot, 2003 Dec, 66(12), 2302 - 6 A rapid most-probable-number-based enzyme-linked immunosorbent assay for the detection and enumeration of Salmonella Typhimurium in poultry wastewater; Goodridge C et al.; The rapid and accurate detection and enumeration of low levels of Salmonella Typhimurium in food processing facilities are critical components of an effective hazard analysis critical control point program . The objective of this study was to develop a rapid (8 h) most probable number (MPN)-enzyme-linked immunosorbent assay (ELISA) for the detection and enumeration of Salmonella Typhimurium in wastewater . The specific objectives were to (i) characterize poly- and monoclonal Salmonella Typhimurium-specific antibodies in order to select the most specific and sensitive antibody for Salmonella Typhimurium detection, and (ii) validate the MPN assay through a correlation between the 8-h MPN-ELISA and the traditional 48-h Salmonella Typhimurium MPN method in poultry scald water . Poultry scald water samples were spiked with 10 and 50 CFU/ml of Salmonella Typhimurium . The traditional MPN method used a 48-h enrichment period followed by an analysis, while the MPN-ELISA used a 5-h enrichment period followed by a 3-h ELISA analysis . No differences (P < 0.05) were found between the traditional MPN and the MPN-ELISA, indicating the promise of the MPN-ELISA for the rapid detection and enumeration of Salmonella Typhimurium within an 8-h shift . This abbreviated assay will permit increased product sampling and more rapid movement of food between production and processing, resulting in reduced spoilage and quality losses. Trends Microbiol, 2003 Dec, 11(12), 562 - 9 Translating tissue culture results into animal models: the case of Salmonella typhimurium; Hurley BP et al.; Investigators use both in vitro and in vivo models to better understand infectious disease processes . Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models . In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses . Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models . Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process. Int J Antimicrob Agents, 2003 Dec, 22(6), 613 - 7 Activity of combinations of ceftazidime, imipenem and pefloxacin against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa; El-Sukhon SN et al.; The chequerboard technique was used to look for synergistic combinations of ceftazidime, imipenem and pefloxacin . The synergistic combinations were used in vivo in mice experimentally infected with Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa . In vitro ceftazidime/imipenem, ceftazidime/pefloxacin and pefloxacin/imipenem combinations showed synergistic effects against Staphylococcus aureus and S . typhimurium and additive effects against P . aeruginosa . Only the ceftazidime/pefloxacin combination was synergistic against E . coli while the ceftazidime/imipenem and pefloxacin/imipenem combinations resulted in additive effects . In vivo, combination of ceftazidime/imipenem against E . coli infection and the pefloxacin/imipenem combination against S . typhimurium infection were protective. Mutat Res, 2003 Dec 9, 542(1-2), 89 - 97 Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism; Park KK et al.; Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression . A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage . Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities . However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds . Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms . In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction . Porphyrins exert a dose-related inhibition of his(+) reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH) . DNA strand breaks by ROS derived from H(2)O(2)/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H(2)O(2)/UV also were inhibited markedly by porphyrins in a concentration-dependent manner . Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H(2)O(2) formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA . These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion. Mol Immunol, 2004 Jan, 40(10), 671 - 9 Activation of innate immune responses by IL-2-expressing Salmonella typhimurium is independent of Toll-like receptor 4; al-Ramadi BK et al.; We previously demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium, engineered to express IL-2 (strain GIDIL2), is cleared more rapidly than its parental, non-cytokine-expressing, strain (designated BRD509) from the reticuloendothelial system of susceptible BALB/c mice . This early clearance correlated with the induction of a strong innate immune response within a few hours of administration of GIDIL2 organisms . In the present study, we wished to assess the contribution of LPS recognition to GIDIL2-induced immune responses using Toll-like receptor 4 (TLR4) mutant C3H/HeJ mice . In contrast to LPS responder mice, both BRD509 and GIDIL2 strains persisted at higher levels in LPS non-responder animals . However, the GIDIL2 bacterial loads in the peritoneal cavity and spleen, recovered over a period of 21 days post infection, were consistently lower than the corresponding CFUs of the BRD509 strain . Direct evidence for the induction of innate immunity was shown by demonstrating increased NK cell cytotoxicity, NOS2 gene expression, and nitric oxide synthesis by peritoneal cells obtained as early as 2h after infection with GIDIL2, but not BRD509, organisms . Unlike BALB/c mice, however, these responses failed to afford any protection against virulent challenge in C3H/HeJ mice . Taken together, our data demonstrate that despite the induction of innate immune responses by IL2-expressing organisms, this was not sufficient to induce protection in TLR4 mutant mice. J Immunol, 2003 Dec 1, 171(11), 6046 - 51 Human C-reactive protein does not protect against acute lipopolysaccharide challenge in mice; Hirschfield GM et al.; The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo . Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS . However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically . Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E . coli O55:B5 or Salmonella typhimurium . Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed . Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls . Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo. Lett Appl Microbiol, 2003, 37(6), 448 - 51 Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium; Olasupo NA et al.; AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium . METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C . As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria . Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm . Typhimurium and E . coli, respectively . After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl . However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities . On the contrary, combination of nisin with diacetyl against Salm . Typhimurium resulted in an antagonism of diacetyl activity . CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E . coli and Salm . Typhimurium. J Appl Microbiol, 2003, 95(6), 1226 - 34 The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig; Delsol AA et al.; AIMS: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracycline-resistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig . METHODS AND RESULTS: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E . coli . Higher numbers of S . Typhimurium DT104 were shed from treated pigs than untreated pigs . This lasted up to 6 weeks post-treatment in the high-dose group . In this group, there was a 30% increase in E . coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l-1 and a 10% increase in E . coli with an MIC > 50 mg l-1 during and 2 weeks post-treatment . This effect was less-pronounced in the low-dose group . PCR identified the predominant tetracycline resistance genes in the E . coli as tetA, tetB and tetC . The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 microg g-1 faeces during treatment . CONCLUSION: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time . SIGNIFICANCE AND IMPACT OF THE STUDY: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man. Helicobacter, 2003 Dec, 8(6), 613 - 25 Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice; Chen M et al.; AIM: To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction . MATERIALS AND METHODS: The H . pylori catalase gene was introduced into attenuated S . typhimurium strain SL3261 . C57BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate-buffered saline (PBS) . Mice were sacrificed 4 weeks after immunization and 5 weeks after H . pylori challenge, respectively . RESULTS: All PBS control mice were infected . Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H . pylori infection . Serum anti-H . pylori IgG2a levels of S . typhimurium-immunized mice were higher than those of PBS controls, both before and after H . pylori challenge, while there were no differences for IgG1 and IgA . Similarly, mRNA expression of interleukin (IL)-2, IL-12 and interferon-gamma in the gastric mucosa of S . typhimurium-immunized mice was significantly higher than that of PBS controls both before and after challenge . Moreover, S . typhimurium-immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge . IL-4 and IL-10 were not detected in any of the three groups . IL-6 expression was increased in the PBS group compared with the S . typhimurium-immunized groups after challenge . CONCLUSIONS: This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S . typhimurium strain offers protection against H . pylori infection . This protective immunity was mediated through a predominantly Th1-type response and was associated with post-immunization gastritis. Anal Chem, 2003 Aug 15, 75(16), 4330 - 4 Procedures for preparing Escherichia coli O157:H7 immunoliposome and its application in liposome immunoassay; Ho JA et al.; Although Escherichia coli serotype O157:H7 was identified as a human pathogen in the ninth decade of the twentieth century, it has become recognized as a major foodborne pathogen . In the United States, the severity of E . coli O157:H7 infection in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety . In laboratory diagnosis, most microbiologic assays rely on a single phenotype to selectively isolate this pathogen . However, the process is labor- and time-consuming . It is important eventually to develop new assay procedures to detect them . Immunoliposomes, anti-E . coli O157:H7 antibody-tagged liposomes, encapsulating a visible dye, sulforhodamine B, were used in the present study for the development of a field-portable colorimetric immunoassay to detect E . coli O157:H7 . The N-succinimidyl-S-acetylthioacetate derivative of the antibodies (anti-E . coli O157: H7) was first conjugated through the reactive N-(kappa-maleimidoundecanoyloxy) sulfosuccinimide ester derivative of dipalmitoylphosphatidylethanolamine and subsequently incorported into liposomes to form the immunoliposomes . A plastic-backed nitrocellulose strip with two immobilized zones is the basis for a sandwich assay to detect E . coli O157:H7 . The first zone is the antigen capture zone (AC zone), which is used in a sandwich (noncompetitive) assay format; the other is the biotin capture zone (BC zone), which is used as a positive control for the strip . During the capillary migration of the wicking reagent containing 50 microL of immunoliposomes and 90 microL of the test sample, E . coli O157:H7 with surface-bound immunoliposomes is captured at the AC zone, while the unbound immunoliposomes migrate and bind to the antibiotin antibodies coated on BC zone . The color density of the AC zone were directly proportional to the amount of E . coli O157:H7 in the test sample . The detection limit of the current assay with heat-killed E . coli O157:H7 was approximately 2500 cells . The selectivity of the newly developed biosensor system was investigated, and pathogens, including Salmonella typhimurium and Listeria genus specific, were proven to have no interference with the detection of E . coli O157:H7. Anal Chem, 2003 Sep 1, 75(17), 4591 - 8 Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection; Koh CG et al.; An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification . The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation . Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors . In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions . After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation . An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence . Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences . The limit of detection is about six copies of target DNA. Toxicol In Vitro, 2004 Feb, 18(1), 109 - 14 Mutagenicity of paepalantine dimer and glycoside derivatives from Paepalanthus bromelioides; Varanda EA et al.; The first isocoumarin isolated from the methylene chloride extract of Paepalanthus bromelioides, named paepalantine (isocoumarin 1), was found to have antimicrobial activity; but, it is mutagenic clastogenic and cytotoxic . Two other isocoumarins, paepalantine-9-O-beta-D-glucopyranoside (isocoumarin 2) and paepalantine-9-O-beta-D-allopyranosyl(1 --> 6) glucopyranoside (isocoumarin 3) were isolated from the ethanolic extract . A fourth new isocoumarin, also isolated from the methylene chloride extract of the capitula of P . bromelioides, was characterized as an 8-8' dimer of paepalantine and denominated isocoumarin 4 . The abilities of isocoumarins 2, 3 and 4 to induce mutations in Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 were investigated . Mutagenic activity was observed in strain TA97a treated with isocoumarin 2 in the presence of S9 mixture . The substitution of H at position 9 by glucose or glucose-allose caused reductions in the mutagenic activities of paepalantine, indicating this to be an important site for these properties. Med Microbiol Immunol (Berl) . 2003 Nov 20; {Epub ahead of print} Induction of cytokine mRNA expression in U937 cells by Salmonella typhimurium porins is regulated by different phosphorylation pathways; Galdiero M et al.; Lipopolysaccharide (LPS) and porins of Gram-negative outer membranes are the main pathogenic factors implicated in the clinical syndrome of septic shock . The biological activity of porins and LPS are similar, but they occur by different mechanisms . It seems that porins act through different intracellular pathways with respect to LPS . In this study we analyzed the role of several inhibitors of the MEK/ERK signal pathway on the induction of proinflammatory and immunological cytokines in U937 cell line stimulated by Salmonella typhimurium porins and compared it to the cytokine induction after LPS stimulation . We investigated the effects of p38 MAP kinase inhibitor SB-203580, MEK/ERK kinase inhibitor PD-098059 and Raf-1 inhibitor forskolin, and demonstrated that they modulate cytokine mRNA expression in a different manner as a consequence of the use of porins or LPS as stimuli . TNF-alpha and IL-1beta mRNA expression is decreased by PD-098059 after stimulation with LPS but not with porins in differentiated U937 cells . IL-10 mRNA expression is inhibited by SB-203580 and PD-098059 after stimulation with porins in U937 cells . IL-6 and IL-8 mRNA expression is not changed by PD-098059 or SB-203580, after stimulation either with porins or LPS . Furthermore, mRNA expression of the studied cytokines, except for GM-CSF, is not changed using forskolin. J Food Prot, 2003 Nov, 66(11), 2138 - 40 Detection of Salmonella from chicken rinses and chicken hot dogs with the automated BAX PCR system; Bailey JS et al.; The BAX system with automated PCR detection was compared with standard cultural procedures for the detection of naturally occurring and spiked Salmonella in 183 chicken carcass rinses and 90 chicken hot dogs . The automated assay procedure consists of overnight growth (16 to 18 h) of the sample in buffered peptone broth at 35 degrees C, transfer of the sample to lysis tubes, incubation and lysis of the cells, transfer of the sample to PCR tubes, and placement of tubes into the cycler-detector, which runs automatically . The automated PCR detection assay takes about 4 h after 16 to 24 h of overnight preenrichment . The culture procedure consists of preerichment, enrichment, plating, and serological confirmation and takes about 72 h . Three trials involving 10 to 31 samples were carried out for each product . Some samples were spiked with Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Montevideo, and Salmonella Enteritidis at 1 to 250 cells per ml of rinse or 1 to 250 cells per g of meat . For unspiked chicken rinses, Salmonella was detected in 2 of 61 samples with the automated system and in 1 of 61 samples with the culture method . Salmonella was recovered from 111 of 122 spiked samples with the automated PCR system and from 113 of 122 spiked samples with the culture method . For chicken hot dogs, Salmonella was detected in all 60 of the spiked samples with both the automated PCR and the culture procedures . For the 30 unspiked samples, Salmonella was recovered from 19 samples with the automated PCR system and from 10 samples with the culture method . The automated PCR system provided reliable Salmonella screening of chicken product samples within 24 h. J Food Prot, 2003 Nov, 66(11), 2093 - 6 Effect of gamma irradiation on the survival of pathogens in kwamegi, a traditional Korean semidried seafood; Chawla SP et al.; Kwamegi (semidried raw Pacific saury) is traditional seafood available in Korea . It has water activity in the range of 0.90 to 0.95 . Spoilage and the growth of most pathogenic bacteria is retarded because of low water activity, low temperature, and packaging . However, it is contaminated with bacteria of public health significance and poses a hazard to the consumer because it is consumed raw without any cooking . The effectiveness of these hurdles in preventing the growth of Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, and Escherichia coli and the efficacy of irradiation treatment in eliminating these bacteria from kwamegi using inoculated pack studies was examined . Radiation sensitivity of S . aureus, B . cereus, Salmonella Typhimurium, and E . coli in kwamegi was investigated . D10-values of these organisms in kwamegi were 590 +/- 13.6, 640 +/- 14.9, 560 +/- 45.4, and 550 +/- 8.6 Gy, respectively . The growth of all four test organisms inoculated into these foods during 4 weeks of storage at an ambient winter temperature (ranging from -5 degrees C to +5 degrees C) was recorded . All four pathogens (inoculated at 10(6) CFU/g) were eliminated by irradiation at 4 kGy . These studies unequivocally demonstrate that irradiation, with a combination of low water activity and low temperature, results in microbiologically safe kwamegi. J Food Prot, 2003 Nov, 66(11), 1996 - 2004 Evaluation of a 24-hour bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken carcass rinses; Valdivieso-Garcia A et al.; A bioluminescent enzyme immunoassay (BEIA), using Salmonella-specific monoclonal antibody M183 for capture and biotinylated monoclonal antibody M183 for detection, was developed with InteLite AB streptavidin-biotinylated firefly luciferase complex as a reporter . Salmonella cultures were preenriched in buffered peptone water with shaking for 6 h at 37 degrees C and then selectively enriched in Muller-Kauffmann tetrathionate (MKTT) broth and modified semisolid Rappaport-Vassiliadis (MSRV) medium for 16 h at 42 degrees C . After enrichment, the total test time for the BEIA was 1.5 h . The analytical sensitivity of the BEIA ranged from 6.0 x 10(2) CFU/ml to 1.2 x 10(5) CFU/ml in MKTT and from 1.4 x 10(5) to 2.3 x 10(6) CFU/ml in MSRV using six Salmonella serovars prevalent in Canada . With enrichment cultures, the BEIA detected 1 CFU of Salmonella Typhimurium and Salmonella Enteritidis in 25 ml of chicken rinses . Representative strains of 10 Salmonella serovars were detected, and cross-reactivity was not observed with 25 non-Salmonella foodborne bacteria . The BEIA performance was assessed by testing 420 poultry samples, which were analyzed in parallel with the standard MSRV culture method . The BEIA detected 117 (27.88%) Salmonella-positive samples, whereas the standard MSRV culture method detected 124 (29.5%) . The BEIA had a sensitivity of 64.5% and a specificity of 87.5% compared to the standard MSRV culture method . However, similar specificities and sensitivities were obtained when the standard MSRV culture method was compared to the BEIA (sensitivity = 68.4% and specificity = 85.5%) . Neither method detected 100% of the Salmonella found in the samples tested, and statistical analyses indicated no significant difference between the two methods . In summary, the BEIA offers another alternative for the detection of Salmonella, with the additional advantage of providing a 24-h test for detecting Salmonella in chicken carcass rinses . The results obtained in this research indicate that tests are still needed for the isolation and detection of Salmonella that will establish the true prevalence of Salmonella in chicken samples. Biosens Bioelectron, 2003 Dec 30, 19(5), 495 - 502 Detection of viable Salmonella typhimurium by impedance measurement of electrode capacitance and medium resistance; Yang L et al.; Three-electrode electrochemical impedance technique was investigated for detection of Salmonella typhimurium by monitoring the growth of bacteria in selenite cystine (SC) broth supplemented with trimethylamine oxide hydrochloride (TMAO.HCl) and mannitol (M) . The change in the system impedance during the growth of bacteria was studied using frequency spectral scanning . It was found that the impedance at low frequencies (<10 kHz) mainly came from the double-charged layer capacitance, reflecting the changes at the electrode interface and the adsorption on the electrode surface . While at high frequencies (>10 kHz), the system impedance mainly depended on the medium resistance . The adsorption of bacteria on the electrode surface was detected by measuring low frequency impedance, and verified with Faradic impedance spectroscopy . Enumeration of S . typhimurium using a low frequency (1 Hz) capacitance measurement and a high frequency (1 MHz) resistance measurement were compared . The detection times were determined for quantitative analysis based on the growth curves of bacteria referring to either the medium resistance or electrode capacitance . The regression equations for the detection times (t(d), h) and the initial cell number (N, cells.ml(-1)) were t(d)=-1.24logN+13.4 with R(2)=0.98 and t(d)=-1.40logN+14.46 with R(2)=0.97 for the medium resistance and electrode capacitance methods, respectively. Int J Food Microbiol, 2003 Dec 31, 89(2-3), 195 - 203 Batch growth of Salmonella typhimurium LT2: stoichiometry and factors leading to cessation of growth; Wilson PD et al.; Salmonella typhimurium LT2 was grown in batch culture (trypticase soy broth, with 0.3%(w/v) yeast extract, 1% (w/v) glucose and 0.5% (w/v/) NaCl, 20 degrees C) at a range of initial pH (4.4, 4.8, 5.0 and 7.0) . The consumption of oxygen and glucose was found to be independent of initial pH, and stoichiometric with growth . Mean yield coefficients of 6.9 x 10(-15) and 15.5 x 10(-15) mol oxygen/cell were estimated . Calculation of the instantaneous state of carbon during the cultivation showed stoichiometric conversion of glucose into biomass, carbon dioxide and organic acids . The concentration of the undissociated form of the primary acidic product (acetic acid) was shown to be the factor limiting growth. Mol Microbiol, 2003 Nov, 50(4), 1411 - 27 Lsr-mediated transport and processing of AI-2 in Salmonella typhimurium; Taga ME et al.; The LuxS-dependent autoinducer AI-2 is proposed to function in interspecies cell-cell communication in bacteria . In Salmonella typhimurium, AI-2 is produced and released during exponential growth and is subsequently imported into the bacteria via the Lsr (luxS regulated) ATP binding cassette (ABC) transporter . AI-2 induces transcription of the lsrACDBFGE operon, the first four genes of which encode the Lsr transport apparatus . In this report, we identify and characterize LsrK, a new protein that is required for the regulation of the lsr operon and the AI-2 uptake process . LsrK is a kinase that phosphorylates AI-2 upon entry into the cell . Our data indicate that phosphorylation of AI-2 results in its sequestration in the cytoplasm . We suggest that phospho-AI-2 is the inducer responsible for inactivation of LsrR, the repressor of the lsr operon . We also show that two previously uncharacterized members of the lsr operon, LsrF and LsrG, are necessary for the further processing of phospho-AI-2 . Transport and processing of AI-2 could be required for removing the quorum-sensing signal, conveying the signal to an internal detector and/or scavenging boron. Clin Microbiol Infect, 2003 Nov, 9(11), 1104 - 11 Porins and lipopolysaccharide from Salmonella typhimurium regulate the expression of CD80 and CD86 molecules on B cells and macrophages but not CD28 and CD152 on T cells; Galdiero M et al.; OBJECTIVE: The aim of this study was to evaluate the effect of porins from Salmonella typhimurium on costimulatory molecules such as CD80/CD86 and CD28/CD152 . The interactions between these molecules are able to influence the immune response through the regulation of cytokines release which, on their own, are able to regulate the immunological response by a feedback mechanism . METHODS: S . typhimurium strain SH5014 (a rough lipopolysaccharide (LPS) producing strain) was used as the source of porins and LPS . Peripheral blood mononuclear cells were obtained from healthy adult donors . THP1 cells were obtained from ATCC (Rockville, MD, USA) . Immunofluorescence and flow cytometry were performed using a FACS IV (Becton-Dickinson, Mountain View, CA, USA) . RESULTS: Our results show that porins of S . typhimurium increase the expression of CD86 and the expression of CD80 both on B lymphocytes and macrophages, while the expression of CD28 and CD152 on T lymphocytes was unaltered . The expression of CD80 and CD86 is dose-dependent and starts after 24 h post treatment, peaks at 48 h and goes back to the basal value after 72 h . CONCLUSIONS: S . typhimurium porins are able to induce a high expression of costimulatory molecules (CD80 and CD86) on lymphocytes and macrophages. Neurosci Lett, 2003 Nov 27, 352(1), 57 - 60 Human serum amyloid P component attenuates the bacterial lipopolysaccharide-induced increase in blood-brain barrier permeability in mice; Veszelka S et al.; Endotoxin challenge leads to septic shock, multi-organ failure and death in mice . Permeability of the blood-brain barrier (BBB) is increased by endotoxemia . Serum amyloid P component (SAP) is a lipopolysaccharide (LPS)-binding protein that can modulate the host reactions during infections . It is controversial whether SAP can protect from LPS toxicity in vivo or not . We have tested the effect of human SAP on BBB permeability of Salmonella typhimurium LPS-injected mice . The animals showed signs of sickness behaviour including immobility, anorexia, and diarrhoea . Intraperitoneally administered LPS increased the BBB permeability for sodium fluorescein for about 4-fold, and for albumin for more than 2-fold in brain cortex . SAP, given intravenously, had no effect on basal BBB permeability for albumin, although it decreased sodium fluorescein extravasation to brain tissue . In LPS-treated mice, SAP administration alleviated the symptoms of septic shock, and significantly inhibited the enhanced BBB permeability for both tracers . Our data indicate that human SAP may counteract the toxic effects of LPS during septic shock. Appl Spectrosc, 2003 Sep, 57(9), 1138 - 44 Use of fluorescently labeled phage in the detection and identification of bacterial species; Mosier-Boss PA et al.; Phages are viruses whose hosts are bacterial cells . They identify their hosts by specific receptor molecules on the outside of the host cell . Once the phages find their specific receptors, they bind to the bacterial cell and inject their nucleic acid inside the cell . The binding between phage and host can be so specific that only certain strains of a single species can be infected . In this communication, the specificity of phage P22 for Salmonella typhimurium LT2 is exploited to allow the detection of Salmonella in the presence of other bacterial species . In particular, the dsDNA of P22 is bound to SYBR gold, a highly sensitive, fluorescent nucleic acid stain . When multiple phages infect the same cell, the fluorescence emissions of the phage DNA inside the cell allow it to be imaged using an epifluorescence microscope . The advantages of using phages as the bacterial recognition element in a sensor over antibodies are discussed. Food Addit Contam, 2003 Oct, 20(10), 940 - 8 Antimutagenic activity of phenolic compounds, oligosaccharides and quinolizidinic alkaloids from Lupinus campestris seeds; Martinez CJ et al.; There are some foods that contain mutagenic or carcinogenic agents, some of which occur naturally and others that may be formed during preparation or cooking . Several foods such as legumes, also contain natural antimutagens and/or anticarcinogens . Lupine is one such legume that contains high amounts of protein (40%) and oils (14%) . About 90 species of lupine have been reported throughout Mexico . However, the use of this crop as a source of food has been limited by the presence of antinutritional agents such as phenolic compounds (PC), carbohydrates (CH) and quinolizidinic alkaloids (Qas) . It has also been suggested that consuming these compounds can affect human health and may even reduce the risk of disease . The objective of this work was to determine the effect of PC, CH and Qas, isolated and quantified from Lupinus campestris on the mutagenicity of 1-nitropyrene (1-NP) as a model mutagen and we used the Salmonella typhimurium tester strain YG1024 by the Kado microsuspension method . The results indicate that L . campestris seeds have 11 mg (+)catechin equivalent g(-1) seed coat; 120.3 mg g(-1) seeds and 2.13 mg g(-1) seeds of PC, CH and Qas, respectively . 1-NP mutagenicity was inhibited by 86% for PC, 76% for CH and 75% for Qas at concentrations of 200, 512 and 13.6 microg/tube, respectively. Am J Chin Med, 2003, 31(4), 523 - 32 Antioxidative and antimutagenic activities of healthy herbal drinks from Chinese medicinal herbs; Chen W et al.; Twenty-nine Chinese medicinal herbs and three healthy herbal drinks made of those herbs in a food processing pilot plant were tested for their antioxidative, free radical scavenging, mutagenic and antimutagenic activities . Water extracts of herbs (with few exceptions) and herbal drinks showed free radical scavenging activity . All water extracts of herbs and herbal drinks showed no mutagenicity toward Salmonella typhimurium tester strains TA98 and TA100 used in the Ames mutagenic tests . In the antimutagenic tests, the mutagenic activity of 4-nitroquinoline N-oxide (NQNO) toward S . typhimurium TA98 was markedly inhibited by water extracts of herbs and herbal drinks . Based on the results, it is suggested that the herbal drinks manufactured in pilot-plant scale are safe and can be served as health-promoting drinks for the public. Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2091 - 9 Antimutagenic activity of flavonoids from Chrysanthemum morifolium; Miyazawa M et al.; A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) . The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water . The ethyl acetate fraction showed a suppressive effect . Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy . Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test . Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml . The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml . These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes . These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation . In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S . typhimurium TA100. Br J Cancer, 2003 Nov 3, 89(9), 1796 - 801 Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours; Dresselaers T et al.; The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD) . The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116) . The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.) . Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy . The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue . No 5-FU metabolites were detectable in vivo in the tumours . However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites . Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu . with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites . Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase. Drug Chem Toxicol, 2003 Nov, 26(4), 295 - 308 Genotoxic activities of drug-nitrite interaction products; Ozhan G et al.; At first 28 orally administered drugs, considered to be potentially nitrosatable on the basis of their chemical structure, have been nitrosated with nitrite under simulated stomach conditions . A maximum daily dose of each drug was incubated with a nitrite concentration that can be found after a normal meal in the stomach at 37 degrees C over 1 h . Reaction was started at pH 6.8-7.0 and stopped at pH 2.0, so we had the same pH change that occurs in the stomach . Secondly the genotoxic activities of drug-nitrite interaction products were tested by the umu-test with Salmonella typhimurium TA 1535/pSK1002 as tester strain in the presence and absence of metabolic activation . By the umu-test, among the nitrosation products of drugs examined 22 products showed genotoxicity at different levels . Other six products showed negative results by the umu-test. Nat Immunol, 2003 Dec, 4(12), 1230 - 7 Epub 2003 Oct 26. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection; Brigl M et al.; CD1d-restricted natural killer T (NKT) cells are important for host defense against a variety of microbial pathogens . How and when these T cells become activated physiologically during infection remains unknown . Our data support a model in which NKT cells use a unique activation mechanism not requiring their recognition of microbial antigens . Instead, weak responses to CD1d-presented self antigens were amplified by interleukin 12 made by dendritic cells in response to microbial products, resulting in potent interferon-gamma secretion . NKT cells were among the first lymphocytes to respond during Salmonella typhimurium infection, and their activation in vivo also depended on interleukin 12 and CD1d recognition . We propose this mechanism of activation as a major pathway responsible for the rapid activation of NKT cells in different microbial infections. Carcinogenesis, 2004 Feb, 25(2), 203 - 9 Epub 2003 Oct 24. Grapefruit juice intake does not enhance but rather protects against aflatoxin B1-induced liver DNA damage through a reduction in hepatic CYP3A activity; Miyata M et al.; Influence of grapefruit juice intake on aflatoxin B1 (AFB1)-induced liver DNA damage was examined using a Comet assay in F344 rats given 5 mg/kg AFB1 by gavage . Rats allowed free access to grapefruit juice for 5 days prior to AFB1 administration resulted in clearly reduced DNA damage in liver, to 65% of the level in rats that did not receive grapefruit juice . Furthermore, rats treated with grapefruit juice extract (100 mg/kg per os) for 5 days prior to AFB1 treatment also reduced the DNA damage to 74% of the level in rats that did not receive grapefruit juice . No significant differences in the portal blood and liver concentrations of AFB1 were observed between grapefruit juice intake rats and the controls . In an Ames assay with AFB1 using Salmonella typhimurium TA98, lower numbers of revertant colonies were detected with hepatic microsomes prepared from rats administered grapefruit juice, compared with those from control rats . Microsomal testosterone 6beta-hydroxylation was also lower with rats given grapefruit juice than with control rats . Immunoblot analyses showed a significant decrease in hepatic CYP3A content, but not CYP1A and CYP2C content, in microsomes of grapefruit juice-treated rats than in non-treated rats . No significant difference in hepatic glutathione S-transferase (GST) activity and glutathione content was observed in the two groups . GSTA5 protein was not detected in hepatic cytosol of the two groups . In microsomal systems, grapefruit juice extract inhibited AFB1-induced mutagenesis in the presence of a microsomal activation system from livers of humans as well as rats . These results suggest that grapefruit juice intake suppresses AFB1-induced liver DNA damage through inactivation of the metabolic activation potency for AFB1 in rat liver. Biochem Biophys Res Commun, 2003 Nov 7, 311(1), 193 - 201 Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+); Simanshu DK et al.; Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle . The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB) . Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S . typhimurium, determined at 2.1 and 2.3A, respectively . The structure closely resembles that of the Escherichia coli enzyme . Unlike the E . coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB . Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate . Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E . coli native PrpB . In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered . Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes . Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism. J Food Prot, 2003 Oct, 66(10), 1916 - 9 Effect of freezing, irradiation, and frozen storage on survival of Salmonella in concentrated orange juice; Niemira BA et al.; Six strains of Salmonella (Anatum F4317, Dublin 15480, Enteritidis 13076, Enteritidis WY15159, Stanley H0588, and Typhimurium 14028) were individually inoculated into orange juice concentrate (OJC) and frozen to -20 degrees C . The frozen samples were treated with 0 (nonirradiated), 0.5, 1.0, or 2.0 kGy of gamma radiation and held frozen for 1 h, and the surviving bacterial population was assessed . The strains showed significant variability in their response to freezing and to freezing in combination with irradiation . The response was dose dependent . Relative to the nonfrozen, nonirradiated control, the reduction following the highest dose (2.0 kGy) ranged from 1.29 log CFU/ml (Salmonella Typhimurium) to 2.17 log CFU/ml (Salmonella Stanley) . Samples of OJC inoculated with Salmonella Enteritidis WY15159 and irradiated were stored at -20 degrees C for 1, 2, 7, or 14 days, and the surviving population was determined . Relative to the nonfrozen, nonirradiated control, after 14 days, the population was reduced by 1.2 log CFU/ml in the nonirradiated samples and by 3.3 log CFU/ml following treatment with 2.0 kGy . The combination of frozen storage plus irradiation resulted in greater overall reductions than either process alone. Radiat Res, 2003 Nov, 160(5), 584 - 92 Influence of ginger rhizome (Zingiber officinale Rosc) on survival, glutathione and lipid peroxidation in mice after whole-body exposure to gamma radiation; Jagetia GC et al.; The radioprotective effect of the hydroalcoholic extract of ginger rhizome, Zingiber officinale (ZOE), was studied . Mice were given 10 mg/kg ZOE intraperitoneally once daily for five consecutive days before exposure to 6-12 Gy of gamma radiation and were monitored daily up to 30 days postirradiation for the development of symptoms of radiation sickness and mortality . Pretreatment of mice with ZOE reduced the severity of radiation sickness and the mortality at all doses . The ZOE treatment protected mice from GI syndrome as well as bone marrow syndrome . The dose reduction factor for ZOE was found to be 1.15 . The optimum protective dose of 10 mg/kg ZOE was 1/50 of the LD50 (500 mg/kg) . Irradiation of the animals resulted in a dose-dependent elevation in the lipid peroxidation and depletion of GSH on day 31 postirradiation; both effects were lessened by pretreatment with ZOE . ZOE also had a dose-dependent antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Candida albicans. Mutat Res, 2003 Sep 29, 530(1-2), 19 - 26 Photomutagenicity of cosmetic ingredient chemicals azulene and guaiazulene; Wang L et al.; The photomutagenicity of the popular skin conditioning agents azulene and guaiazulene were tested in Salmonella typhimurium TA98, TA100 and TA102 . Following irradiation with UVA and/or visible light, both azulene and guaiazulene exhibited mutagenicity 4-5-fold higher than the spontaneous background mutation . In contrary, naphthalene, a structural isomer of azulene, was not photomutagenic under the same conditions . Azulene was photomutagenic when irradiated with UVA light alone, visible light alone, or a combination of UVA and visible light . Azulene and guaiazulene are not mutagenic when the experiment is conducted with the exclusion of light . Therefore, extreme care must be taken when using cosmetic products with azulene/guaiazulene as ingredients since after applying these products on the skin, exposure to sunlight is inevitable. Food Chem Toxicol, 2003 Dec, 41(12), 1771 - 80 Toxicological evaluation of propane expanded tobacco; Theophilus EH et al.; A tiered testing strategy has been developed to evaluate the potential for tobacco processes, ingredients, and other technological developments to increase or decrease the biological activity resulting from burning tobacco . The strategy is based on comparative chemical and biological testing . Propane expanded tobacco is an example of a processed tobacco used in the modern manufacture of cigarettes . Test cigarettes containing propane expanded tobacco were compared to control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11) . The toxicological evaluation included chemistry studies using mainstream cigarette smoke (determination of selected constituent yields), in vitro studies using cigarette smoke condensate (Ames study in Salmonella typhimurium and sister chromatid exchange study in Chinese hamster ovary cells) and in vivo studies (13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats and 30-week dermal tumor promotion study of cigarette smoke condensate in SENCAR mice) . Although statistically significant differences in several smoke constituents were observed, most constituents from cigarettes containing 100% propane expanded tobacco were within market survey ranges . Furthermore, biological tests indicated that the cigarettes containing propane or Freon-11 expanded tobacco were not significantly different. Avian Dis, 2003 Jul-Sep, 47(3), 656 - 61 Use of a live attenuated Salmonella typhimurium vaccine to protect hens against Salmonella enteritidis infection while undergoing molt; Holt PS et al.; Previous studies demonstrated that Salmonella enteritidis infections in hens undergoing molt via feed withdrawal were more severe than in full fed hens . We conducted two trials to determine if immunizing specific-pathogen-free, Salmonella-culture-negative hens via aerosol exposure to MeganVacl, a commercially available attenuated Salmonella typhimurium vaccine, would reduce transmission of S . enteritidis from infected hens to uninfected but contact-exposed hens during a molt . In trial 1, one group of hens received two aerosol doses of vaccine 2 wk apart whereas a second group of hens remained nonvaccinated . In trial 2, the vaccinated group received only one dose of vaccine . Two weeks after the final immunization, feed was removed from all the hens, and on day 4, the center hen in rows of 11 hens received a dose of 3 x 10(5) (trial 1) or 1.3 x 10(6) (trial 2) . Transmission to the unchallenged hens was followed 3, 10, 17, and 24 days later . Vaccination reduced the horizontal spread of S . enteritidis in vaccinated hens compared with their nonvaccinated counterparts, with vaccinated hens shedding significantly less S . enteritidis on day 10 postchallenge in trial 1 and on days 3, 10, 17, and 24 in trial 2 . Recovery of S . enteritidis from ovaries was significantly reduced in the vaccinated hens in trial 1 and from livers/spleens, ovaries, and cecum in trial 2 . These studies indicate that immunization of hens with a live S . typhimurium vaccine could help reduce S . enteritidis problems during a molt situation. J Agric Food Chem, 2003 Oct 22, 51(22), 6456 - 60 Prenylated flavanones isolated from flowers of Azadirachta indica (the neem tree) as antimutagenic constituents against heterocyclic amines; Nakahara K et al.; Four prenylated flavanones were isolated from the methanol extract of the flowers of Azadirachta indica (the neem tree) as potent antimutagens against Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole) in the Salmonella typhimurium TA98 assay by activity-guided fractionation . Spectroscopic properties revealed that those compounds were 5,7,4'-trihydroxy-8-prenylflavanone (1), 5,4'-dihydroxy-7-methoxy-8-prenylflavanone (2), 5,7,4'-trihydroxy-3',8-diprenylflavanone (3), and 5,7,4'-trihydroxy-3',5'-diprenylflavanone (4) . All isolated compounds were found for the first time in this plant . The antimutagenic IC(50) values of compounds 1-4 were 2.7 +/- 0.1, 3.7 +/- 0.1, 11.1 +/- 0.1, and 18.6 +/- 0.1 microM in the preincubation mixture, respectively . These compounds also similarly inhibited the mutagenicity of Trp-P-2 (3-amino-1-methyl-5H-pyrido{4,3-b}indole) and PhIP (2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine) . All of the compounds 1-4 strongly inhibited ethoxyresorufin O-dealkylation activity of cytochrome P450 1A isoforms, which catalyze N-hydroxylation of heterocyclic amines . However, compounds 1-4 did not show significant inhibition against the direct-acting mutagen NaN(3) . Thus, the antimutagenic effect of compounds 1-4 would be mainly based on the inhibition of the enzymatic activation of heterocyclic amines. J Agric Food Chem, 2003 Oct 22, 51(22), 6413 - 22 Antimutagenic activity of phenylpropanoids from clove (Syzygium aromaticum); Miyazawa M et al.; Phenylpropanoids that possess antimutagenic activity were isolated from the buds of clove (Syzygium aromaticum) . The isolated compounds suppressed the expression of the umu gene following the induction of SOS response in the Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens . The suppressive compounds were mainly localized in the ethyl acetate extract fraction of the processed clove . This ethyl acetate fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compounds . Electron impact mass spectrometry, IR, and (1)H and (13)C NMR spectroscopy were then used to delineate the structures of the compounds that confer the observed antimutagenic activity . The secondary suppressive compounds were identified as dehydrodieugenol (1) and trans-coniferyl aldehyde (2) . When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, compound 1 suppressed 58% of the umu gene expression as compared to the controls at a concentration of 0.60 micromol/mL, with an ID(50) (50% inhibitory dose) value of 0.48 micromol/mL, and compound 2 suppressed 63% of the umu gene expression as compared to the controls at a concentration of 1.20 micromol/mL, with an ID(50) value of 0.76 micromol/mL . Additionally, compounds 1 and 2 were tested for their ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver metabolizing enzymes, and aflatoxin B(1) (AfB(1)) and 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which require liver metabolizing enzymes and activated Trp-P-1 and UV irradiation . Compounds 1 and 2 showed dramatic reductions in their mutagenic potential of all of the aforementioned chemicals or treatment . For the search of the structure-activity relationship, the derivatives of 1 and 2 (1a and 2a-c) were also assayed with all mutagens . Finally, the antimutagenic activities of compounds 1, 1a, 2, and 2a-c against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using the S . typhimurium TA100 strain. Ann Ig, 2003 Jul-Aug, 15(4), 277 - 302 {Genotoxic and ecotoxic effects of urban waste water disinfected with sodium hypochlorite or peracetic acid}; Crebelli R et al.; Genotoxic and ecotoxicologic effects of urban wastewater disinfected with sodium hypochlorite or peracetic acid were analyzed . The formation of genotoxic species was studied by determining clastogenic and mutagenic activity of aqueous samples and their extracts with in vivo and in vitro tests, respectively . In particular, we have applied citogenetic tests to Allium cepa roots and Tradescantia inflorescence (Allium cepa test and Tradescantia/micronuclei test) and reversion test to Salmonella typhimurium according to the microsuspension procedure (Kado test) . The latter is the method of choice for the analysis of complex matrices due to its high sensitivity and specificity . The mutagenic activity of disinfected effluents was similar to the corresponding untreated wastewater both sampled in four different periods . Therefore, the disinfection process did not seem to contribute to aquatic mutagenicity in the examined range of biocide concentration . The potential toxicity of disinfected wastewater for aquatic organisms was evaluated using Daphnia magna . The acute toxicity of peracetic acid in sewage was 0.4 mg/L (24 h E(L)C50) . By comparing this value with peracetic acid concentrations detected in effluents from a pilot plant it is expected that treated wastewater would show acute toxic effects on aquatic organisms . Dissociation compounds (hydrogen peroxide and acetic acid) and possible by-products of peracetic acid did not seem to contribute significantly to the toxicity of sewage treated with peracetic acid. Biotechnol Adv, 1990, 8(1), 121 - 30 Protein phosphorylation in microorganisms; Janecek J et al.; The covalent modification of proteins by phosphorylation constitutes a major regulatory mechanism . It was first recognized in mammalian tissues . A conclusive evidence for the occurrence of protein phosphorylation and protein kinases in coliform bacteria was obtained in 1978 . Several phosphate labeled proteins were found when Salmonella typhimurium was pulse-labeled with 32p(i) and solubilized bacterial contents were analyzed by SDS-polyacrylamide gel electrophoresis . In streptomycetes protein phosphorylation has not yet been demonstrated . We found that Streptomyces albus possesses a protein kinase activity . This in vitro protein phosphorylation is cAMP-independent. J Environ Pathol Toxicol Oncol, 2003, 22(2), 101 - 9 Genotoxicity of textile dyes evaluated with Ames test and rec-assay; Bakshi DK et al.; We evaluated for mutagenicity 14 commercial textile dyes used extensively in the northern part of India using both the Ames Salmonella typhimurium microsome reversion test as well as the recombination-repair (rec)-assay . The Ames test revealed that 57.14% of dyes were mutagenic and acting directly . The rec-assay detected 50% of dyes to be mutagenic; of these, 71.43% were direct acting, whereas 28.57% required Aroclor-induced exogenous metabolic activation . Used together, the two tests detected 78.57% of the dyes to be mutagenic, and a 50% correlation was found between these two tests . Groupwise, three out of four azo dyes and all five anthraquinone dyes were found to be mutagenic by the Ames assay; the rec-assay detected methine/polymethine (1 out of 3), an oxazine, and a triphenylmethane dye to be mutagenic, besides the azo (1 out of 4) and the anthraquinone (3 out of 5) dyes . The structure-activity analysis attributed the mutagenicity of dyes to the structural alerts such as phenylenediamine, amino and nitro-groups, methylation, CH=CH, and chloro groups; whereas deamination, bulkier groups, and sulfonation may be responsible for diminishing mutagenicity. J Trace Elem Med Biol, 2003, 17(2), 85 - 90 Element analysis and biological studies on ten oriental spices using XRF and Ames test; Al-Bataina BA et al.; Ten oriental spices were analyzed for their element composition using X-ray fluorescence (XRF): nutmeg (Myristica fragrans), coriander (Coriandrum sativum), safflower (Carthamus tinctorius), caraway (Carum carvi), Sicilian sumac (Rhus coriaria), aniseed (Anisum vulgare), black pepper (Piper nigrum), cardamom (Elettaria cardamomum), cumin (Cuminum cyminum) and nigella (Nigella sativum) . The spices were found to contain the following elements: Mg, Al, Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Cu and Zn, with varying concentrations . Mutagenic studies using Salmonella typhimurium strains TA97a, TA98, TA100, and TA102 showed that the above spices have no base pair substitution mutagenic activity . However, a weak frameshift mutagenicity has been shown by nutmeg and a very weak oxidative mutagenic action has been revealed by cumin. Nat Prod Res, 2003 Oct, 17(5), 319 - 23 Suppressive effect of the SOS-inducing activity of chemical mutagen by citric acid esters from Prunus mume Sieb . Et Zucc . using the Salmonella typhimurium TA1535/pSK1002 umu test; Miyazawa M et al.; A methanol extract from Prunus mume Sieb . Et Zucc . showed a suppressive effect of the SOS-inducing activity on the mutagen 3-amino-1,4-dimethyl-SH-pyrido{4,3-b}indole(Trp-P-1) in the Salmonella typhimurium TA1535/pSK1002 umu test . The methanol extract was re-extracted with hexane, dichloromethane, ethyl acetate, butanol and water . The dichloromethane and ethyl acetate fractions showed suppressive effect . Suppressive compounds were isolated by silica gel column chromatography and identified as trimethyl citrate (1) and dimethyl citrate (2) by GC-MS, IR and 1H and 13C-NMR spectroscopy . Compounds 1 and 2 suppressed 51 and 39% of the SOS-inducing activity at a concentration of 2.0 micromol/mL. J Ethnopharmacol, 2003 Nov, 89(1), 81 - 90 The development of an in vitro screening strategy for topically applied products; Horan I et al.; The objective of this study was the pharmaco-toxicological understanding of the constituents of an authenticated herbal mixture . The mixture was prepared by maceration in ethanol and subsequent dilution to produce a topically applied lotion, for which the intended target conditions are psoriasis and eczema . A three-tiered in vitro screening strategy was adopted for evaluating this product, comprising cytotoxicity assays; mutagenicity screening and therapeutic evaluation . Viability assays performed with dilutions of both the herbal concentrate and final product on organotypic cell lines indicated that neither preparation acted as an irritant . Genotoxicity screening using six strains of Salmonella typhimurium showed no mutagenic potential, and furthermore significant anti-microbial activity was evident . Therapeutic evaluation involved assessing the antioxidant potential of the extract, which can be correlated to an anti-inflammatory effect . Nitroblue-tetrazolium (NBT) assay results indicate that the extract can reduce superoxide anion generation by 45% . The extract also increased cell viability on exposure to hydrogen peroxide by 28%, illustrating its dismutation potential . A 3-D skin culture system, EpiDerm, released 3000 microg/ml upon exposure to the extract, implying that the components enhance arachidonic acid metabolism . Overall, it may be concluded that the herbal extract is sufficiently non-toxic for dermal application and possesses anti-inflammatory activity. Cardiovasc Res, 2003 Oct 15, 60(1), 119 - 30 Impairment of glucose metabolism in hearts from rats treated with endotoxin; Tessier JP et al.; OBJECTIVE: In patients and animals with sepsis or critical illness, the mechanical function of the heart is often impaired . Although these conditions are accompanied by dramatic metabolic and hormonal changes, little is known about alterations of cardiac metabolism . In this study, we assessed the impact of an endotoxin-induced inflammation on cardiac glucose utilization . METHODS: Bacterial endotoxin (1 mg/kg lipopolysaccharide from Salmonella typhimurium, LPS) was injected intravenously to rats . Six hours after LPS application, hearts were isolated and perfused in the Langendorff mode . RESULTS: Left ventricular pressure was reduced by 50% in hearts from LPS-treated rats, compared to those from saline-injected control animals . With glucose as the sole fuel, there was no difference in glycolysis between the groups . However, on addition of beta-hydroxybutyrate (an alternative fuel which inhibits phosphofructokinase via an increased citrate level), the glycolytic rate in the LPS group was 44 and 48% lower (in basal, and insulin-stimulated conditions, respectively; P<0.01) than in control hearts . At the end of perfusions with beta-hydroxybutyrate and insulin, the cardiac citrate content was 40% higher in LPS vs . controls (P<0.001) . In addition to the reduced glycolysis, the insulin-dependent increase of cardiac glycogen was 77% smaller in LPS hearts . The difference between LPS and control glycolysis was abolished if the hearts were perfused with the ceramidase inhibitor N-oleyl-ethanolamine (5 microM), and also with the cyclooxygenase-2 inhibitor NS-398 (10 microM), or the thromboxane A2 receptor antagonist SQ-29548 (1 microM) . CONCLUSION: The inflammatory reaction caused by endotoxin impairs cardiac glucose metabolism (and in particular, the action of insulin) in at least two ways: through the exacerbation of the counterregulatory effect of alternative fuels on glycolysis, and through a reduction in net glycogen synthesis . Impairment of glycolysis may be mediated by a sphingomyelin derivative, and COX-2-derived thromboxane A2. Int J Antimicrob Agents, 2003 Oct, 22(4), 395 - 405 Salmonella bloodstream infections: report from the SENTRY Antimicrobial Surveillance Program (1997-2001); Stephen JM et al.; Salmonella spp . are significant bloodstream pathogens and are routinely monitored for antimicrobial resistance by the SENTRY Antimicrobial Surveillance Program . Six hundred and one bloodstream infection (BSI) isolates of Salmonella spp., collected over a 5-year period (1997-2001) were tested for their susceptibility against 20 antimicrobial agents, comparing year and geographical region . Salmonella enterica serotype Typhi was the most frequently identified 'species' (43% of identified strains), although 'unspeciated' strains predominated overall (54.2%) . The rank order for six selected drugs tested by their MIC(90) values and percentage susceptibility was: ceftriaxone (< or =0.25 mg/l; 99.5% susceptible)>ciprofloxacin (0.12 mg/l; 99.3%)> trimethoprim/sulphamethoxazole (< or =0.5 mg/l; 92.7%)>amoxycillin/clavulanate (16 mg/l; 89.7%)>ampicillin (>16 mg/l; 81.0%)>tetracycline (>8 mg/l; 79.4%) . Most isolates remained highly susceptible to all 20 agents examined, with the exception of Salmonella Typhimurium (only 35.3% susceptible to tetracycline, 41.2% to ampicillin, and 61.8% to amoxycillin/clavulanate) . DT104 resistance phenotypes were noted in 3.4 and nearly 60.0% of unspeciated Salmonella and S . Typhimurium, respectively . Unexpectedly, the highest overall susceptibility rates were recorded in Latin America . Fluoroquinolone resistance was observed and nalidixic acid screening MICs (< or =8 mg/l) predicted full susceptibility to ciprofloxacin . Five-year results from the SENTRY Program show no clear trend toward greater resistances in Salmonella spp . BSIs for the commonly used antimicrobial classes . With the exception of S . Typhimurium DT104, most Salmonella spp . remain highly susceptible to the tested antimicrobials that maybe utilized for Salmonella BSI. Microbiol Res, 2003, 158(3), 259 - 64 Antimicrobial potentiality of a new non-antibiotic: the cardiovascular drug oxyfedrine hydrochloride; Mazumdar K et al.; Ten cardiovascular drugs, having diverse pharmacological action, were screened for possible antimicrobial property against known eight sensitive bacteria, belonging to Gram positive and Gram negative types . Although five drugs failed to show antimicrobial activity and three had moderate antimicrobial action, oxyfedrine HCl and dobutamine were seen to possess pronounced antimicrobial property . Oxyfedrine was further tested in vitro against 471 strains of bacteria from two Gram positive and fourteen Gram negative genera . The minimum inhibitory concentration (MIC) of oxyfedrine was determined by agar dilution method, which ranged from 50-200 microg/ml in most of the strains, while some strains were inhibited at even lower concentrations . In animal experiments, this compound was capable of offering significant protection to Swiss strain of white mice, challenged with 50 median lethal dose (MLD) of a virulent strain of Salmonella typhimurium at concentrations of 15, 30 and 60 microg/mouse . The in vivo results were highly significant according to chi-square test. Lupus, 2003, 12(9), 710 - 3 Salmonella typhimurium mediastinal abscess in a patient with systemic lupus erythematosus; Joshua F et al.; We describe a case of a Salmonella typhimurium mediastinal abscess in a patient with systemic lupus erythematosus (SLE) . Patients with SLE are predisposed to nontyphoidal salmonella infection with a high incidence of bacteraemia and abscess formation . Our case is the first report of a mediastinal abscess from Salmonella typhimurium in an SLE patient and highlights the need for thorough assessment and treatment of SLE patients who have this organism identified. Biotechnol Lett, 2003 Aug, 25(16), 1369 - 74 Comparison of different approaches for the incorporation of non-radioactive labels into polymerase chain reaction products; Gauthier M et al.; Different methods for labelling polymerase chain reaction (PCR) products with non-radioactive labels for their detection by hybridization with immobilized DNA probes were compared . The use of digoxigenin (DIG) as a label provided greater sensitivity than biotin in a PCR system targeting the invA gene from Salmonella typhimurium . Incorporation of digoxigenin into amplicons in the form of 5'-DIG-labelled oligonucleotide primers resulted in better assay signals and was more economical than DIG-labelled dUTP. Nature, 2003 Sep 25, 425(6956), 402 - 6 Phagosomes are competent organelles for antigen cross-presentation; Houde M et al.; The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections . It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules . Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation . The cellular mechanisms allowing the cross-presentation pathway are poorly understood . Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum. Mol Microbiol, 2003 Oct, 50(1), 219 - 30 Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides; Bader MW et al.; Cationic antimicrobial peptides (CAMP) represent a conserved and highly effective component of innate immunity . During infection, the Gram-negative pathogen Salmonella typhimurium induces different mechanisms of CAMP resistance that promote pathogenesis in animals . This study shows that exposure of S . typhimurium to sublethal concentrations of CAMP activates the PhoP/PhoQ and RpoS virulence regulons, while repressing the transcription of genes required for flagella synthesis and the invasion-associated type III secretion system . We further demonstrate that growth of S . typhimurium in low doses of the alpha-helical peptide C18G induces resistance to CAMP of different structural classes . Inducible resistance depends on the presence of PhoP, indicating that the PhoP/PhoQ system can sense sublethal concentrations of cationic antimicrobial peptides . Growth of S . typhimurium in the presence of CAMP also leads to RpoS-dependent protection against hydrogen peroxide . Because bacterial resistance to oxidative stress and CAMP are induced during infection of animals, CAMP may be an important signal recognized by bacteria on colonization of animal tissues. Asian Pac J Cancer Prev, 2003 Jul-Sep, 4(3), 193 - 8 Antimutagenic effects of black tea in the Salmonella typhimurium reverse mutation assay; Taneja P et al.; Black tea (Camellia sinensis) is one of the most widely consumed beverages worldwide . Its chemopreventive effects are well documented in the literature . In the present set of investigations antimutagenic effects of aqueous black tea extract (ATE) and black tea polyphenols (BTP) were evaluated in the Ames test using Salmonella typhimurium tester strains TA 98 and TA 100 . Addition of benzo(a)pyrene (BaP) and cyclophosphamide (CP), two well known mutagens, at the concentrations of 20 and 15 microg/plate, respectively, in an S-9 metabolically activated system resulted in significant induction of his+ revertant colonies . However, addition of 500 microl 1, 2 and 4% ATE to the BaP and CP treated plates resulted in a dose dependent inhibition in the number of his+ revertant colonies . Furthermore in another set of experiments, supplementation with BTP at the concentrations of 100, 200 and 400 microg/plate also led to a significant inhibition in BaP and CP induced colony formation . The antimutagenic activity of BTP was found to be higher than that of ATE, which may be attributable to the higher amount of polyphenolic ingredients . Hence the study revealed that black tea has a protective efficacy in suppressing BaP and CP induced mutagenicity in a microbial test system. Am J Physiol Lung Cell Mol Physiol, 2004 Jan, 286(1), L106 - 11 Epub 2003 Sep 22. Granulocyte/macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung; Pelaez A et al.; Chronic alcohol abuse increases the risk of developing acute lung injury approximately threefold in septic patients, and ethanol ingestion for 6 wk in rats impairs alveolar epithelial barrier function both in vitro and in vivo . Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a trophic factor for the alveolar epithelium, and a recent phase II clinical study suggests that GM-CSF therapy decreases sepsis-mediated lung injury . Therefore, we hypothesized that GM-CSF treatment could improve ethanol-mediated defects in the alveolar epithelium during acute stresses such as endotoxemia . In this study, we determined that recombinant rat GM-CSF improved lung liquid clearance (as reflected by lung tissue wet:dry ratios) in ethanol-fed rats anesthetized and then challenged with 2 ml of saline via a tracheostomy tube . Furthermore, GM-CSF treatment improved lung liquid clearance and decreased epithelial protein leak in both control-fed and ethanol-fed rats after 6 h of endotoxemia induced by Salmonella typhimurium lipopolysaccharide given intraperitoneally, but with the greater net effect seen in the ethanol-fed rats . Our previous studies indicate that chronic ethanol ingestion decreases lung liquid clearance by increasing intercellular permeability . Consistent with this, GM-CSF treatment in vitro decreased permeability of alveolar epithelial monolayers derived from both control-fed and ethanol-fed rats . As in the endotoxemia model in vivo, the effect of GM-CSF was most dramatic in the ethanol group . Together, these results indicate that GM-CSF treatment has previously unrecognized effects in promoting alveolar epithelial barrier integrity and that these salutary effects may be particularly relevant in the setting of chronic alcohol abuse. Int J Med Microbiol, 2003 Aug, 293(4), 273 - 85 Occurrence and regulation of the multicellular morphotype in Salmonella serovars important in human disease; Romling U et al.; Multicellular behavior in Salmonella Typhimurium ATCC14028 called the rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli fimbriae . Over 90% of S . Typhimurium and S . Enteritidis strains from human disease, food and animals expressed the rdar morphotype at 28 degrees C . Regulation of the rdar morphotype occurred via the response regulator ompR, which activated transcription of csgD required for production of cellulose and curli fimbriae . Serovar-specific regulation of csgD required rpoS in S . Typhimurium, but was partially independent of rpoS in S . Enteritidis . Rarely, strain-specific temperature-deregulated expression of the rdar morphotype was observed . The host-restricted serovars S . Typhimurium var . Copenhagen phage type DT2 and DT99, Salmonella Typhi and Salmonella Choleraesuis did not express the rdar morphotype, while in Salmonella Gallinarum cellulose expression at 37 degrees C was seen in some strains . Therefore, the expression pattern of the rdar morphotype is serovar specific and correlates with a disease phenotype breaching the intestinal epithelial cell lining. J Food Prot, 2003 Sep, 66(9), 1543 - 9 Interaction of a free-living soil nematode, Caenorhabditis elegans, with surrogates of foodborne pathogenic bacteria; Anderson GL et al.; Free-living nematodes may harbor, protect, and disperse bacteria, including those ingested and passed in viable form in feces . These nematodes are potential vectors for human pathogens and may play a role in foodborne diseases associated with fruits and vegetables eaten raw . In this study, we evaluated the associations between a free-living soil nematode, Caenorhabditis elegans, and Escherichia coli, an avirulent strain of Salmonella Typhimurium, Listeria welshimeri, and Bacillus cereus . On an agar medium, young adult worms quickly moved toward colonies of all four bacteria; over 90% of 3-day-old adult worms entered colonies within 16 min after inoculation . After 48 h, worms moved in and out of colonies of L . welshimeri and B . cereus but remained associated with E . coli and Salmonella Typhimurium colonies for at least 96 h . Young adult worms fed on cells of the four bacteria suspended in K medium . Worms survived and reproduced with the use of nutrients derived from all test bacteria, as determined for eggs laid by second-generation worms after culturing for 96 h . Development was slightly slower for worms fed gram-positive bacteria than for worms fed gram-negative bacteria . Worms that fed for 24 h on bacterial lawns formed on tryptic soy agar dispersed bacteria over a 3-h period when they were transferred to a bacteria-free agar surface . The results of this study suggest that C . elegans and perhaps other free-living nematodes are potential vectors for both gram-positive and gram-negative bacteria, including foodborne pathogens in soil. Curr Opin Infect Dis, 2003 Oct, 16(5), 467 - 72 Antimicrobial drug resistance in Salmonella enterica; Parry CM; PURPOSE OF REVIEW: This review addresses the changing patterns of antimicrobial resistance in Salmonella . RECENT FINDINGS: Resistance to chloramphenicol, amicillin and cotrimoxazole is common in Salmonella Typhi and Paratyphi A in Asia and a few countries of Africa . In some countries, the isolation of multidrug resistant strains appears to be declining . R-type ACSSuT Salmonella Typhimurium DT104 is common in animal and human infections in many industrialized countries . Strains with additional trimethoprim and low-level ciprofloxacin resistance are increasingly seen . Resistance is appearing in new Typhimurium phage types, such as DT204b, and is common in serotypes Hadar and Virchow but not Enteritidis . A variety of Ambler class A and class C beta-lactamase enzymes have now been described causing extended spectrum cephalosporin resistance in different Salmonella serotypes . The overall level of extended spectrum cephalosporin resistance currently appears low . Low-level ciprofloxacin resistance, associated with point mutations in the gyrA gene, is inceasingly common in typhoidal and non-typhoidal serotypes isolated from humans and animals and has been associated with treatment failures . Sporadic reports describe human infections with non-Typhi Salmonella that are fully fluoroquinolone resistant . There is increasing support for the call to revise the fluoroquinolone breakpoints for Salmonella . A study from Denmark suggested that infections with drug resistant Salmonellae are associated with a poorer outcome than drug susceptible infections . SUMMARY: Resistance is increasing to several critical antimicrobials used to treat invasive salmonellosis including extended spectrum cephalosporins and quinolones . In resource poor countries, such drug resistant Salmonella infections may become effectively untreatable. Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1849 - 52 Epub 2003 Sep 19. Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase of Salmonella typhimurium; Makde RD et al.; The aphA gene of Salmonella enterica sv . Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-) . The recombinant AphA protein was purified to homogeneity . The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 112.4, b = 130.2, c = 139.6 A . Consistent with the self-rotation function, there are two tetramers in the asymmetric unit, indicating a solvent content of approximately 54% . The crystals are composed of biologically active AphA molecules. J Immunol, 2003 Oct 1, 171(7), 3668 - 74 Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella; Zeng H et al.; The gastroenteritis-causing pathogen Salmonella typhimurium induces profound transcriptional changes in intestinal epithelia resulting in the recruitment of neutrophils whose presence is the histopathologic hallmark of salmonellosis . Here we used cDNA microarray expression profiling to define the molecular determinants that mediate such changes in model intestinal epithelia . Enteropathogenic Salmonella induced a classical proinflammatory gene expression program similar to that activated by the canonical proinflammatory agonist TNF-alpha . Nonproinflammatory bacteria, both commensals (Escherichia coli) and systemic pathogens (S . typhi), did not activate this expression profile . While S . typhimurium strains lacking the SPI-1-encoded type III system were fully proinflammatory, strains lacking the genes for the flagellar structural component flagellin were nearly devoid of proinflammatory signaling . Lastly, the epithelial proinflammatory response could be largely recapitulated by basolateral addition of purified flagellin . Thus, S . typhimurium flagellin is the major molecular trigger by which this pathogen activates gut epithelial proinflammatory gene expression. Cancer Res, 2003 Sep 1, 63(17), 5188 - 93 Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors; Forbes NS et al.; Blood-borne therapeutics, which rely on diffusion and convection for delivery, often do not accumulate in effective concentrations distant from vasculature and are therefore unable to eradicate all cells within a tumor . Motile bacteria have the potential to overcome the diffusion and pressure gradients that prevent passive materials from penetrating into poorly perfused regions of tumors . Here, we test several proposed mechanisms of Salmonella typhimurium accumulation in tumors, including: (a) entrapment in the chaotic vasculature of tumors; (b) attraction to specific tumor microenvironments; and (c) preferential replication within specific microenvironments . After systemic injection of S . typhimurium into tumor-bearing mice, we used intravital microscopy and histological techniques to quantify their interaction with tumor vasculature . Immediately after injection, few S . typhimurium (<4 in 10,000) adhered to tumor vasculature; most remained passively suspended in the blood . Despite this low initial adhesion, approximately 10,000-fold more S . typhimurium accumulated in tumors than any other organ 1 week after the injection, thus demonstrating their specificity . However, within the tumors, we found that most bacteria were located in necrotic tissue as large colonies far (750 micro m) from functional vasculature . Together, these results suggest that S . typhimurium has limited ability to adhere to tumor vasculature and migrate within tumors and only survives in tissue that becomes necrotic . Although S . typhimurium is a promising delivery vehicle because of its tumor specificity, increasing its intra-tumoral motility should improve its therapeutic effectiveness. Res Microbiol, 2003 Sep, 154(7), 510 - 20 Comparison of the structure and regulation of the udp gene of Vibrio cholerae, Yersinia pseudotuberculosis, Salmonella typhimurium, and Escherichia coli; Zolotukhina M et al.; The nucleotide sequences of the udp gene encoding uridine phosphorylase of Yersinia pseudotuberculosis and Vibrio cholerae are presented and compared with the udp sequences of Salmonella typhimurium and Escherichia coli . Both genes contain 759 bases and encode a 253 amino acid polypeptide, which is the same as for E . coli and S . typhimurium . The amino acid sequence derived from S . typhimurium gene was more similar to the derived E . coli sequence, with only a 7 amino acid difference . The Y . pseudotuberculosis and V . cholerae uridine phosphorylases presented a higher degree of divergence in their amino acid sequence as compared to the corresponding E . coli amino acid sequence, with 20 and 64 changes, respectively . The promoter regions of the udp gene for S . typhimurium (udpPSt), Y . pseudotuberculosis (udpPYp) and V . cholerae (udpPVc) were identified by primer extension analysis . Comparative analysis of the udpP promoter region from Y . pseudotuberculosis, V . cholerae, S . typhimurium and E . coli revealed that location, spacing and orientation of putative binding sites for CRP protein are highly conserved, whereas CytR protein recognition sequences of udpPYp and udpPVc deviate markedly from the E . coli and S . typhimurium CytR binding site . In vitro studies demonstrated that the CytR protein from E . coli shows different affinity for each promoter region analyzed . According to this, the degree of CytR derepression after introduction of heterologous promoters into E . coli cells is different. Res Microbiol, 2003 Sep, 154(7), 457 - 65 The tripartite tricarboxylate transporter (TTT) family; Winnen B et al.; Extracytoplasmic solute binding receptors are constituents of primary and secondary active transport systems . Previous studies have shown that the constituents of two such families (ABC and TRAP-T) occur in bacteria and archaea and have undergone minimal shuffling of constituents between systems during evolutionary history . We here show that a third family of binding receptor-dependent transporters, the tripartite tricarboxylate transporter (TTT) family, the prototype of which is the TctABC system of Salmonella typhimurium, occurs in many bacteria but not in archaea or eukaryotes . Phylogenetic analyses suggest that these systems have evolved from a primordial tripartite system with only two out of 39 possible examples of shuffling of constituents between systems . The occurrence of TctA homologues in many bacteria and archaea that apparently lack corresponding TctB and TctC homologues suggests that the appearance of tripartite systems was a relatively recent evolutionary invention that occurred after the divergence of archaea and eukaryotes from bacteria. Phytomedicine, 2003, 10(6-7), 575 - 82 Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens; Shishu et al.; Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and 2-amino-6-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate . DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz{a}anthracene (DMBA)-induced mammary tumors in rats . But there are no reports regarding its antimutagenic properties against cooked food mutagens . Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S . typhimurium . These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent. Res Vet Sci, 2003 Dec, 75(3), 185 - 93 The effects of oral and intramuscular administration and dose escalation of enrofloxacin on the selection of quinolone resistance among Salmonella and coliforms in pigs; Wiuff C et al.; The effect of route of administration and dose of enrofloxacin (Baytril) on the development of fluoroquinolone resistance in Salmonella and Escherichia coli in the intestinal tract of pigs was investigated . Healthy pigs at the age of 8-10 weeks were infected with a mixture of susceptible wild-type (MICciprofloxacin = 0.03 microg/ml) and a mutant Salmonella typhimurium with reduced susceptibility to fluoroquinolones (MICciprofloxacin = 0.5 microg/ml) (in the ratio 99:1) and treated with 2.5 mg/kg bwt enrofloxacin by either intramuscular (i.m.) or oral (p.o.) administration at time points either 4 or 24 h after the infection . The treatment via the intramuscular route of administration (24 h after the infection) was carried out with elevated doses of 7.5 and 15 mg/kg bwt as well . Emergence of resistance during a 3-day treatment period and persistence up to 13 days after treatment, was monitored by counting the resistant and total number of coliforms and Salmonella in faeces of the pigs . High frequencies of fluoroquinolone resistance developed rapidly among the coliform flora independent of route of administration, dose or time of initiation of the treatment . Selection for resistance among the artificially introduced Salmonella was reduced by using the intramuscular route and by escalating the dose 3 or 6 times the recommended dose of 2.5 mg/kg bwt, which also resulted in shortening of the period, in which the pigs were shedding Salmonella . The resistance among the coliform flora persisted for at least 2 weeks . The Salmonella infection was cleared in all cases during the 2 weeks independent of frequency of resistance . The study showed that resistance is very easily selected by treatment with enrofloxacin at the recommended dose 2.5 mg/kg bwt, but also that the intensity of selection can be reduced by using intramuscular dosing (instead of oral dosing) and by escalating that i.m . dose . The results obtained with Salmonella also showed that even very small changes in the active drug concentrations might completely change the intensity of selection. J Agric Food Chem, 2003 Sep 24, 51(20), 5916 - 22 Potential chemopreventive properties of anthocyanin-rich aqueous extracts from in vitro produced tissue of sweetpotato (Ipomoea batatas L.); Konczak-Islam I et al.; Anthocyanin-rich aqueous extracts from cell suspension cultures of a high anthocyanin-producing sweetpotato PL (purple line) cell line grown under two different media conditions, MM (multiplication medium) and APM (high anthocyanin-producing medium) and from the cell line's donor tissue, field-grown storage root (SR) of sweetpotato, cv . Ayamurasaki, were evaluated for antioxidative (DPPH test), antimutagenic (Salmonella/reversion assay; mutagen, Trp-P-1), and antiproliferative (human promyelocytic leukaemia cells HL-60) activities . Both cell line extracts MM and APM exhibited higher radical scavenging activities (RSA), 3.8- and 1.4-fold, respectively, than the SR extract . The antimutagenic activity of all extracts was found to be dose-dependent . At a dose of 1 mg/plate, the highest activity exhibited APM (73% inhibition of Trp-P-1-induced reverse mutation of Salmonella typhimurium TA98), followed by MM (54% inhibition) and SR (36% inhibition) . The MM extract was the strongest inhibitor of the proliferation of human promyelocytic leukemia cells . At a concentration of 1.6 mg/mL medium during 24 h, it suppressed the growth of 47% of HL-60 cells . A significantly lower growth suppression effect displayed APM and SR extracts (21 and 25%, respectively) . Total anthocyanin levels and anthocyanin composition in evaluated samples seem to be related to their activities . The MM extract, which exhibited the highest RSA and antiproliferation activities, contained the highest level of anthocyanins . Among them, nonacylated cyanidin 3-sophoroside-5-glucoside dominated . It is speculated that the presence of this anthocyanin contributed toward enhanced activities of MM extract. Indian J Exp Biol, 2002 Dec, 40(12), 1365 - 72 Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food; Shishu et al.; Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and 2-amino-6-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate . In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S . typhimurium . However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC . More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens . Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate . These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food. Mutat Res, 2003 Sep 9, 540(1), 67 - 77 Mutagenic activity of airborne particulate matter as an indicative measure of atmospheric pollution; Ducatti A et al.; Mutagenic activity of organic extracts of airborne particulate matter at four different sites within the urban area of the city of Porto Alegre, Brazil, was investigated using the Salmonella/microsome assay, with the Kado microsuspension method . The extracts were obtained by sonication, sequentially extracted according to polarity, with cyclohexane (CX) and dichloromethane (DCM) solvents . The different fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, without S9 mix metabolic activation . A positive frameshift mutagenic response was observed for non-polar (CX) and/or moderately polar (DCM) compounds at the different sites . The responses varied at different seasons of the year, and the highest revertants per m3 (rev/m3) values were observed at the site subject to the strongest influence of automotive vehicles (site 3) in spring (17.13 rev/m3) in DCM fractions, and in summer (13.01 rev/m3) in CX fractions . The responses observed for the TA98NR and TA98/1,8-DNP6 strains suggest the contribution of nitrocompounds to the mutagenic activity observed . Although there appears to be an indicative association between the increased mass per unit volume of air (TSP) and the mutagenicity of organic extracts of airborne particulate matter in the present study, the Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples that present TPS values acceptable by the environmental quality standards established by law. Mutat Res, 2003 Sep 9, 540(1), 1 - 18 Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102; Edenharder R et al.; Mutagenicity induced by tert-butyl hydroperoxide (BHP) or cumene hydroperoxide (CHP) in Salmonella typhimurium TA102 was effectively reduced by flavonols with 3',4'-hydroxyl groups such as fisetin, quercetin, rutin, isoquercitrin, hyperoxide, myricetin, myricitrin, robinetin, and to a lesser extent also by morin and kaempferol (ID50=0.25-1.05 micromol per plate) . With the exception of isorhamnetin, rhamnetin, morin, and kaempferol, closely similar results were obtained with both peroxides . Hydrogenation of the double bond between carbons 2 and 3 (dihydroquercetin, dihydrorobinetin) as well as the additional elimination of the carbonyl function at carbon 4 (catechins) resulted in a loss of antimutagenicity with the notable exception of catechin itself . Again, all flavones and flavanones tested were inactive except luteolin, luteolin-7-glucoside, diosmetin, and naringenin . The typical radical scavenger butylated hydroxytoluene also showed strong antimutagenicity against CHP (ID50=5.4 micromol per plate) and BHP (ID50=11.4 micromol per plate) . Other lipophilic scavengers such as alpha-tocopherol and N,N'-diphenyl-1,4-phenylenediamine exerted only moderate effects, the hydrophilic scavenger trolox was inactive . The metal chelating agent 1,10-phenanthroline strongly reduced mutagenicities induced by CHP and BHP (ID50=2.75 and 2.5 micromol per plate) at low concentrations but induced mutagenic activities at higher concentrations . The iron chelator deferoxamine mesylate, however, was less effective in both respects . The copper chelator neocuproine effectively inhibited mutagenicity induced by BHP (ID50=39.7 micromol per plate) and CHP (ID50=25.9 micrommol per plate), the iron chelator 2,2'-dipyridyl was less potent (ID50=6.25 mmol per plate against BHP, 0.42 mmol per plate against CHP) . In the absence of BHP and CHP, yet not in the presence of these hydroperoxides, quercetin, rutin, catechin, epicatechin, and naringenin induced strong mutagenic activities in S . typhimurium TA102 . Radical scavenging activities of flavonoids against peroxyl radicals generated from 2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH) as measured in the haemolysis test, confirmed that in general flavonoids with di- or trihydroxy hydroxyl functions especially in positions 3', 4', 5' are effective radical scavengers . In this test system, however, luteolin was the most potent compound, followed by epicatechin and eriodictyol . Again, isorhamnetin was a potent inhibitor of lysis of red blood cells despite the presence of a 3'-OCH3 function . Radical scavenging activities of flavonoids against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in principle obeyed the rules outlined above . Flavanones, tamarixetin, and rhamnetin, however, were only weakly active against DPPH, while isorhamnetin was again a potent compound . From these results we conclude that in the Salmonella/reversion assay with strain TA102 antimutagenic activities of flavonoids against the peroxide mutagens CHP and BHP are mainly caused by radical scavenging effects. Plant Physiol, 2003 Sep, 133(1), 348 - 60 The gene expression and enzyme activity of plant 3-deoxy-D-manno-2-octulosonic acid-8-phosphate synthase are preferentially associated with cell division in a cell cycle-dependent manner; Delmas F et al.; 3-deoxy-D-manno-2-octulosonic acid-8-phosphate (Kdo-8-P) synthase catalyzes the condensation of phosphoenolpyruvate with D-arabinose-5-phosphate to yield Kdo-8-P . Kdo-8-P is the phosphorylated precursor of Kdo, a rare sugar only found in the rhamnogalacturonan II pectic fraction of the primary cell walls of higher plants and of cell wall polysaccharides of some green algae . A cDNA named LekdsA (accession no . AJ294902) encoding tomato (Lycopersicon esculentum) Kdo-8-P synthase has been isolated . The recombinant protein rescued a kdsA thermosensitive mutant of Salmonella typhimurium impaired in the synthesis of a functional Kdo-8-P synthase . Using site-directed mutagenesis of LekdsA cDNA, the tomato Kdo-8-P synthase was shown to possess the same essential amino acids that form the active sites in the bacterial enzymes . The tomato kdsA gene expression and the relevant Kdo-8-P synthase activity were preferentially associated to dividing cells, in the course of the early development of tomato fruit and in meristematic tissues . Furthermore, the transcription of the kdsA gene was found to oscillate during the cell cycle in tobacco (Nicotiana tabacum) Bright-Yellow 2 synchronized cells with a maximum during mitosis. J Basic Microbiol, 2003, 43(5), 367 - 75 Enhancing bioremoval of textile dyes by eight fungal strains from media supplemented with gelatine wastes and sucrose; Abd El-Rahim WM et al.; Eight Aspergillus strains were found to be successful in removing textile dyes from liquid media . These fungal strains were grown on medium containing: gelatine wastes and sucrose, as sources of nitrogen and carbon to test the possible speed up of the dyes removing while fungus biomass is building up in the media . The growth of fungal strains ranged from 10 to 110 mg biomass dry weight/100 ml medium . This growth induced high decolorization percentages, which ranged 33-95% within eight days . Two textile dyes Direct brown and Polar red were included in the study . The growth of the fungal strains as well as decolorization percentage of the dyes increased after 5, 6, and 8 days from incubation time with most tested strains . With Direct brown dye the strains number 2, 5, 31 and 37 recorded the highest percentage of decolorization (91, 92, 93 and 95 respectively) after incubation for 6 days . Fungal strains Aspergillus 5 and 31 gave the highest mycelium dry weight being 110 mg . Most of fungal strains induced 86 to 95 percentage of decolorization after 6 days incubation with Polar red dye . The possible toxicity of the remaining supernatant media after fungal biomass removal was tested by Ames test to assess the residual mutagenic agents remaining after dye removal, using three strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538) . The results showed that the toxicity of the dyes, measured by Ames test could be removed by the dye absorption on the fungal biomass. Structure (Camb), 2003 Sep, 11(9), 1097 - 110 Structure of the oxygen sensor in Bacillus subtilis: signal transduction of chemotaxis by control of symmetry; Zhang W et al.; Much is now known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium . The mechanism of chemotaxis of Bacillus subtilis is, in a sense, reversed . Attractant binding strengthens the activity of histidine kinase in B . subtilis, instead of an inhibition reaction . The HemAT from B . subtilis can detect oxygen and transmit the signal to regulatory proteins that control the direction of flagella rotation . We have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at 2.15 A and 2.7 A resolution, respectively . The liganded structure reveals a highly symmetrical organization . Tyrosine70 shows distinct conformational changes on one subunit when ligands are removed . Our study suggests that disruption of the symmetry of HemAT plays an important role in initiating the chemotaxis signaling transduction cascade. J Leukoc Biol, 2003 Oct, 74(4), 620 - 9 Epub 2003 Jul 15. Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho-GTPases and Akt; Forsberg M et al.; In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival . Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages . We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants . Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion . However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by approximately 70% . Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac . In contrast to phagocytosis of opsonized bacteria, invasion by S . typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K . Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella . These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis. FEMS Microbiol Lett, 2003 Aug 29, 225(2), 299 - 304 Monoclonal antibody against O:5 Salmonella antigen cross-reacts with unidentified lipopolysaccharide epitope of Salmonella serogroup O:8 (C(2)-C(3)); Mitov I et al.; Monoclonal antibody (MAb) 8aC10 against Salmonella O:5 antigen was obtained after immunization of BALB/c mice with live attenuated mutant of Salmonella typhimurium . Antigen specificity of the MAb was characterized by ELISA, immunoblotting, passive hemagglutination (PHA), passive hemolysis and agglutination tests . In ELISA, PHA and immunoblotting the MAb reacted only with lipopolysaccharides (LPS) of Salmonella strains from group O:4 (B), expressing O:5 antigen . The MAb agglutinated in addition Salmonella strains with O:8 antigen from group C(2)-C(3) but did not react with purified LPS . These results demonstrate O:5 specificity of MAb 8aC10 . Cross-agglutination with group C(2)-C(3) suggests the presence of similar but not identical epitope in O:8 expressing strains, which is possibly localized onto O-acetyl-abequose and abequose residues bound with a alpha-1-->3 linkage to the basic polysaccharide backbone of Salmonella LPS with O:5 and O:8 antigen respectively. Rheumatology (Oxford), 2004 Feb, 43(2), 148 - 55 Epub 2003 Aug 29. Comparative immune responses to candidate arthritogenic bacteria do not confirm a dominant role for Klebsiella pneumonia in the pathogenesis of familial ankylosing spondylitis; Stone MA et al.; OBJECTIVE: Using humoral immune responses, Klebsiella pneumoniae has been implicated as a candidate microbial trigger in ankylosing spondylitis (AS) by several investigators but refuted by others . The objective of this case-control study was to compare the cellular (T-cell proliferation) and humoral (IgG and IgA, by ELISA) immune responses of affected individuals in multiplex AS families with those of unaffected family members and normal healthy controls in order to find out whether affected individuals exhibit a predominant immune response to K . pneumoniae . METHODS: Twenty-five families with two or more individuals affected with AS and 34 normal healthy controls matched with the affected family members for age, sex and ethnicity were enrolled in the study . All affected (n = 57) and unaffected (n = 37) family members had a detailed clinical evaluation . Peripheral blood was drawn to determine T-lymphocyte proliferation and the IgG and IgA (by ELISA analysis) immune responses to K . pneumoniae, Salmonella typhimurium, Yersinia enterocolitica and Chlamydia trachomatis . Immune responses to each of the four candidate organisms were compared in affected and unaffected individuals . Each individual was classified by the predominant antigenic immune response that they showed when comparison was made among the same concentrations of the four candidate microbial antigens . This stratification was then used (i) to compare immune responses in affected and unaffected family members and (ii) to compare clinical characteristics of affected family members . RESULTS: There was no difference in mean stimulation indices or antibody responses between affected and unaffected family members for each of the candidate organisms . In terms of predominant cellular immune responses to these organisms, there was no difference between affected and unaffected family members with respect to K . pneumoniae, C . trachomatis or Y . enterocolitica . However, a higher percentage of affected family members (25.9%) exhibited a predominant response to S . typhimurium compared with unaffected family members (5.9%, P < 0.02) . In assessing antibody titres, K . pneumoniae was the predominant amongst these four organisms, but there was no difference between affected family members, unaffected family members and normal healthy controls . There was no relationship between immune responses and clinical characteristics . CONCLUSION: Our analysis of affected and unaffected family members in familial AS demonstrated no significant differences with respect to cellular or humoral immune responses to K . pneumoniae and three control microbes . In addition, K . pneumoniae did not exhibit a predominant immune response in affected individuals . Thus we find no supportive evidence to implicate a causal role for K . pneumoniae in familial AS. Microbiology, 2003 Sep, 149(Pt 9), 2385 - 96 The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system; Garmendia J et al.; The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells . The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication . Several of these effectors, including SifA and SifB, are encoded outside SPI-2 . The two-component regulatory system SsrA-SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors . The expression of SsrA-B is in turn regulated by the OmpR-EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter . Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA-B or OmpR-EnvZ systems . In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA-B and OmpR-EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB) . Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+ was determined . SsrA-B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals . OmpR-EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA-B system to mediate their effect on SPI-2 TTSS gene expression. Mutat Res, 2003 Aug 5, 539(1-2), 203 - 6 The mutagenic activity of the S-nitrosoglutathione/glutathione system in Salmonella typhimurium TA1535; Abu-Shakra A; S-nitrosoglutathine (GSNO) and reduced glutathione (GSH) were tested for mutagenicity against strain Salmonella typhimurium TA1535 in the Ames Standard plate incorporation assay . Neither GSNO not GSH were mutagenic when tested alone . In combination, the GSNO/GSH system induced a positive mutagenic response that ranged from 3 to 20 x over background at concentrations of 10 to 50 micromol (micromol)per plate, respectively . This mutagenic response can be attributable to the generation nitric oxide, among the many other reactive products generated by the reaction of GSNO with GSH. Mutat Res, 2003 Aug 5, 539(1-2), 157 - 66 Mutagenicity of the glutathione and cysteine S-conjugates of the haloalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene and trichlorofluoroethene in the Ames test in comparison with the tetrachloroethene-analogues; Dreessen B et al.; The nephrotoxic and nephrocarcinogenic potential of the haloalkenes is associated with the conjugation of the chemicals to L-glutathione . Subsequent processing of the haloalkene glutathione S-conjugates via the cysteine conjugate beta-lyase pathway in the mammalian kidney yields nephrotoxic and mutagenic species . To investigate whether S-conjugates of the model chlorofluoroalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene (CAS # 431-52-7) and trichlorofluoroethene (CAS # 359-29-5) show comparable effects, we have synthesised the respective cysteine and glutathione S-conjugates and subjected them to the Ames test . The cysteine and glutathione S-conjugates of tetrachloroethene (CAS # 127-18-4), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,2-trichlorovinyl)glutathione (TCVG) were used as positive controls and reference substances . S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine (DCTFPC) and S-(2,2-dichloro-1-fluorovinyl)-L-cysteine (DCFVC) showed clear dose-dependent mutagenic effects with the Salmonella typhimurium tester strains TA100 and TA98 . Using TCVC as a reference substance the following ranking in mutagenic response was established: TCVC>DCTFPC>DCFVC . S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) and S-(2,2-dichloro-1-fluorovinyl)glutathione (DCFVG) showed potent dose-dependent mutagenic effects with the S . typhimurium tester strain TA100 in the presence of a rat kidney S9-protein fraction; tests carried out in the absence of the bioactivation system resulted only in background rates of revertants . Using TCVG as a reference substance the following ranking in mutagenic response was established: TCVG=DCTFPG>DCFVG.The data obtained provide a basis for further studies on the mutagenic and presumable carcinogenic potential of the substances. Vopr Virusol, 2003 Jul-Aug, 48(4), 16 - 20 {Immune response in oral and rectal immunization by the attenuated strain of salmonella carrying the HIV DNA-vaccine}; Karpenko LI et al.; The recombinant strain of Salmonella typhimurium SL7207/pBMC-env, carrying a plasmid containing the gene of protein HIV-1 gp-160, was obtained under the monitoring by CMV-promoter . The above strain was used in the rectal and oral immunization of BALB/c mice . HIV-specific antibodies were detected in serum after a one-time immunization; such antibodies were able to inhibit the viral replication in vitro . Furthermore, the shaping-up of the specific cytotoxic and of proliferative responses was registered . Finally, the rectal immunization by cells of the Salmonella recombinant strain can be regarded as a promising delivery system of DNA-vaccine (pBMC-env), and it is more effective versus the oral immunization variant. Poult Sci, 2003 Aug, 82(8), 1337 - 42 D and z-values for Listeria monocytogenes and Salmonella typhimurium in packaged low-fat ready-to-eat turkey bologna subjected to a surface pasteurization treatment; McCormick K et al.; The D-values of Listeria monocytogenes and Salmonella Typhimurium at various surface pasteurization temperatures were determined for low-fat turkey bologna . Four cm2 meat squares were sterilized by irradiation prior to inoculation with 0.1 mL of a 10(8) cfu/mL culture, aseptically packaged in a linear low-density polyethylene pouch, and vacuum-sealed . Thermal treatments were administered by submerging packages in a heated water bath maintained at various set temperatures . At an 85 degrees C water bath temperature, no L . monocytogenes cells were detected (<10(2)) after 10 s of exposure, whereas at 61 degrees C cells viable were detected (>10(2)) up to 10 min of heating . No S . Typhimurium cells (<10(2)) were detected after 10 s at 70 degrees C, but cells survived up to 7 min at 60 degrees C . The D-values for L . monocytogenes at 61 and 65 degrees C were 124 and 16.2 s, respectively; whereas S . Typhimurium D-values were 278 s at 57 and 81 s at 60 degrees C . Z-values were 4.44 and 5.56 degrees C, respectively, for L . monocytogenes and S . Typhimurium . This study demonstrated that significant reductions in bacterial populations and complete inactivation of S . Typhimurium and L . monocytogenes cells can be achieved using an in-package thermal pasteurization process. Biochem J, 2003 Nov 1, 375(Pt 3), 745 - 52 Serine acetyltransferase of Escherichia coli: substrate specificity and feedback control by cysteine; Hindson VJ; Although SAT (serine acetyltransferase) of Escherichia coli, which catalyses the first step in cysteine synthesis, proceeds via a random-order ternary complex reaction mechanism {Hindson and Shaw (2003) Biochemistry 42, 3113-3119}, it has been suggested that the nearly identical enzyme from Salmonella typhimurium might involve an acetyl-enzyme intermediate {Leu and Cook (1994) Protein Peptide Lett . 1, 157-162} . In this study the alternative acetyl acceptor threonine and the alternative acyl donor, propionyl-CoA were used to further investigate the reaction mechanism of SAT from E . coli . Steady-state kinetic data and dead-end inhibition studies were again diagnostic of a random-order ternary complex reaction mechanism for alternative substrates . Since earlier kinetic studies with SAT from S . typhimurium suggested that cysteine competes with acetyl-CoA for binding, rather than serine with which it is isostructural, the specificity of the serine-binding pocket was assessed with three substrate mimics; beta-hydroxypropionic acid, glycine and ethanolamine . The data show that SAT interacts productively with the amino and hydroxymethyl moieties of serine, whereas the carboxyl group provides an essential contribution to binding strongly, supporting a view that cysteine will interact productively at the serine-binding site . Furthermore, since the hydroxymethyl contact region of the serine-binding site appears able to accommodate the methylene and acetyl moeties of threonine and O -acetyl-serine respectively, the site is unlikely to provide obligatory short-range contacts with the hydroxyl group of serine, a prerequisite for exclusion of cysteine . Such a proposal is supported by the results of micro-calorimetric studies which show that cysteine competes with serine for binding to SAT rather than with CoA . It follows that tight binding of cysteine at the serine-binding site near the catalytic centre may be the effector of a substantial reduction in the affinity of SAT for CoA, yielding the observed pattern of steady-state inhibition and the mechanism by which cysteine mediates effective end-product control of its synthesis. J Food Prot, 2003 Aug, 66(8), 1490 - 4 Spread of bacterial pathogens during preparation of freshly squeezed orange juice; Martinez-Gonzales NE et al.; To study the potential of three bacterial pathogens to cross-contaminate orange juice during extraction, normal operation conditions during juice preparation at food service establishments were simulated . The spread of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes from inoculated oranges to work surfaces and to the final product was determined . The transference of these three bacterial pathogens to orange juice made from uninoculated oranges with the use of contaminated utensils was also studied . Fresh oranges were inoculated with a marker strain of rifampicin-resistant Salmonella Typhimurium, E . coli O157:H7, or L . monocytogenes . Final pathogen levels in juice were compared as a function of the use of electric or mechanical juice extractors to squeeze orange juice from inoculated oranges . Pathogen populations on different contact surfaces during orange juice extraction were determined on sulfite-phenol red-rifampicin plates for Salmonella Typhimurium and E . coli O157:H7 and on tryptic soy agar supplemented with 0.1 g of rifampicin per liter for L . monocytogenes . After inoculation, the average pathogen counts for the orange rind surface were 2.3 log10 CFU/cm2 for Salmonella Typhimurium, 3.6 log10 CFU/cm2 for E . coli O157:H7, and 4.4 log10 CFU/cm2 for L . monocytogenes . This contamination was spread over all utensils used in orange juice squeezing . Mean pathogen counts for the cutting board, the knife, and the extractor ranged from -0.3 to 2.1 log10 CFU/cm2, and the juice contained 1.0 log10 CFU of Salmonella Typhimurium per ml, 2.3 log10 CFU of E . coli O157:H7 per ml, and 2.7 log10 CFU of L . monocytogenes per ml . Contact with contaminated surfaces resulted in the presence of all pathogens in orange juice made from uninoculated oranges . These results give emphasis to the importance of fresh oranges as a source of pathogens in orange juice. J Food Prot, 2003 Aug, 66(8), 1379 - 84 Stability of electrolyzed oxidizing water and its efficacy against cell suspensions of Salmonella typhimurium and Listeria monocytogenes; Fabrizio KA et al.; Electrolyzed oxidizing (EO) water has proved to be effective against foodborne pathogens attached to cutting boards and poultry surfaces and against spoilage organisms on vegetables; however, its levels of effectiveness against Listeria monocytogenes and Salmonella Typhimurium in cell suspensions have not been compared with those of other treatments . In this study, the oxidation reduction potentials (ORPs), chlorine concentrations, and pHs of acidic and basic EO water were monitored for 3 days at 4 and 25 degrees C after generation . There were no differences between the pHs or ORPs of acidic and basic EO waters stored at 4 or 25 degrees C . However, the free chlorine concentration in acidic EO water stored at 4 degrees C increased after 24 h . In contrast, the free chlorine concentration in acidic EO water stored at 25 degrees C decreased after one day . Cell suspensions of Salmonella Typhimurium and L . monocytogenes were treated with distilled water, chlorinated water (20 ppm), acidified chlorinated water (20 ppm, 4.5 pH), acidic EO water (EOA), basic EO water (EOB), or acidic EO water that was "aged" at 4 degrees C for 24 h (AEOA) for up to 15 min at either 4 or 25 degrees C . The largest reductions observed were those following treatments carried out at 25 degrees C . EOA and AEOA treatments at both temperatures significantly reduced Salmonella Typhimurium populations by > 8 log10 CFU/ml . EOA and AEOA treatments effectively reduced L . monocytogenes populations by > 8 log10 CFU/ml at 25degrees C . These results demonstrate the stability of EO water under different conditions and that EO water effectively reduced Salmonella Typhimurium and L . monocytogenes populations in cell suspensions. J Food Prot, 2003 Aug, 66(8), 1360 - 7 Sensitization of outer-membrane mutants of Salmonella typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under high pressure; Masschalck B et al.; High pressure can sensitize gram-negative bacteria to antimicrobial peptides or proteins through the permeabilization of their outer membranes; however, the range of compounds to which sensitivity is induced is species and strain dependent . We studied the role of outer-membrane properties in this sensitization by making use of a series of rough and deep rough mutants of Salmonella enterica serovar Typhimurium that show an increased degree of lipopolysaccharide (LPS) truncation, along with Pseudomonas aeruginosa PhoP and PhoQ mutants with altered outer-membrane properties . The outer-membrane properties of P . aernginosa were also modulated through the use of different Mg2- concentrations in the growth medium . Each of these strains was challenged under high pressure (15 min at 270 MPa for Salmonella Typhimurium and 15 min at 100 MPa for P . aerttginosa) in phosphate buffer with lysozyme (100 microg/ml), nisin (100 IU/ml), lactoferricin (20 microg/ml), and HEL96-116 (100 microg/ml), a synthetic lysozyme-derived peptide, and sensitization levels were compared . The results obtained indicated that outer-membrane properties affected high-pressure sensitization differently for different compounds . LPS truncation in Salmonella Typhimurium was correlated with increased sensitization to lysozyme (up to 1.5 log10 units) and nisin (up to 1.2 log10 units) but with decreased sensitization to lactoferricin under pressure . For P . aeruginosa, the pattern of sensitization to lactoferricin and nisin resembled that of polymyxin B at atmospheric pressure, suggesting that pressure induces the self-promoted uptake of both peptides . Sensitization to HEL96-116 was not affected by outer-membrane properties for either organism . Hence, outer-membrane permeabilization by high pressure cannot be explained by a single unifying mechanism and is dependent on the organism, the outer-membrane properties, and the nature of the antimicrobial compound . On the basis of these findings, the use of antimicrobial cocktails targeting different bacteria and fractions of bacterial populations may enhance the efficacy of high pressure as a preservation treatment. Environ Mol Mutagen, 2003, 42(2), 85 - 90 In vitro assessment of mutagenicity and clastogenicity of BDE-99, a pentabrominated diphenyl ether flame retardant; Evandri MG et al.; Polybrominated diphenyl ethers (PBDEs), which are widely used as flame retardants, are considered persistent organic pollutants . To date, the available toxicological data on PBDEs are limited and were primarily obtained by studying technical blends . The present study was undertaken to investigate the genotoxicity of the pure congener 2,2',4,4',5-brominated diphenyl ether (BDE-99), one of the major isomers present in penta-commercial products . Bacterial reverse mutation assays in Salmonella typhimurium strains TA98 and TA100 and in Escherichia coli WP2 uvrA, and the Allium cepa chromosome aberration test were carried out to evaluate mutagenicity and clastogenicity . The experimental design also involved testing a well-known polychlorinated biphenyl (PCB) mixture, Aroclor(R) 1254, which is structurally related to PBDEs . BDE-99 was negative in the bacterial mutagenicity assays, with and without S9 mix . Also, the frequency of structural chromosome aberrations was not significantly higher than the control and no signs of cytotoxicity were observed in BDE-99-treated A . cepa . Aroclor(R) 1254 was not mutagenic, but it induced a significant increase in chromosomal aberrations in A . cepa . In conclusion, BDE-99 was not mutagenic in S . typhimurium or E . coli, or clastogenic in A . cepa; however, the possibility that PBDEs might act through an epigenetic mechanism cannot be excluded . J Immunol, 2003 Sep 1, 171(5), 2594 - 601 C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide; Liu D et al.; C1 inhibitor (C1INH) is beneficial in animal models of endotoxemia and sepsis . However, the mechanism(s) of C1INH protection remain(s) ill-defined . In this study, we demonstrated that both active C1INH and reactive center-cleaved, inactive C1INH protected mice from lethal Gram-negative endotoxemia . Both forms of C1INH blocked the LPS-binding protein-dependent binding of Salmonella typhimurium LPS to the murine macrophage cell line, RAW 264.7, and suppressed LPS-induced TNF-alpha mRNA expression . Inhibition of LPS binding to RAW 264.7 cells was reversed with anti-C1INH Ab and was more efficient when C1INH was incubated first with LPS rather than with the cells . C1INH also suppressed LPS-induced up-regulation of TNF-alpha mRNA in whole human blood . The interaction of C1INH with LPS was directly demonstrated both by ELISA and by nondenaturing PAGE, but deletion of the amino-terminal 97-aa residues abrogated this binding . Therefore, C1INH, in addition to its function as a serine protease inhibitor, has a novel anti-inflammatory function mediated via its heavily glycosylated amino-terminal non-serpin domain. Int J Food Microbiol, 2003 Oct 15, 87(1-2), 45 - 53 Predicting thermal inactivation in media of different pH of Salmonella grown at different temperatures; Manas P et al.; The influence of the growth temperature and the pH of the heating medium on the heat resistance at different temperatures of Salmonella typhimurium ATCC 13311 was studied and described mathematically . The shift of the growth temperature from 10 to 37 degrees C increased heat resistance of S . typhimurium fourfold . The pH of the heating medium at which heat resistance was maximum was pH 6 for cells grown at 37 degrees C, but changed with growth temperature . The alkalinization of the heating medium from pH 6 to pH 7.7 decreased the heat resistance of cells grown at 37 degrees C by a factor of 3 . Neither the growth temperature nor the pH modified the z values significantly (4.9 degrees C) . The decimal reduction times at different treatment temperatures, in buffers of different pH of cells of S . typhimurium grown at different temperatures, were accurately described by a mathematical equation (correlation coefficient of 0.97) . This equation was also tested for Salmonella senftenberg 775W (ATCC 43845) and Salmonella enteritidis ATCC 13076, strains in which the correlation coefficients between the observed and the theoretically calculated values were 0.91 and 0.98, respectively. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1656 - 8 Epub 2003 Aug 19. X-ray crystallographic characterization and phasing of an NtrC homologue; Sallai L et al.; The ZraR (HydG) protein is a 441-amino-acid protein with three functional domains and is homologous to the general nitrogen-regulatory protein NtrC that regulates nitrogen assimilation in many bacteria . The AAA and DNA-binding domains (residues 141-441) of the ZraR protein from Salmonella typhimurium were crystallized using the sitting-drop vapour-diffusion method . X-ray diffraction data from the native crystal have been collected to 3.0 A resolution . Initial phasing was successfully performed by the SIRAS method using derivativatized crystals soaked in 1 mM ethylmercuric phosphate . Preliminary structural analysis shows the presence of a hexamer in the asymmetric unit . Model building is in progress. Cell Microbiol, 2003 Sep, 5(9), 601 - 11 Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections; van der Sar AM et al.; Bacterial virulence is best studied in animal models . However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals . In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium . Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days . A low dose of wild-type S . typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels . Lipopolysaccharide (LPS) mutants of S . typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells . However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation . Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity . In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10213 - 8 Epub 2003 Aug 13. A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake; Hicks DB et al.; The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function . A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI . Most Bacillus species, and some other bacteria, possess atpZ homologues . AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses . Deletion of atpZ, atpI, or atpZI from B . pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5 . Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake . atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux . We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins . Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation. J Appl Microbiol, 2003, 95(3), 563 - 75 Evaluation of the pH-dependent, stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7; Samelis J et al.; AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant . METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S . Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively . Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min . Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L . monocytogenes and Salmonella . No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed . Compared with 30 degrees C cultures, growth and acid adaptation of L . monocytogenes and S . Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase . E . coli O157:H7 had the greatest AR, relying less on acid adaptation . A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells . CONCLUSIONS: Culturing L . monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates . Low storage temperatures may substantially decrease the stationary-phase ATR of L . monocytogenes and S . Typhimurium DT104, but their effect on ATR of E . coli O157:H7 appears to be far less dramatic. Bull Exp Biol Med, 2003 May, 135(5), 452 - 5 Stimulation of mouse resistance to bacterial infection with muramyl dipeptide glycoside; Kalyuzhin OV et al.; We studied the capacity of 9 new muramyl dipeptide glycosides to stimulate mouse resistance to experimental sepsis induced by intraperitoneal injection of salmonella typhimurium culture . Preventive intraperitoneal injections of muramyl dipeptide beta-glycosides better improved survival of infected animals compared to the original (unmodified) muramyl dipeptide and muramyl dipeptide alpha-glycosides . The most effective drug muramyl dipeptide beta-heptylglycoside injected during sepsis development also reduced animal mortality, decreased bacterial contamination of the viscera, and increased phagocytic activity of peritoneal macrophages in infected animals. Chem Biol Interact, 2003 Jul 25, 146(1), 39 - 49 {35S}-labeling of the Salmonella typhimurium glutathione pool to assess glutathione-mediated DNA binding by 1,2-dibromoethane; Ross MK et al.; Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures . In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation . However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g . conjugates of 1,2-dibromoethane) . The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a {35S}-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of {35S}-label to DNA . The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates . Bacteria were grown to mid-log phase and then incubated with {35S}-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached . At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium . The {35S}-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify {35S}-binding to DNA . The amount of {35S}-label that was covalently bound to DNA in the GSTT1-1-expressing Salmonella strain (33.2 nmol/mg DNA) was sevenfold greater than that of the control strain that does not express GSTT1-1 . Neutral thermal hydrolysis of the DNA yielded a single {35S}-labeled adduct with a similar t(R) as S-{2-(N(7)-guanyl)ethyl}GSH, following HPLC analysis of the hydrolysate . This adduct accounted for 95% of the total {35S}-label bound to DNA . Thus, this {35S}-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels . This is also, to our knowledge, the first demonstration of de novo incorporation of {35S}-L-cysteine into the bacterial GSH pool. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Aug, 35(8), 695 - 701 Antigen-expressed recombinant Salmonella typhimurium driven by an in vivo-activated promoter is capable of inducing cellular immune response in transgenic mice; Wang HW et al.; To explore the approaches and mechanisms for reversing the immune tolerance in transgenic mouse, and the pathogenicity of hepatitis G virus (HGV), the promoter of phoP-activated gene (P(pagC)) of Salmonella typhimurium was used as a transcriptionally regulating element to construct an attenuated S . typhimurium expressing HGV NS3 . The recombinant S . typhimurium was orally administered to HGV transgenic mice . As the results, HGV antigen in serum and liver as well as HGV mRNA in liver were decreased significantly, although the serum anti-HGV NS3 remained undetectable as the control transgenic mice . The spleen cell proliferation, in vitro HGV NS3 specific CTL, and IFN-gamma assays with the primed cultured splenocytes indicated the induction of Th1 immune responses in those administered transgenic mice . Adoptive transfer of fractionated primed spleen cells to the transgenic mice showed that T lymphocytes were responsible for, maybe through IFN-gamma, the down-regulation of HGV mRNA transcription . Histological examination found no significant inflammatory changes in liver of the transgenic mice . These findings suggested that the oral inoculation of the HGV NS3-expressed attenuated S . typhimurium driven by an in vivo-activated promoter should be a simple and effective approach for potential treatment of chronic viral infection. Berl Munch Tierarztl Wochenschr, 2003 Jul-Aug, 116(7-8), 274 - 80 {Distribution of immunoglobulin isotypes and Salmonella antibodies in blood serum and meat juice of pigs}; Steinbach G et al.; Using the close linear regression between the logarithm of the dilution degree of a sample and the logarithm of the extinction measured in an ELISA both the relative concentrations of immunoglobulines of the isotypes IgG, IgM and IgA and of the LPS antibodies against S . Typhimurium of the different isotypes in blood sera and meat juice of 15 slaughtered pigs were detected and compared . Furthermore the total concentration of antibodies against LPS of S . Typhimurium according to the "meat juice ELISA" were compared . Distribution of immunoglobulines between serum and meat juice revealed individual differences between the animals as well as between the different immunoglobulin-isotypes . Within the same isotype the ratio of the concentrations of anti-LPS Salmonella Typhimurium antibodies between serum and meat juice was significantly closer than relating the whole of immunoglobulines of the referred isotype . In order to detect pig herds with a high level of Salmonella exposure a comparison of the 1:30 diluted meat juice samples with the 1:400 diluted blood sera is justified, however, for detailed epidemiological or scientific studies there is a need to consider the existing differences between the immunoglobuline-isotypes as well as between the specificity of antibodies and of total immunoglobulines . While the concentration of Salmonella antibodies of the isotypes IgG1, IgG2 and IgA showed a clear and statistically significant correlation between both one below the other and with the total amount of Salmonella antibodies, this connection could not be established for the total amount of immunoglobulines of different isotypes and the IgM-antibodies. Biochem Soc Trans, 2003 Aug, 31(Pt 4), 795 - 800 Expression of BPI (bactericidal/permeability-increasing protein) in human mucosal epithelia; Levy O et al.; Among the antimicrobial proteins and peptides of humans is the cationic 55 kDa bactericidal/permeability-increasing protein (BPI), which possesses antibacterial, endotoxin-neutralizing and opsonic activity against Gram-negative bacteria . Although identified originally as an abundant constituent of neutrophil granules, we have recently identified functional expression of BPI by human mucosal epithelia . BPI expression was markedly up-regulated by exposure of epithelia to lipoxins, endogenous anti-inflammatory eicosanoids that are generated in vivo in the context of aspirin treatment (aspirin-triggered lipoxins) . Epithelial BPI was found to be surface expressed and fully functional, as measured by antibacterial activity against Salmonella typhimurium as well as lipopolysaccharide (LPS; endotoxin)-neutralizing activity . These results suggest a role for BPI as an effector of epithelial antibacterial activity and as a modulator of epithelial responses to LPS . Both BPI and the lipoxins are currently the subject of intensive biopharmaceutical development, raising the possibility that therapeutic use of BPI or modulation of epithelial BPI expression may be a useful adjunctive therapy for conditions in which epithelial inflammation is associated with Gram-negative infections and/or endotoxin. Avian Dis, 2003 Apr-Jun, 47(2), 493 - 8 Outbreak of salmonellosis in a zoologic collection of lorikeets and lories (Trichoglossus, Lorius, and Eos spp.); Ward MP et al.; During August 2001, a syndrome characterized by acute lethargy and dyspnea was observed in a population of 45 lorikeets and lories in an open-air zoologic exhibit . The first death occurred on August 10, and within the next 12 days, nine more birds died (22% mortality rate) . Hepatomegaly, reddening and congestion of the lungs, and injection of the serosal surface of the intestines were the common gross findings . Histologic changes, including fibrinonecrotic hepatitis and splenitis, bacterial emboli (liver, spleen, lung, kidney, proventriculus), pulmonary congestion and hemorrhage, and enteritis, were indicative of an acute, overwhelming bacterial septicemia . Salmonella typhimurium, with the same antibiogram, was isolated from four birds . Several birds had attacked and killed a snake on July 24, and Salmonella serogroup B (untypeable) was isolated from intestine and kidney samples of a garter snake caught in the open-air exhibit on August 28 . Salmonella was also isolated from environmental samples of the exhibit but not from food preparation areas . After antimicrobial therapy, Salmonella spp . was not isolated from the surviving birds . The source of Salmonella in this outbreak remains unknown, but infection either directly or indirectly from snakes in the exhibit is possible . Contact between captive psittacine populations and reptiles should be avoided to prevent the risk of salmonellosis. Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 3 - 7 {Alteration of phagocyte functional activity depending on the presence of pR50 plasmid in bacteria}; Bondarenko VM et al.; The relationship between the presence, or the absence, of conjugative plasmid pR50 detected in Klebsiella oxytoca 89, in the isogenic pairs of attenuated strains Shigella flexneri 2a 516 Near and Salmonella typhimurium 129 Rifr and the regeneration of the active forms of oxygen by mouse peritoneal phagocytizing cells was studied . As indicated by the data obtained in the course of the experiment, plasmids pR50 could influence the chemiluminescent response of phagocytes . The inhibition of the synthesis of oxygen metabolites was found to occur at the primary stage of the infectious process, that later this synthesis increased, that facilitated the survival of the animals infected with the cultures carrying plasmid pR50. Prog Nucleic Acid Res Mol Biol, 2003, 73, 43 - 68 DNA supercoiling and transcription control: a model from the study of suppression of the leu-500 mutation in Salmonella typhimurium topA- strains; Wu HY et al.; DNA supercoiling is known to modulate gene expression . The functional relationship between DNA supercoiling and transcription initiation has been established genetically and biochemically . The molecular mechanism whereby DNA supercoiling regulates gene expression remains unclear however . Quite commonly, the same gene responds to the same DNA supercoiling change differently when the gene is positioned at different locations . Such strong positional effects on gene expression suggest that rather than the overall DNA supercoiling change, the variation of DNA supercoiling at a local site might be important for transcription control . We have started to understand the local DNA supercoiling dynamic on the chromosome . As a primary source of local DNA supercoiling fluctuation, transcription-driven DNA supercoiling is important in determining the chromosome supercoiling dynamic and theoretically, therefore, for transcription control as well . Indeed, by studying the coordinated expression of genes in the ilvIH-leuO-leuABCD gene cluster, we found that transcription-driven DNA supercoiling governs the expression of a group of functionally related genes in a sequential manner . Based on the findings in this model system, we put forward the possible mechanisms whereby DNA supercoiling plays its role in transcription control. Cancer Lett, 2003 Jul 18, 197(1-2), 211 - 7 Vaccination with minigenes encoding for novel 'self' antigens are effective in DNA-vaccination against neuroblastoma; Huebener N et al.; The induction of T-cell mediated immunity against neuroblastoma is a challenge due to poor immunogenicity of this malignancy . Here, we demonstrate the induction of protective immunity in a syngeneic murine neuroblastoma model following vaccination with minigenes comprising of three novel natural MHC class I ligands . First, after immunoprecipitation of MHC class I from NXS2 cells, peptides were eluted and examined in tandem-MS analysis which lead to the identification of three novel natural MHC class I peptide ligands, TEALPVKLI from ribonucleotide reductase M2, NEYIMSLI from Ser/Thr protein phosphatase 2A and FEMVSTLI with unknown origin . Second, we constructed two different minigenes, one encoding for the three novel epitopes and the second for three known mTH derived epitopes with high predicted binding affinity to MHC class I by cloning them into the mammalian expression vector pCMV-3FUB . This lead to constructs with an ubiquitin-tag upstream the inserted epitopes in order to facilitate proteasomal degradation . Furthermore the epitopes were separated by a spacer peptide (AAY), which proved to be a preferential proteasome cleavage site . Third, we demonstrate the induction of protective immunity against neuroblastoma using an attenuated strain of Salmonella typhimurium as a carrier harboring pCMV 3FUb vectors encoding for the two minigenes . These findings establish proof of concept that disruption of self tolerance against neuroblastoma associated epitopes may be an effective adjuvant therapeutic strategy. Folia Microbiol (Praha), 2003, 48(3), 403 - 7 Systemic and local cytokine response of young piglets to oral infection with Salmonella enterica serotype Typhimurium; Trebichavsky I et al.; One-week-old breast-fed miniature piglets were orally infected either with virulent LT2 strain or with a non-virulent SF1591 rough mutant of Salmonella Typhimurium for 1 d . Both microorganisms were cultivated from mesenteric lymph nodes but not from the blood of infected piglets . Interleukins (IL) 1 beta, 8, 18, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were quantified by ELISA in plasma and washes of a terminal part of the small bowel . In plasma, cytokines were mostly missing in non-infected piglets and either missing or low in infected piglets . In the gut of non-infected piglets, IL-1 beta, IL-8 and IL-18 were detected whereas TNF-alpha and IFN-gamma were mostly missing . IFN-gamma levels highly increased (p < 0.05) after infection with nonvirulent salmonellae . The variability of cytokine levels in the gut of suckling piglets is discussed. Environ Mol Mutagen, 2003, 42(1), 26 - 36 Comparison of mutagenicity and calf thymus DNA adducts formed by the particulate and semivolatile fractions of vehicle exhausts; Pohjola SK et al.; In this study we compared the ability of extractable organic material from particulate and semivolatile fractions of gasoline emission to induce mutations in bacteria and form adducts with calf thymus (CT) DNA with corresponding data obtained from diesel exhaust . Exhaust particles from gasoline-powered passenger cars were collected on filters and semivolatile compounds were collected on polyurethane foam (PUF) . The mutagenicity of the soluble organic fraction (SOF) was determined in Salmonella typhimurium strain TA98 and the DNA binding of aromatic compounds in the extracts was assessed by in vitro incubations with CT DNA and rat liver S9 (oxidative activation) or xanthine oxidase (reductive activation) followed by butanol-enhanced (32)P-postlabeling analysis . Semivolatile fractions of gasoline emission collected on PUF formed more CT DNA adducts than filter extracts under all reaction conditions, but showed a lower mutagenic potential than the corresponding particulate samples . This suggests that the capacity of PUF to collect exhaust particle-derived compounds and/or the efficiency of xanthine oxidase and enzymes in the rat liver S9 to activate these compounds to DNA binding metabolites was higher than expected . Gasoline extracts, benzo{a}pyrene and diesel particulate matter (SRM 1650) formed more S9-mediated DNA adducts as their dose increased, although a linear dose-response was not observed for the gasoline exhausts . Lower concentrations of gasoline and diesel extracts bound to DNA with greater efficiency than did 8-fold higher doses, suggesting complex interactions and/or an inhibition of S9 enzyme activities by the high doses . Diesel extracts formed higher levels of adducts than gasoline extracts, especially with the reductive activation system, suggesting that diesel extracts contain high levels of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) . The higher direct-acting Salmonella mutagenicity in diesel extracts in comparison with gasoline extracts is consistent with diesel extracts containing higher concentrations of nitro-PAHs . The results of this study indicate that diesel extracts are more mutagenic and form more DNA adducts than gasoline extracts and that the effects of extract dose on DNA adduct formation are complex . J Biol Chem, 2003 Sep 26, 278(39), 38094 - 103 Epub 2003 Jul 17. LeuO-mediated transcriptional derepression; Chen CC et al.; To understand the coordination of gene expression in the Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster, we had previously identified a 72-bp AT-rich (78% A+T) DNA sequence element, AT4, which was capable of silencing transcription in a promoter nonspecific manner . LeuO protein provided in trans relieved (derepressed) AT4-mediated gene silencing (transcriptional repression), but underlying mechanisms remained unclear . In the present communication, the 72-bp DNA sequence element is further dissected into two functional elements, AT7 and AT8 . LeuO binds to the 25-bp AT7, which lies closest to the leuO promoter in the AT4 DNA . After deletion of the AT7 DNA sequence responsible for LeuO binding from AT4, the remaining 47-bp AT-rich (85% A+T) DNA sequence, termed AT8, retains the full bi-directional gene-silencing activity, which is no longer relieved by LeuO . LeuO-mediated transcriptional derepression is restored when the LeuO binding site, AT7, is placed within close proximity to the gene silencer AT8 . As a pair of functionally coupled transcription elements, the presence of an equal copy number of AT7 and AT8 within proximity is important for the transcription control . The characterization provides clues for future elucidation of the molecular details whereby LeuO negates the gene-silencing activity. J Food Prot, 2003 Jul, 66(7), 1292 - 303 The role of gastric acid in preventing foodborne disease and how bacteria overcome acid conditions; Smith JL; The secretion of hydrochloric acid by the stomach plays an important role in protecting the body against pathogens ingested with food or water . A gastric fluid pH of 1 to 2 is deleterious to many microbial pathogens; however, the neutralization of gastric acid by antacids or the inhibition of acid secretion by various drugs may increase the risk of food- or waterborne illnesses . Peptic ulcer disease is often treated by decreasing or eliminating gastric acid secretion, and such treatment blocks the protective antibacterial action of gastric fluid . The majority of peptic ulcer disease cases originate from Helicobacter pylori infections . Treatment of H . pylori-induced peptic ulcers with antibiotics reduces the need for drugs that inhibit gastric acid secretion and thereby diminishes the risk of food- and waterborne illness for peptic ulcer disease patients . Many bacterial pathogens, such as Escherichia coli, Salmonella Typhimurium, and H . pylori, can circumvent the acid conditions of the stomach by developing adaptive mechanisms that allow these bacteria to survive in acid environments . As a consequence, these bacteria can survive acidic stomach conditions and pass into the intestinal tract, where they can induce gastroenteritis. Mol Microbiol, 2003 Aug, 49(3), 639 - 54 Complex regulation of csgD promoter activity by global regulatory proteins; Gerstel U et al.; The starvation-induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose . To analyse regulatory elements of csgD promoter (PcsgD) response genetic studies combined with in vitro experiments were performed . Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints . While ompR is required for PcsgD expression, binding of OmpR-P to D2 centred immediately upstream of D1 at position -70.5 is proposed to repress PcsgD activity . The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF) . Subsequently, two IHF-binding sites were identified up- and downstream of PcsgD . IHF competes with OmpR-P for binding at its upstream site IHF1, which overlaps with D3-D6 and thereby modulates the response to microaerophilic conditions . A complex regulatory network involving IHF, H-NS and OmpR is proposed whereby the nucleo-complex composition in the csgD-csgBA intergenic region is altered in response to oxygen tension. J Pediatr Surg, 2003 Jul, 38(7), 1075 - 9 Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model; Soto LJ 3rd et al.; PURPOSE: The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model . METHODS: A single, oral 10(8) cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases . Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival . RESULTS: Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls . The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes . Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days . All long-term survivors were devoid of hepatic tumor . CONCLUSIONS: Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit . Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8850 - 5 Epub 2003 Jul 11. Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine; Luo Y et al.; Protection against breast cancer was achieved with a DNA vaccine against murine transcription factor Fos-related antigen 1, which is overexpressed in aggressively proliferating D2F2 murine breast carcinoma . Growth of primary s.c . tumor and dissemination of pulmonary metastases was markedly suppressed by this oral DNA vaccine, carried by attenuated Salmonella typhimurium, encoding murine Fos-related antigen 1, fused with mutant polyubiquitin, and cotransformed with secretory murine IL-18 . The life span of 60% of vaccinated mice was tripled in the absence of detectable tumor growth after lethal tumor cell challenge . Immunological mechanisms involved activation of T, natural killer, and dendritic cells, as indicated by up-regulation of their activation markers and costimulatory molecules . Markedly increased specific target cell lysis was mediated by both MHC class I-restricted CD8+ T cells and natural killer cells isolated from splenocytes of vaccinated mice, including a significant release of proinflammatory cytokines IFN-gamma and IL-2 . Importantly, fluorescence analysis of fibroblast growth factor 2 and tumor cell-induced vessel growth in Matrigel plugs demonstrated marked suppression of angiogenesis only in vaccinated animals . Taken together, this multifunctional DNA vaccine proved effective in protecting against growth and metastases of breast cancer by combining the action of immune effector cells with suppression of tumor angiogenesis. Arch Toxicol, 2003 Sep, 77(9), 533 - 42 Epub 2003 Jul 11. Supplemental role of the Ames mutation assay and gap junction intercellular communication in studies of possible carcinogenic compounds from diesel exhaust particles; Rivedal E et al.; This study presents a new strategy for the carcinogenic evaluation of complex chemical mixtures based on genotoxic and nongenotoxic assays . We studied the ability of organic extracts of diesel exhaust particles (DEP) to induce point mutations in five different Salmonella typhimurium strains (Ames test) and to inhibit gap junction intercellular communication (GJIC) in rat liver epithelial cell lines . A crude extract of DEP was prepared by extraction with dichloromethane (DCM), and fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitro-PAH, dinitro-PAH, and polar compounds . Statistical experimental design, multivariate data analysis, and modeling were used to quantify the mutagenicity of individual and combined DEP fractions in the Ames assay . Quantitative determination of GJIC was carried out using a recently described combination of scrape loading and digital image analysis . Both assays responded to the DEP extract, but the responses were due to different fractions . The nitro-PAH fraction showed the strongest mutagenic potential, followed by the dinitro-PAH fraction . The effect on GJIC was due to the fraction containing the polar components, followed by the dinitro-PAH fraction . The extract was found to induce both basepair substitutions and frameshift mutations, through activation by bacterial nitroreductases . Hyperphosphorylation of connexin43, the major connexin in the epithelial cell lines, was less evident for DEP extract than for other communication inhibitors such as phorbol esters and growth factors, and consequently inhibitors of the protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathway were unable to counteract the inhibition by DEP extract . Since the Ames test is a well accepted method to screen for substances with genotoxic activity while inhibition of GJIC is associated with effect of tumor promoters and nongenotoxic carcinogens, it is not surprising but encouraging and interesting that the present data indicate that the two endpoints supplement each other as screening tests and in the evaluation of hazardous compounds in complex mixtures. Int J Toxicol, 2003 May-Jun, 22(3), 233 - 51 A review of the genotoxicity of triallate; Healy CE et al.; Triallate is a selective herbicidal chemical used for control of wild oats in wheat . It has an extensive genotoxicity database that includes a variety of in vitro and in vivo studies . The chemical has produced mixed results in in vitro assay systems . It was genotoxic in bacterial mutation Ames assays, predominantly in Salmonella typhimurium strains TA100 and TA1535 in the presence of S9 . Weaker responses have been observed in TA100 and TA1535 in the absence of S9 . Mixed results have been observed in strain TA98, whereas no genotoxicity has been observed in strains TA1537 and TA1538 . The presence and absence of S9 and its source seem to play a role in the bacterial response to the chemical . There have also been conflicting results in other test systems using other bacterial genera, yeast, and mammalian cells . Chromosome effects assays (sister-chromatid exchange and cytogenetics assays) have produced mixed results with S9 but no genotoxicity without S9 . Triallate has not produced any genotoxicity in in vitro DNA damage or unscheduled DNA synthesis assays using EUE cells, human lymphocytes, and rat and mouse hepatocytes . In a series of in vivo genotoxicity assays (cytogenetics, micronucleus, dominant lethal, and unscheduled DNA synthesis), there has been no indication of any adverse genotoxic effect . Metabolism data indicate that the probable explanation for the differences observed between the in vitro studies with S9 and without S9 and between the in vitro and the in vivo studies is the production of a mutagenic intermediate in vitro at high doses of triallate is expected to be at most only transiently present in in vivo studies . The weight of evidence strongly suggests that triallate is not likely to exert mutagenic activity in vivo due to toxicokinetics and metabolic processes leading to detoxification. Avian Pathol, 2003 Jun, 32(3), 277 - 83 Monitoring of pigeon paramyxovirus type-1 in organs of pigeons naturally infected with Salmonella Typhimurium; Barbezange C et al.; An experimental pigeon paramyxovirus (pPMV-1) infection was followed by reverse transcription-nested polymerase chain reaction for 31 days after infection, in 16 organs of inoculated or contact pigeons naturally infected with Salmonella Typhimurium . With two exceptions, both groups presented similar results . Typical nervous signs and a green diarrhoea were observed . The spread of pPMV-1 was relatively quick, all organs being largely positive at 4 days after inoculation or contact . The lung, spleen, caecal tonsils, kidneys and brain, for which almost all tested samples remained positive during most of the experiment, seemed to be the principal targets for virus persistence . However, the virus was significantly recovered later in the brain parts and for longer in the trachea of the contact pigeons than of the inoculated ones. Int J Occup Environ Health, 2003 Apr-Jun, 9(2), 138 - 46 Absence of carcinogenic activity in Fischer rats and B6C3F1 mice following 103-week inhalation exposures to toluene; Huff J; Toluene, methylbenzene, is used to back-blend gasoline, as a chemical intermediate, and as a solvent; more than 7 million tonnes are produced each year in the United States . Following 14-15-week toxicity studies to estimate appropriate exposure concentrations for the carcinogenesis bioassays, toxicology and carcinogenesis studies of toluene (>99% pure) were conducted by whole-body inhalation exposures of F344/N rats and B6C3F1 mice of each sex for 15 months or two years . Toluene levels were 0 (chamber controls), 600, and 1,200 ppm for rats and 0, 120, 600, and 1200 ppm for mice . Exposures were 6.5 hr/day 5 days/wk . Genetic toxicology studies using Salmonella typhimurium, mouse L5178Y lymphoma cells, and Chinese hamster ovary cells were negative . No chemically related neoplasm was found in male rats, and one nasal, two kidney, and two forestomach neoplasms observed in female rats were considered not to be associated with the toluene exposure . For mice, no biologically important increase was observed for any nonneoplastic or neoplastic lesion . Studies by others had reported carcinogenicity of toluene, especially for total malignant tumors. Arthritis Rheum, 2003 Jul, 48(7), 1853 - 7 Salmonella septicemia in rheumatoid arthritis patients receiving anti-tumor necrosis factor therapy: association with decreased interferon-gamma production and Toll-like receptor 4 expression; Netea MG et al.; OBJECTIVE: Patients treated with antibodies to tumor necrosis factor alpha (TNFalpha) have an increased susceptibility to intracellular infections . We describe 2 patients with rheumatoid arthritis (RA) who developed Salmonella septicemia during anti-TNF treatment . The aim of this study was to identify the mechanisms involved in the increased susceptibility of anti-TNF-treated patients to intracellular microorganisms . METHODS: We evaluated an additional 6 RA patients receiving anti-TNF antibodies, 5 RA patients not receiving anti-TNF therapy, and 6 age- and sex-matched healthy volunteers . The in vitro production of cytokines (interleukin-1beta {IL-1beta}, IL-6, interferon-gamma {IFNgamma}, and IL-10) upon bacterial stimulation of whole blood and the expression of Toll-like receptor 4 (TLR-4) on dendritic cells from RA patients treated with infliximab, RA patients not treated with infliximab, and healthy controls were compared . RESULTS: Stimulation with heat-killed Salmonella typhimurium or Candida albicans led to a significantly decreased production of IFNgamma, but not to a decreased production of IL-10, IL-beta, or IL-6, in anti-TNF-treated RA patients compared with RA patients who were not receiving anti-TNF antibodies and compared with healthy controls . TNF-blocking treatment ex vivo significantly inhibited TLR-4 expression on dendritic cells from RA patients and healthy controls . CONCLUSION: Since recognition of microorganisms by TLR-4 and activation of phagocytes by IFNgamma are essential mechanisms for the defense against intracellular and fungal pathogens, we propose that this pathway is crucial for the increased susceptibility to these microorganisms in patients receiving anti-TNF therapy. J Immunol, 2003 Jul 15, 171(2), 938 - 47 GM-CSF restores innate, but not adaptive, immune responses in glucocorticoid-immunosuppressed human blood in vitro; Xu J et al.; Infection remains the major complication of immunosuppressive therapy in organ transplantation . Therefore, reconstitution of the innate immunity against infections, without activation of the adaptive immune responses, to prevent graft rejection is a clinically desirable status in transplant recipients . We found that GM-CSF restored TNF mRNA and protein expression without inducing IL-2 production and T cell proliferation in glucocorticoid-immunosuppressed blood from either healthy donors or liver transplant patients . Gene array experiments indicated that GM-CSF selectively restored a variety of dexamethasone-suppressed, LPS-inducible genes relevant for innate immunity . A possible explanation for the lack of GM-CSF to restore T cell proliferation is its enhancement of the release of IL-1betaR antagonist, rather than of IL-1beta itself, since exogenously added IL-1beta induced an IL-2-independent Con A-stimulated proliferation of glucocorticoid-immunosuppressed lymphocytes . Finally, to test the in vivo relevance of our findings, we showed that GM-CSF restored the survival of dexamethasone- or cyclosporine A-immunosuppressed mice from an otherwise lethal infection with Salmonella typhimurium . In addition to this increased resistance to infection, GM-CSF did not induce graft rejection of a skin allotransplant in cyclosporine A-immunosuppressed mice . The selective restoration potential of GM-CSF suggests its therapeutic use in improving the resistance against infections upon organ transplantation. Natl Toxicol Program Tech Rep Ser, 2003 May, (508), 1 - 280 Toxicology and carcinogenesis studies of riddelliine (CAS No . 23246-96-0) in F344/N rats and B6C3F1 mice (gavage studies); National Toxicology Program; Riddelliine belongs to a class of toxic pyrrolizidine alkaloids and is isolated from plants of the genera Crotalaria, Amsinckia, and Senecio that grow in the western United States . Cattle, horses, and sheep that ingest these plants succumb to their toxic effects . Riddelliine residues have been found in meat, milk, and honey, and the plants may contaminate human food sources . Riddelliine was nominated for study by the Food and Drug Administration because of its potential for human exposure and its economic impact on the livestock industry and because the toxicity of other pyrrolizidine alkaloids suggests riddelliine may be carcinogenic . Male and female F344/N rats and B6C3F1 mice received riddelliine (approximately 92% pure) by gavage . Female rats and male and female mice were dosed for 2 years; due to high mortality, the study in male rats was terminated at week 72 . In vitro genetic toxicology studies were conducted in Salmonella typhimurium and in cultured Chinese hamster ovary (CHO) cells . In addition, riddelliine was evaluated in vivo for induction of micronuclei in mouse bone marrow and peripheral blood erythrocytes and for induction of S-phase DNA synthesis and unscheduled DNA synthesis in the liver of rats and mice . Riddelliine-induced DNA adduct levels were determined in liver tissue obtained from female rats admininstered riddelliine for 3 or 6 months . 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0 or 1 mg riddelliine/kg body weight in sodium phosphate buffer by gavage 5 days per week; additional groups of 50 female rats received 0.01, 0.033, 0.1, or 0.33 mg/kg . A wide dose range was used in female rats to better characterize the dose-response curve . Females were dosed for 105 weeks; due to high mortality, male rats were terminated at week 72 . All but three 1 mg/kg males died before week 70, and all 1 mg/kg females died before week 97 . Mean body weights of 1 mg/kg males and females were less than those of the vehicle controls throughout most of the study . The only clinical finding related to riddelliine administration was a general debilitation of the animals prior to death . Hemangiosarcomas were present in the liver of 86% of males and 76% of females in the 1 mg/kg groups, and this neoplasm was considered the cause of the large number of early deaths in these groups . The incidences of hepatocellular adenoma and mononuclear cell leukemia in 1 mg/kg males and females were significantly increased . Nonneoplastic lesions related to riddelliine treatment occurred in the liver and kidney of males and females . Analyses of liver tissue from female rats treated with riddelliine for 3 or 6 months yielded eight DNA adducts; these were the same as DNA adducts formed in vitro by the metabolism of riddelliine by human liver microsomes in the presence of calf thymus DNA . 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered riddelliine in sodium phosphate buffer by gavage at doses of 0 or 3 mg/kg, 5 days per week, for 105 weeks; additional groups of 50 male mice received 0.1, 0.3, or 1 mg/kg for 105 weeks . A wide dose range was used in male mice to better characterize the dose-response curve . Survival of males and females administered 3 mg/kg was significantly less than that of the vehicle controls . Mean body weights of 3 mg/kg mice were less than those of the vehicle controls throughout most of the study . Hemangiosarcomas of the liver were present in 62% of males in the 3 mg/kg group . The incidences of hepatocellular neoplasms occurred with negative trends in male mice and were significantly decreased in 3 mg/kg females . The incidences of alveolar/bronchiolar neoplasms in 3 mg/kg females were significantly increased . Nonneoplastic lesions related to riddelliine administration occurred in the liver and kidney of males and females and in the lung and arteries (multiple tissues) of females . GENETIC TOXICOLOGY: Riddelliine was mutagenic in S . typhimurium strain TA100 with, but not without, S9 activation; no significant mutagenic activity was detected in strain TA98 or TA1535,ed in strain TA98 or TA1535, with or without S9 . A small, dose-related increase in mutant colonies seen in strain TA97 with S9 was judged to be equivocal . Riddelliine induced sister chromatid exchanges in cultured CHO cells with and without S9 . Chromosomal aberrations were induced in CHO cells only in the presence of S9 . Following 4 or 13 weeks of daily gavage treatment with riddelliine, no increases in the frequency of micronucleated erythrocytes were noted in the peripheral blood of male or female B6C3F1 mice . Use of a single intraperitoneal injection protocol, however, produced a small but significant increase in the frequency of micronucleated eryth-rocytes in peripheral blood of male Swiss mice 48 hours after injection; bone marrow analysis 24 hours after injection demonstrated a small but insignificant increase in the frequency of micronuclei . Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage . In addition, an S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period . CONCLUSIONS: Under the conditions of these studies, there was clear evidence of carcinogenic activity of riddelliine in male and female F344/N rats based primarily on increased incidences of hemangiosarcoma in the liver . The increased incidences of hepatocellular adenoma and mononuclear cell leukemia in male and female rats were also considered to be treatment related . There was clear evidence of carcinogenic activity of riddelliine in male B6C3F1 mice based on increased incidences of hemangiosarcoma in the liver . There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms . Administration of riddelliine by gavage resulted in nonneoplastic lesions in the liver and kidney of male and female rats; the liver and kidney of male and female mice; and the lung and arteries (multiple tissues) of female mice . Decreased incidences of hepatocellular neoplasms in male and female mice were related to riddelliine administration. Mutagenesis, 2003 Jul, 18(4), 331 - 5 Mutagenicity and cross-linking activity of chloroalkylnitrosamines, possible new antitumor lead compounds; Ishikawa S et al.; The mutagenicity of chlorinated alpha-acetoxy nitrosamines was assayed using nine bacterial strains with various DNA repair abilities and the mechanism of their reaction with DNA was evaluated . Three alpha-acetoxy nitrosamines without a chloro group were used to investigate the effect of the chloro group on mutagenicity . Three nitrosamines having chloroethyl, chloropropyl and chlorobutyl groups were directly mutagenic in all tester strains used in this study, which showed that they damaged DNA in intact bacteria . Compared with Salmonella typhimurium TA1535, mutagenic activity was enhanced in ogt gene-deficient strains (YG7108 and YG7104), suggesting that an O(6)-alkylguanine adduct causes the mutation . The chlorinated nitrosamines showed stronger mutagenicity than non-chlorinated nitrosamines, indicating that alkylating activity was strengthened by the presence of a chloro group . The nitrosamines, especially the chloropropyl homolog, showed clear mutagenicity in the strains with an intact excision repair system, S.typhimurium TA92, TA1975 and G46 . Further, chloropropyl and chlorobutyl homologs showed interstrand cross-linking activity towards plasmid DNA . These results suggest that some chlorinated nitrosamines can act on DNA to form DNA cross-links, as observed in antitumor chloroethylnitrosoureas . Environmental nitrosamines are usually dealt with as potential carcinogens, but introduction of a chloro group has added the possibility of in vivo cross-linking activity, which is a classical and essential mechanism for antitumor agents . Therefore, the novel chlorinated nitrosamines examined in this study are proposed as new bifunctional antitumor lead compounds. Water Res, 2003 Aug, 37(14), 3319 - 26 Use of a Salmonella typhimurium hilA fusion strain to assess effects of environmental fresh water sources on virulence gene expression; Nutt JD et al.; Many fruits and vegetables are irrigated with water from rivers, lakes and even wastewater systems . Irrigation may be a route for the introduction of Salmonella . Our objectives in this study were to determine survivability and virulence expression in a strain of Salmonella typhimurium when exposed to environmental water sources . Virulence expression was measured using a beta-galactosidase assay on a hilA:lacZY fusion strain of S . typhimurium . Water samples for environmental impact studies were taken from a local pond and specific sites along the Rio Grande River, which serves as a source of irrigation water in southern Texas . There was a significant difference (p<0.05) of virulence expression among the water sites . Certain regions along the Rio Grande River yielded greater amounts of beta-galactosidase activity than others . All sites yielded at least a two-fold greater virulence response than S . typhimurium grown in brain heart infusion . Salmonella survivors were enumerated as colony forming units (CFU)/ml as plated on a selective medium for the duration of 1 week and beta-galactosidase assays were performed to determine a possible correlation between culturable cells and virulence gene expression . Bacterial cells remained viable but decreased after 7 days incubation . In conclusion, water sampled at specific locations and at different times water samples exhibited differences in virulence expression in S . typhimurium. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 135 - 45 Effect of in situ expression of human interleukin-6 on antibody responses against Salmonella typhimurium antigens; Li Y et al.; In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyA(s)) . Through stable introduction of a second hIL-6-HlyA(s) expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyA(s) secretion efficiencies were increased by at least 10-fold . As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did plasmids interfere in their stabilities . Increased hIL-6-HlyA(s) expression did not adversely interfere with bacterial growth . Comparative immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses . In mice immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain . Higher sera antibody titers were accompanied by increased numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer's patches, respectively . These data suggest that systemic antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 111 - 9 In vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var . Typhimurium; Dunstan SJ et al.; This study examined the ability of different plasmid vectors encoding H(C) fragment, the non-toxic binding portion of tetanus toxin, to be stably retained by Salmonella enterica var . Typhimurium (Salmonella typhimurium) vaccine strain BRD509 and, upon immunisation, to induce an antibody response against the carried antigen . The H(C) fragment expression cassette containing the transcription/translation signals, H(C) fragment open reading frame and the downstream TrpA terminator, was excised from pTETtac4 and incorporated into the plasmids pIC20H, pBR322, pACYC184 and pRSF1010 . The resulting constructs were transferred into attenuated S . typhimurium, BRD509, and the level of H(C) fragment expression was examined by Western blot analysis . The relative stability of each plasmid in S . typhimurium was determined in vitro in the absence of antibiotic selection, and in vivo following immunisation . The ability of each H(C) fragment-expressing strain to induce lipopolysaccharide- and tetanus toxoid-specific antibody responses was assayed by an enzyme-linked immunosorbent assay . These studies showed that all the vaccine vector constructs, except the S . typhimurium carrying the expression vector based on pIC20H, were able to elicit a high titre immune response . The level of tetanus toxoid-specific antibody induced by S . typhimurium directly correlated with the level of in vitro and in vivo stability of the H(C) fragment expression plasmid carried by the bacterium, and not with an increased copy number of the parent plasmid vector. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 99 - 104 Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization; Kang HY et al.; The subcellular location of a recombinant antigen in recombinant attenuated Salmonella vaccines may influence immunogenicity dependent on exposure of the recombinant antigen to cells involved in systemic immune responses . It has been shown that a recombinant attenuated Salmonella vaccine secreting the recombinant Streptococcus pneumoniae PspA (rPspA) antigen specified by pYA3494 induced protective anti-rPspA-specific immune responses (Kang et al . (2002) Infect . Immun . 70, 1739-1749) . A recombinant plasmid pYA3496 specifying a His(6)-tagged rPspA (His(6)-rPspA) protein (no apparent signal sequence) caused the rPspA antigen to localize to the cytoplasm of Salmonella . Salmonella vaccines carrying pYA3494 or pYA3496 expressed similar amounts of rPspA . After a single oral immunization in BALB/c mice with 10(9) colony-forming units (CFU) of the recombinant Salmonella vaccines carrying pYA3494 or pYA3496, IgG antibody responses were stimulated to both rPspA and Salmonella lipopolysaccharide (LPS) antigens . The anti-rPspA IgG titer induced by Salmonella carrying pYA3494 (1.9 x 10(7)) was 10(4) times higher than induced by Salmonella carrying pYA3496 (<2.4 x 10(3)). Biochem Biophys Res Commun, 2003 Jul 11, 306(4), 1083 - 8 Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium; Khan F et al.; Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity . The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S . typhimurium was more active than that from E . coli and the K(m) values were found to be similar . The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes . A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min . This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction . The apoenzyme of E . coli and S . typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP . The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography. Biochemistry, 2003 Jul 1, 42(25), 7807 - 18 Allosteric communication in the tryptophan synthase bienzyme complex: roles of the beta-subunit aspartate 305-arginine 141 salt bridge; Ferrari D et al.; The allosteric interactions that regulate substrate channeling and catalysis in the tryptophan synthase bienzyme complex from Salmonella typhimurium are triggered by covalent reactions at the beta-site and binding of substrate/product to the alpha-site . The transmission of these allosteric signals between the alpha- and beta-catalytic sites is modulated by an ensemble of weak bonding interactions consisting of salt bridges, hydrogen bonds, and van der Waals contacts that switch the subunits between open and closed conformations . Previous work has identified a scaffolding of salt-bridges extending between the alpha- and beta-sites consisting of alphaAsp 56, betaLys 167, and betaAsp 305 . This work investigates the involvement of yet another salt bridging interaction involving the betaAsp 305-betaArg 141 pair via comparison of the spectroscopic, catalytic, and allosteric properties of the betaD305A and betaR141A mutants with the behavior of the wild-type enzyme . These mutations were found to give bienzyme complexes with impaired allosteric communication . The betaD305A mutant also exhibits altered beta-site substrate reaction specificity, while the catalytic activity of the betaR141A mutant exhibits impaired beta-site catalytic activity . The >25-fold activation of the alpha-site by alpha-aminoacrylate Schiff base formation at the beta-site found in the Na(+) form of the wild-type enzyme is abolished in the Na(+) forms of both mutants . Replacing Na(+) by NH(4)(+) or Cs(+) restores the betaD305A to a wild-type-like behavior, whereas only partial restoration is achieved with the betaR141A mutant . These studies establish that the betaD305-betaR141 salt bridge plays a crucial role both in the formation of the closed conformation of the beta-site and in the transmission of allosteric signals between the alpha- and beta-sites that switch the alpha-site on and off. J Mol Biol, 2003 Jun 27, 330(1), 87 - 97 Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors; Uppsten M et al.; The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors . The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands . Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes . The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation . This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site . R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes . We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound . The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind. Mol Cells, 2003 Apr 30, 15(2), 226 - 32 A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide); Kim CH; Invasion of host cells is essential for the pathogenicity of Salmonella . The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core {Jin et al . (2001); Kim (2002)} . The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis . rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO) . Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature . Complementation of S . typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains . In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells . These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness. Environ Mol Mutagen, 2003, 41(5), 360 - 9 Mutagenicity, antioxidant potential, and antimutagenic activity against hydrogen peroxide of cashew (Anacardium occidentale) apple juice and cajuina; Melo Cavalcante AA et al.; Fresh and processed cashew (Anacardium occidentale) apple juice (CAJ) are among the most popular drinks in Brazil . Besides their nutritional benefits, these juices have antibacterial and antitumor potential . The chemical constituents of both the fresh juice and the processed juice (cajuina) were analyzed and characterized as complex mixtures containing high concentrations of vitamin C, various carotenoids, phenolic compounds, and metals . In the present study, these beverages exhibited direct and rat liver S9-mediated mutagenicity in the Salmonella/microsome assay with strains TA97a, TA98, and TA100, which detect frameshifts and base pair substitution . No mutagenicity was observed with strain TA102, which detects oxidative and alkylating mutagens and active forms of oxygen . Both CAJ and cajuina showed antioxidant activity as determined by a total radical-trapping potential assay . To test whether this antioxidant potential might result in antimutagenesis, we used a variation of the Salmonella/microsome assay that included pre-, co-, and posttreatment of hydrogen peroxide-exposed Salmonella typhimurium strain TA102 with the juices . CAJ and cajuina protected strain TA102 against mutation by oxidative damage in co- and posttreatments . The antimutagenic effects during cotreatment with hydrogen peroxide may be due to scavenging free radicals and complexing extracellular mutagenic compounds . The protective effects in posttreatment may be due to stimulation of repair and/or reversion of DNA damage . The results indicate that CAJ and cajuina have mutagenic, radical-trapping, antimutagenic, and comutagenic activity and that these properties can be related to the chemical constituents of the juices . Mol Gen Mikrobiol Virusol, 2003, (2), 16 - 20 {Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation}; Tiganova IG et al.; The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity . We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes . The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes . An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e . of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E . col controlling the cell division. World J Gastroenterol, 2003 Jun, 9(6), 1191 - 5 Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier; Guo CC et al.; AIM: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice . METHODS: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers . By PCR, the fusion gene of the two epitopes was amplified . The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid . The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salmonella typhimurium . C57BL/6 mice were orally immunized with 1X10(8) cfu Salmonella transfectants . Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS) were used as negative controls . At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELISA . At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a ((3)H)-thymidine incorporation assay . Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine . RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P<0.01; 0.841 vs 0.298, P<0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups . Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation . CONCLUSION: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic . It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects. Bioresour Technol, 2003 Sep, 89(3), 313 - 6 Growth kinetics response of a Salmonella typhimurium poultry marker strain to fresh produce extracts; Nutt JD et al.; The purpose of this research was to assess growth response of a Salmonella typhimurium poultry marker strain to fresh homogenized vegetables . Salmonella growth rates were significantly higher (p<0.05) in jalapeno extracts than any other produce extract examined . Growth rates on samples of broccoli and lettuce extracts were greater (p<0.05) than the respective growth rates on bell pepper and tomato . Broccoli extracts yielded the highest extent of growth (4 h optical density) followed by jalapeno and bell pepper extracts . From this study, it appears that fresh produce extracts have different abilities to significantly alter growth response in Salmonella . This could potentially be explained by the variations of pH, nutrient availability to the bacteria, or unknown components found within fresh produce. J Immunol, 2003 Jun 15, 170(12), 6141 - 50 Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice; Bihl F et al.; Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents . Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane . The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4 . There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS . Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection . The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with SALMONELLA: Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication . However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host. J Immunol, 2003 Jun 15, 170(12), 6133 - 40 Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms; Raupach B et al.; The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms . Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent . We examined whether similar host defense mechanisms are required for growth control of two S . typhimurium mutant strains . Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies . We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains . After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S . typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma . TNFRp55-deficiency restored systemic virulence to both Salmonella mutants . After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not . However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells. Ann N Y Acad Sci, 2003 May, 992, 21 - 9 HPA axis activation and neurochemical responses to bacterial translocation from the gastrointestinal tract; Dunn AJ et al.; Stress can cause migration of indigenous bacterial flora from the gut to the peritoneum, a phenomenon known as bacterial translocation . Destruction of the cell walls of gram-negative bacteria can result in the production of endotoxin (lipopolysaccharide, LPS), which is the likely cause of sepsis . Exogenously administered LPS can activate the hypothalamo-pituitary-adrenal (HPA) axis as well as brain noradrenergic and indoleaminergic systems . Thus, it is possible that activations of these systems associated with laboratory stressors in rats and mice could be attributed to bacterial translocation and LPS production . To test this hypothesis we conducted experiments on the time course of bacterial translocation in response to restraint in mice, while measuring HPA and neurochemical responses . These experiments failed to show good correlations between the occurrence of bacterial translocation and HPA and neurochemical activations, suggesting that the later responses were not linked to bacterial translocation . This conclusion was supported by the observation of normal neurochemical responses to restraint in germ-free mice . In further experiments, translocation of Salmonella typhimurium, a bacterium that readily translocates in unstressed animals, was associated with HPA activation and noradrenergic and indoleaminergic responses, indicating that bacterial translocation can indeed activate the HPA axis and brain amines . However, the above experiments suggest that this is not the mechanism by which restraint activates these systems. Mol Microbiol, 2003 Jun, 48(6), 1633 - 45 Global regulation by CsrA in Salmonella typhimurium; Lawhon SD et al.; CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium . To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray . As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus . The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile . The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism . Reduced expression of reporter fusions in these two operons confirmed the microarray data . Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine . Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane . These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal. Mutat Res, 2003 Jun 19, 527(1-2), 27 - 36 Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102; Grey CE et al.; An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed . Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants) . No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and alpha-tocopherol . Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested . The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism . Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations . It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA . Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals. Mutat Res, 2003 Jun 6, 537(2), 201 - 8 The unusual effect of pKM101 on the mutagenicity of acetaldehyde oxime in Salmonella typhimurium; Prival MJ; Acetaldehyde oxime was found to induce more revertants in Salmonella typhimurium strain TA1535 than in TA100 in the absence of S9 metabolic activation . TA100 was originally constructed from TA1535 by the addition of the plasmid pKM101, carrying mucAB which generally enhances sensitivity to the mutagenic effects of chemicals . The role of pKM101 in lowering the sensitivity to acetaldehyde oxime was explored by: (1) increasing the incubation time of the selective agar plates from 2 to 3 days; (2) using a new strain, isogenic to TA100, constructed by introducing pKM101 into the TA1535 isolate used in these experiments; (3) by testing a strain constructed by inserting into TA1535 a plasmid carrying mucAB but otherwise unrelated to pKM101 . Each of these alterations increased the number of revertants per plate in the presence of acetaldehyde oxime, indicating that the apparent nonmutagenicity of this chemical in TA100 is due to multiple factors. Mutat Res, 2003 Jun 6, 537(2), 183 - 99 An assessment of the genotoxicity of 2-hydroxy-1,4-naphthoquinone, the natural dye ingredient of Henna; Kirkland D et al.; 2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna (Lawsonia inermis) . Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation . HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster . However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24h after intraperitoneal injection . In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ . HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays . It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism . Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests . However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h . There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests . As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism . Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer. Pharmazie, 2003 May, 58(5), 303 - 7 Synthesis, characterization and mutagenicity of new cis-{Pt(2-substituted-benzimidazole)2Cl2} complexes; Gumus F et al.; In this study, four new platinum(II) complexes with the structures cis-{Pt(Ligand)2Cl2} (ligand = 2-(p-methoxy-/or-p-chlorobenzyl or p-methoxyphenyl)benzimidazol (1, 2, 4 respectively) and 5(6)-methyl-2-phenoxymethylbenzimidazole (3) were synthesized and characterized by their elemental analysis, and IR and 1H NMR spectra . The potentials of the Pt(II) complexes for short-term bacterial mutagenicity were tested in reverse-mutation assays using Salmonella typhimurium frame-shift strain T 98 and S . typhimurium TA 100 and TA 102 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution . The tests were performed in the absence of S9 rat liver fraction . Among the complexes tested 1 had no mutagenic activity . Complex 4 was found to be weakly mutagenic in TA 98 only . The Pt(II) complexes 2 and 3 were found to be mutagenic in TA 98, TA 100 and TA 102. Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1081 - 3 Epub 2003 May 23. Expression and preliminary crystallographic studies of R1E, the large subunit of ribonucleotide reductase from Salmonella typhimurium; Uppsten M et al.; The nrdE gene product R1E, the large subunit of the class 1b Salmonella typhimurium ribonucleotide reductase, has been overexpressed, purified and crystallized . Initially, the protein crystallized in two orthorhombic space groups, C222(1) and P2(1)2(1)2, using tartrate and PEG 6000 as precipitants, respectively . Better diffracting crystals belonging to the tetrahedral space group P4(3)2(1)2 were obtained using sodium malonate as precipitant . The P4(3)2(1)2 crystals could only be obtained after seeding from a drop containing C222(1) crystals grown in sodium tartrate . Thus, streak-seeding resulted in crystals of a supergroup to C222(1) . Data to 2.8 A resolution have been collected on the P4(3)2(1)2 crystals which contained one R1E subunit in the asymmetric unit. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 615 - 9 Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function; Sim E et al.; The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens . The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes . This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine . Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes . The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated . These identified a 'Cys-His-Asp' catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue . As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis . The discovery of NAT in M . tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance . Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism . These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target . Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs. Biochem Soc Trans, 2003 Jun, 31(Pt 3), 532 - 6 A structural perspective on the enzymes that convert dTDP-d-glucose into dTDP-l-rhamnose; Dong C et al.; Bacteria have a rich collection of biochemical pathways for the synthesis of complex metabolites . These conversions often involve chemical reactions that are hard to reproduce in the laboratory . An area of considerable interest is in the manipulation and synthesis of carbohydrates . In contrast with amino acids, carbohydrates are densely functionalized (each carbon atom is attached to at least one heteroatom) and this holds out the prospect of discovering novel enzyme mechanisms . The results from the study of the biosynthetic dTDP-L-rhamnose pathway are discussed . dTDP-L-rhamnose is a key intermediate in many pathogenic bacteria, as it is the donor for L-rhamnose, which is found in the cell wall of important human pathogens, such as Mycobacteria tuberculosis and Salmonella typhimurium . All four enzymes have been structurally characterized; in particular, the acquisition of structural data on substrate complexes was extremely useful . The structural data have guided site-directed-mutagenesis studies that have been used to test mechanistic hypotheses . The results shed light on three classes of enzyme mechanism: nucleotide condensation, short-chain dehydrogenase activity and epimerization. J Nutr, 2003 Jun, 133(6), 1845 - 52 Fermentable fiber reduces recovery time and improves intestinal function in piglets following Salmonella typhimurium infection; Correa-Matos NJ et al.; Diarrhea is a leading cause of morbidity and mortality in infants . The addition of fiber to infant formulas reduces recovery time following pathogenic infection in infants > 6 mo old, but effects on neonates are unknown . The hypothesis that fermentable fiber reduces infection-associated symptoms and enhances intestinal structure and function in neonates was examined . Piglets (2 d old) were randomly assigned to receive formula alone (control) or formula containing methylcellulose (MCEL), soy polysaccharides (SPS) or fructooligosaccharides (FOS) for 14 d . On d 7, piglets were further randomly assigned to receive an oral gavage of Salmonella typhimurium or serve as noninfected controls . S . typhimurium infection produced diarrhea in controls and MCEL groups, but not in the SPS and FOS groups . Postinfection physical activity was lower (P = 0.0001) in the controls than in all other groups . Ileal lactase activity was reduced (P < 0.05) following infection in the control group but not in the MCEL, SPS and FOS groups . Ileal mucosal barrier function, measured as resistance, was impaired by infection (P < 0.05) in the control and SPS groups, but was unaltered in the jejunum and colon . Total ion transport and basal short-circuit current were higher (P < 0.05) in jejunum than in ileum and colon, irrespective of diet or infection . SPS and FOS increased (P < 0.05) ileal glutamine transport relative to piglets fed MCEL, irrespective of infection . Because fermentable fiber enhances intestinal function and reduces the severity of S . typhimurium infection-associated symptoms, it may be a cost-effective way in which to reduce the severity of pathogenic infection-associated symptoms in infants. Eur J Med Chem, 2003 May, 38(5), 473 - 80 Synthesis, cytotoxic activity on MCF-7 cell line and mutagenic activity of platinum(II) complexes with 2-substituted benzimidazole ligands; Gumus F et al.; Four Pt(II) complexes with 2-H/or-methyl/or-aminomethylbenzimidazole or 1,2-dimethylbenzimidazole ligands as "non-leaving groups" were synthesized and their antiproliferative properties were tested against the human MCF-7 breast cancer cell line . The mutagenic potentials of the complexes were tested in Salmonella typhimurium strains TA 98 and TA 100 in the absence of S9 rat liver fraction . In general, Pt(II) complexes tested which were found to be less active than cisplatin, exhibited moderate in vitro cytotoxic activity on MCF-7 cell line . Among the complexes tested, Pt(II) complex with 2-aminomethylbenzimidazole ligand was found to be highly mutagenic in S . typhimurium TA 98 and low mutagenic in S . typhimurium TA 100 . Pt(II) complex with 1,2-dimethylbenzimidazole was mutagenic only in S . typhimurium TA 98 . The other two complexes were found to be non-mutagen in both strains. Poult Sci, 2003 May, 82(5), 754 - 9 Evaluation of a method of ultraviolet light sanitation of broiler hatching eggs; Coufal CD et al.; Sanitation of hatching eggs is an important area of research due to the need for an effective, economical, and safe method of egg sanitation . Improved hatching egg sanitation is an important part of an overall pathogen reduction program within integrated poultry operations . This must be accomplished without disturbing the cuticle of the egg, which can decrease hatchability . The ability of ultraviolet (UV) light to kill bacteria on eggshell surfaces has been well documented . To accomplish the task of treating the eggs in a method that could be commercially implemented, a cabinet was constructed in which ultraviolet lamps were placed . A conveyor system was used to carry a plastic hatching egg flat containing 42 eggs through the cabinet for a period of 3 or 4 min . Ultraviolet intensities within the cabinet reached a maximum of 14 mW/cm2 . Experiments were conducted to test the impact of UV light (254 nm) exposure of hatching eggs on aerobic plate counts (APC), inoculated Salmonella typhimurium and inoculated Escherchia coli . In the first three experiments, seven eggs were sampled from a flat passed through the UV chamber . Ultraviolet-treated eggs compared to untreated eggs had APC reductions of 1.3 log, S . typhimurium had a 4 log reduction, and E . coli had a 4 to 5 log reduction . Laboratory trials were also conducted to test the effects of UV irradiation on the cuticle of the egg and hatchability . No significant differences for eggshell conductance or hatchability were found between UV-treated and control eggs . From these trials, it can be concluded that UV irradiation of hatching eggs in a prototype irradiation cabinet can effectively reduce aerobic and pathogenic bacteria on eggshell surfaces without affecting eggshell conductance or hatchability. J Basic Microbiol, 2003, 43(3), 218 - 29 Evaluation of biotoxicity of textile dyes using two bioassays; Moawad H et al.; The toxicity of eight textile dyes was evaluated using two bioassays namely: Ames test and seed germination test . The Ames test is widely used for the evaluation of hazardous mutagenic effect of different chemicals, as a short-term screening test for environmental impact assessment . The eight-textile dyes and Eithidium bromide dye (as positive control) were tested with five "his" Salmonella typhimurium strains: TA 100; TA 98; TA 1535; TA 1537; TA 1538 . Using six concentrations of each dye (2.5 microg/ml, 4.5 microg/ml, 9 microg/ml, 13.5 microg/ml, 18 microg/ml, and 22.5 microg/ml) revealed that, most of the dyes were mutagenic for the test strains used in this study . The high concentrations of dye eliminated microbial colonies due to the high frequency of mutation causing lethal effect on the cells.In this work the phytotoxicity of different soluble textile dyes was estimated by measuring the relative changes in seed germination of four plants: clover, wheat, tomato and lettuce . The changes in shooting percentages and root length as affected by dye were also measured . Seed germination percent and shoot growth as well as root length were recorded after 6 days of exposure to different concentrations of textile dyes in irrigation water . The results show that high concentrations of dyes were more toxic to seed germination as compared with the lower concentrations . However, the low concentrations of the tested dyes adversely affected the shooting percent significantly. J Immunol, 2003 Jun 1, 170(11), 5445 - 54 Stimulation of Toll-like receptor 4 by lipopolysaccharide during cellular invasion by live Salmonella typhimurium is a critical but not exclusive event leading to macrophage responses; Royle MC et al.; Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns . LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear . We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4(-/-) (TLR-4(-/-)) mice . We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-alpha . Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-kappa B is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae . Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531 . Both purified LPS and bacterial invasion caused translocation of NF-kappa B, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points . In conclusion, we show for the first time that production of NO and TNF-alpha is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae. Microbes Infect, 2003 May, 5(6), 527 - 34 Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium; Janssen R et al.; Salmonella typhimurium is an intracellular pathogen that can survive and replicate in macrophages . One of the host defense mechanisms that S . typhimurium encounters upon infection is superoxide produced by the phagocytes' NADPH-oxidase . Salmonella has evolved numerous ways of coping with superoxide in the extracellular environment . In addition, Salmonella has to defend itself against superoxide produced as a by-product of aerobic respiration . Over the last decade, research on bacterial mutants has led to the identification of Salmonella strains that differ from their parental strain in susceptibility to superoxide in vitro . However, the consequences of such mutations for bacterial virulence are highly variable, indicating that superoxide sensitivity per se is not a characteristic that renders Salmonella less virulent . By discussing various bacterial mutants classified according to their in vitro sensitivity to superoxide, we will exemplify the complex mechanisms that Salmonella has evolved to cope with superoxide stress. FEMS Microbiol Lett, 2003 May 16, 222(1), 75 - 82 Immunochemical detection of Salmonella group B, D and E using an optical surface plasmon resonance biosensor; Bokken GC et al.; A surface plasmon resonance biosensor (Biacore) was used to detect Salmonella through antibodies reacting with Salmonella group A, B, D and E (Kauffmann-White typing) . In the assay designed, anti-Salmonella antibodies immobilized to the biosensor surface were allowed to bind injected bacteria followed by a pulse with soluble anti-Salmonella immunoglobulins to intensify the signal . No significant interference was found for (mixtures of) 30 non-Salmonella serovars at 10(9) CFU ml(-1) . A total of 53 Salmonella serovars were successfully detected at 1 x 10(7) CFU ml(-1), except those of groups C, G, L and P, as expected . The cut-off point was determined with an equicellular mixture of Salmonella enteritidis and Salmonella typhimurium at a final amount of 1.7 x 10(3) CFU per test portion . Although further work is needed to cover the detection of all relevant Salmonella serovars in food-producing animals and food products, this work demonstrates the merits of this alternative biosensor approach in terms of automation, sensitivity, specificity, simple handling and limited hands-on time. Int J Med Microbiol, 2003 Apr, 293(1), 95 - 106 Transfer of eukaryotic expression plasmids to mammalian hosts by attenuated Salmonella spp; Weiss S; Transkingdom transfer of DNA from bacteria to other organisms, well established for bacteria, yeast and plants, was recently also extended to mammalian host cells . Attenuated intracellular bacteria or non-pathogenic bacteria equipped with adhesion and invasion properties have been demonstrated to transfer eukaryotic expression plasmids in vitro and in vivo . Here the mucosal application of attenuated Salmonella enterica spp . as DNA carrier for the induction of immune responses towards protein antigens encoded by expression plasmids, their use to complement genetic defects or deliver immunotherapeutic proteins is reviewed . Plasmid transfer has been reported for Salmonella typhimurium, S . typhi and S . choleraesuis so far but clearly other Salmonella strains should be able to transfer expression plasmids as well . Transfer of DNA is effected most likely by bacterial death within the host cell resulting from metabolic attenuation . Since these bacteria remain in the phagocytic vacuole it is unclear how the DNA from such dying bacteria is delivered to the nucleus of infected cells . Nevertheless, the efficiency that has been observed was astonishingly high, reaching close to 100% under certain conditions . Gene transfer in vivo was mainly directed towards vaccination strategies either as vaccination against infectious microorganisms or model tumors . Interestingly, in some cases tolerance against autologous antigens could be broken . In general, this type of immunization was more efficacious than either direct application of antigen, vaccination with naked DNA or using the same bacterium as a heterologous carrier expressing the antigen via a prokaryotic promoter . The ease of generating such vehicles for gene transfer combined with technology validated for mass vaccination programs and the efficacy of induction of protective immune responses makes Salmonella as carrier for mucosal DNA vaccination a highly attractive area for further research and development. J Math Biol, 2003 May, 46(5), 461 - 78 Cattaneo models for chemosensitive movement: numerical solution and pattern formation; Dolak Y et al.; We derive models for chemosensitive movement based on Cattaneo's law of heat propagation with finite speed . We apply the model to pattern formation as observed in experiments with Dictyostelium discoideum, with Salmonella typhimurium and with Escherichia coli . For Salmonella typhimurium we make predictions on pattern formation which can be tested in experiments . We discuss the relations of the Cattaneo models to classical models and we develop an effective numerical scheme. Pharmazie, 2003 Apr, 58(4), 263 - 7 Evaluation of the mutagenicity of antimalarial products isolated from Solanum nudum (Solanaceae); Pabon A et al.; Diosgenone is a major component of the hexane extract from the plant Solanum nudum (Solanaceae) . The products from degraded and acetylated diosgenone that showed in vitro antimalarial activity against the FCB-2 strain of Plasmodium falciparum and methanol, dichloromethane and ethereal extracts of Solanum nudum were tested for their mutagenic activity using the Ames test with the TA-97a, TA-98, TA-100 and TA-102 strains of Salmonella typhimurium . These compounds were not mutagenic at the tested concentrations. Natl Toxicol Program Tech Rep Ser, 1990 Feb, 313, 1 - 140 NTP Toxicology and Carcinogenesis Studies of Mirex (1,1a,2,2,3,3a,4,5,5,5a,5b,6-Dodecachlorooctahydro-1,3,4- metheno-1H-cyclouta{cd}pentalene) (CAS No . 2385-85-5) in F344/N Rats (Feed Studies); National Toxicology Program ; Mirex (95% pure), formerly used a systemic insecticide and as a fire retardant, was studied for toxicologic and carcinogenic effects by administering diets containing 0, 0.1, 1.0, 10, 25, or 50 ppm mirex to groups of 52 F344/N rats of each sex for 104 weeks . Doses selected for the 2-year studies were based primarily on the effects on body weights and survival of rats in a 26-week study . During the first 6 months of the 2-year study, because of good survival and the absence of observable toxic effects in female rats, additional groups (termed second study) of 52 F344/N female rats were started at higher dietary concentrations of 0, 50, and 100 ppm mirex . Based on feed consumption data, the estimated average intake per day was 0, 0.007, 0.075, 0.75, 1.95, and 3.85 mg mirex/kg body weight for male rats and female rats in the first study, and 0, 3.9, and 7.7 mg/kg for female rats in the additional study . Body Weights, Feed Consumption, and Survival in Two-Year Studies: Mean body weights of male rats that received 25 or 50 ppm mirex were 5%-18% lower than those of the controls throughout most of the study; mean body weights of female rats that received 50 or 100 ppm mirex were 4%-18% lower than those of the controls after week 40; mean body weights of groups receiving 0.1, 1.0, or 10 ppm were similar to those of controls . Feed consumption by dosed male rats was 83%-91% that by controls, and that by dosed female rats was 86%-99% that by controls . The top dietary exposure groups of rats received the equivalent of 3.85 mg mirex/kg body weight, whereas the 100-ppm group of female rats (second study) averaged 7.7 mg/kg . At the end of the study, survival of male rats that received 25 or 50 ppm of mirex was lower than that of controls, whereas survival of all dosed groups of female rats was similar to that of controls (male: control, 44/52; 0.1 ppm, 37/52; 1 ppm, 36/52; 10 ppm, 37/52; 25 ppm, 19/52; 50 ppm, 15/52; female-- first study: 38/52; 38/52; 35/52; 41/52; 35/52; female-- second study: control, 44/52; 50 ppm, 44/52; 100 ppm, 39/52) . Nonneoplastic and Neoplastic Effects in the Two-Year Studies: The most notable compound-related effects were observed in the liver of male and female rats . Fatty metamorphosis, cytomegaly, angiectasis (males only), and necrosis of the liver were observed at increased incidences in dosed rats . The incidences of of neoplastic nodules of the liver were dose related, and in the 10-, 25-, and 50-ppm groups of males and the 50- and 100-ppm groups of females (second study), they were markedly greater than those in controls (52/group-- male: control, 3; 0.1 ppm, 5; 1 ppm, 5;10 ppm, 14; 25 ppm, 15; 50 ppm, 26; female (second study): control, 2; 50 ppm, 23; 100 ppm, 30) . In the first study in female rats, the incidences of neoplastic nodules were not significantly different between control and dosed groups (10; 5; 4; 5; 9; 7) . The 10 neoplastic nodules of the liver seen in the control group (19%) was significantly greater than the mean incidence observed historically (57/2,015; 2.8%) . The incidences of hepatocellular carcinomas in control and dosed groups were relatively low and were not significantly different between groups . The incidences of pheochromocytomas of the adrenal gland occurred with a positive trend in male rats (8/51; 7/52; 13/52; 11/52; 18/51, 19/51); the incidences in the 25- and 50-ppm male rats were greater than that in controls; malignant pheochromocytomas were observed in 2 controls and in 2 mirex-exposed male rats . The incidence of pheochromocytomas in 50-ppm female rats in the first study was marginally greater than that in controls (control, 1/51; 50 ppm, 6/52); this borderline increase was not observed in the second female rat study and thus is not considered to be due to the dietary administration of mirex . Nephropathy occurred at similar incidences in control and mirex-exposed groups of male and female rats; however, the severity of this nonneoplastic lesion was judged to be slightly greater in the groups given 25, 50, or 100 ppm mirex (male: severe vs . moderate in controls; femas given 25, 50, or 100 ppm mirex (male: severe vs . moderate in controls; female: moderate to severe vs . moderate) . Hyperplasia of the transitional epithelium of the kidney pelvis was observed in dosed male rats (0/51; 2/51; 2/52; 5/52; 14/51; 9/52) . Transitional cell papillomas of the renal pelvis in male rats occurred with a positive trend (P&lt;0.02) (0/51; 0/51; 0/52; 1/51; 3/52) . The highest incidence previously observed in untreated male F344/N rats in NTP studies is 1/48, and the mean historical incidence is 5/1,968 (0.3&percnt;) . In both the first and second studies in female rats, the incidence of mononuclear cell leukemia showed dosed-related increases (first study: 8/52; 8/52; 11/52; 14/52; 18/52; 18/52; second study: 6/52; 9/52; 14/52) . When the data from both studies are combined, the incidences are significantly increased in the 10-, 25-, 50-, and 100-ppm groups . The mean historical incidence is 19&percnt; (375/2,021) . For the thyroid gland, there was a positive trend for follicular cell neoplasms in male rats (0/51; 1/50; 0/47; 1/47; 0/35; 4/49) and a negative trend for C-cell neoplasms in male rats (8/51; 6/50; 4/47; 7/47; 3/35; 0/49) and infemale rats in the first study (12/50; 13/50; 7/48; 9/47; 6/48; 2/46) . Neither observation is considered to be associated with the dietary administration of mirex . Genetic Toxicology: Mirex was not mutagenic in the Salmonella typhimurium-microsome assay when tested in a preincubation protocol in the presence or absence of exogenous metabolic activation in strains TA98, TA100, TA1535, or TA1537 . Mirex did not induce either sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of S9 . Conclusions: Under the conditions of these 2-year feed studies of mirex, there is clear evidence of carcinogenic activity for male and female F344/N rats, as primarily indicated by marked increased incidences of benign neoplastic nodules of the liver, as well as by increased incidences of pheochromocytomas of the adrenal gland and transitional cell papillomas of the kidney in males and by increased incidences of mononuclear cell leukemia in females . Nonneoplastic effects induced by mirex include cytomegaly, fatty metamorphosis, angiectasis (males only), and cellular necrosis in the liver . Synonyms and Trade Names: 1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachlorooctahydro-1,3,4-metheno-1H-cyclobta{cd}pentalene; hexachloropentadiene dimer; dodecachloropentacyclodecane; perchloropentacyclodecane; hexachlorocyclopentadiene dimer; Dechloranereg.; Ferriamicidereg. Natl Toxicol Program Tech Rep Ser, 1986 Oct, 314, 1 - 202 NTP Toxicology and Carcinogenesis Studies of Methyl Methacrylate (CAS No . 80-62-6) in F344/N Rats and B6C3F1 Mice (Inhalation Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of methyl methacrylate, a liquid chemical intermediate used in the plastics industry in the manufacture of plexiglass and other acrylic products, were conducted by exposing groups of F344/N rats and B6C3F1 mice by inhalation for 14 weeks and 2 years . In the 14-week studies, groups of 10 male and 10 female rats and mice were exposed to methyl methacrylate at concentrations of up to 5,000 ppm . All male and female rats and eight male and eight female mice exposed at 5,000 ppm died, one male and nine female rats and four male and no female mice exposed at 3,000 ppm died, and one male and three female rats and two male and one female mouse exposed at 2,000 ppm died; all rats and mice exposed at 500 or 1,000 ppm survived . Compared with the controls, the body weights of the exposed male and female rats and mice were lower . Compound-related lesions included inflammation associated with necrosis and loss of olfactory epithelium in the nasal turbinates in both male and female rats; malacia and gliosis in female rats; inflammation of the nasal turbinates and nasal epithelium metaplasia in both male and female mice; and renal cortical necrosis, renal cortical tubular degeneration, renal focal mineralization, and liver necrosis in male mice . Based on these results, 2-year inhalation toxicology and carcinogenesis studies were conducted in which groups of 50 male rats were exposed to methyl methacrylate at 0, 500, or 1,000 ppm; female rats at 0, 250, or 500 ppm; and male and female mice at 0, 500, or 1,000 ppm . In the 2-year studies, the body weights of the low dose and high dose male and female rats were within 10% of those of the controls . There was no difference in survival between the dosed male and female rats and the controls . Incidences of inflammation of the nasal cavity and degeneration of the olfactory sensory epithelium were greater in the dosed male and female rats than in the controls, with lesions seen in virtually all high dose animals . An increased incidence of mononuclear cell leukemia was observed in female rats exposed to methyl methacrylate at 500 ppm compared with the controls (control, 11/50; 250 ppm, 13/50; 500 ppm, 20/50) . This increase was not significant by life table tests, the method of analysis most appropriate for this fatal neoplasm . The mean body weights of dosed male and female mice were 5%-8% lower than those of the controls at the end of the 2-year studies . However, during most of the second year of the studies, body weights of dosed male mice and high dose female mice were 10%-18% lower than those of the controls . Survival rates of the dosed and control mice were similar . Incidences of inflammation and epithelial hyperplasia of the nasal cavity and degeneration of the olfactory sensory epithelium were significantly greater in all dosed groups of male and female mice compared with those of the controls . Compound-related neoplastic lesions were not found in the dosed mice . Significant dose-related decreases were observed in the incidences of pituitary gland and preputial gland tumors in male rats, alveolar/bronchiolar adenomas or carcinomas (combined) in male mice, hepatocellular adenomas in both male and female mice, and pituitary gland adenomas or adenocarcinomas (combined) and uterine adenocarcinomas in female mice . Methyl methacrylate was not mutagenic in strains TA100, TA1535, TA97, or TA98 of Salmonella typhimurium in the presence or absence of male rat or hamster liver S9 when assayed by a preincubational protocol but gave a positive response in L5178Y/TK+/- mouse lymphoma cells in the presence or absence of male rat liver S9 . In cultured Chinese hamster ovary cells, methyl methacrylate produced a reproducible, dose-related increase in the frequency of sister-chromatid exchanges, both with and without rat liver S9 . A slight, dose-related increase in chromosomal aberrations was also induced in cultured Chinese hamster ovary cells in the absence of S9; in the presence of S9, an increase in the frequency of aberrations was seen only at the highest, near-lethalas seen only at the highest, near-lethal dose of 5 mg/ml . An audit of the experimental data was conducted for the 2-year carcinogenesis studies on methyl methacrylate . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenicity of methyl methacrylate for male F344/N rats exposed at 500 or 1,000 ppm, for female F344/N rats exposed at 250 or 500 ppm, or for male and female B6C3F1 mice exposed at 500 or 1,000 ppm . Inhalation of methyl methacrylate was associated with inflammation of the nasal cavity and degeneration of the olfactory sensory epithelium in male and female rats and mice; epithelial hyperplasia of the nasal cavity was also observed in exposed mice . Synonyms: acrylic acid, 2-methyl, methyl ester; methacrylic acid, methyl ester; methyl a-methylacrylate; methyl methylacrylate; methyl-2-methylpropenoate; methyl-2-methyl-2-propenoate; 2-methyl-2-propenoic acid methyl ester; MME Natl Toxicol Program Tech Rep Ser, 1987 Jan, 315, 1 - 192 NTP Toxicology and Carcinogenesis Studies of Oxytetracycline Hydrochloride (CAS No . 2058-46-0) in F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Toxicology and carcinogenesis studies were conducted on oxytetracycline hydrochloride (greater than 98.8% pure), a broad-spectrum antibiotic . Groups of F344/N rats and B6C3F1 mice were fed diets containing oxytetracycline hydrochloride for a series of 14-day, 13-week, and 2-year studies . In the 14-day studies, no compound-related gross pathologic effects were seen in rats or mice (groups of five animals per sex per species) given up to 100,000 ppm in their feed . The final mean body weight of male rats receiving in feed was 27% lower than that of the controls . Final mean body weights of mice that received 25,000, 50,000, or 100,000 ppm were lower (male: 11%; 16%; 17%; female: 6%; 5%; 17%) than those of the controls . In the 13-week studies, groups of 10 male and 10 female rats and mice were fed diets containing up to 50,000 ppm in feed, and no chemically related gross or histopathologic effects were observed in mice of either sex or in female rats . In male rats, fatty metamorphosis of minimal severity was diagnosed in the liver of 5/10 animals at 6,300, 12,500, and 50,000 ppm and in 2/10 animals at 3,100 and 25,000 ppm . None was seen in the controls . Oxytetracycline levels in bones of rats and mice (as determined fluorometrically) at the end of the 13-week studies increased with dose, the highest levels (3-10 times background levels) being observed at 50,000 ppm . The 2-year toxicology and carcinogenesis studies were conducted by administering diets containing 0, 25,000, or 50,000 ppm oxytetracycline hydrochloride to groups of 50 male and 50 female rats and diets containing 0, 6,300, or 12,500 ppm oxytetracycline hydrochloride to groups of 50 male and 50 female mice for 103 weeks . The highest dose selected for rats was considered to be the maximum level that would not affect the nutritional value of dosed feed . The dietary concentrations correspond to the following approximate doses: rats-- 0, 1,000, or 2,000 mg/kg body weight per day; mice-- 0, 650, or 1,400 mg/kg per day . Mean body weights were approximately 5%-8% lower than those of controls in high dose male rats during weeks 4-47, in high dose male mice after week 31, and in high dose female mice after week 26 . The mean body weights of dosed female rats and low dose male and female mice were comparable to those of controls . The survival of control male rats was lower than that of the high dose group (22/50 vs 38/50) . No significant differences in survival were observed between the remaining groups of rats or between any groups of mice . Pheochromocytomas of the adrenal gland occurred with positive trends in male rats (control, 10/50; low dose, 18/50; high dose, 24/50), and the incidence in the high dose group was greater than that in the controls . Two additional control males and one additional low dose male had malignant pheochromocytomas . The incidence of adrenalgland medullary hyperplasia was elevated slightly but not significantly in dosed male rats (7/50; 14/50; 9/50) . Adenomas and adenomas and adenocarcinomas (combined) of the pituitary gland in female rats occurred with positive trends, and the incidences in the high dose group were greater than that in the controls (adenomas: 19/50; 17/50; 30/50; adenomas or adenocarcinomas {combined}: 20/50; 24/50; 32/50) . The incidence of pituitary gland hyperplasia was slightly decreased in dosed female rats (16/50; 10/50; 11/50) . No compound-related increases in nonneoplastic or neoplastic lesions were observed in male or female mice . Oxytetracycline hydrochloride was not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, or TA98 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 when assayed according to the preincubational protocol . Oxytetracycline hydrochloride was mutagenic in L5178Y/TK+/- mouse lymphoma cells in the presence but not in the absence of Aroclor 1254-induced male rat liver S9 . In cultured Chinese hamster ovary cells, oxytetracycline was weakly positive in inducing sister-chromatid exchanges both with and without Aroclor 1254-induced mang sister-chromatid exchanges both with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 but did not induce chromosomal aberrations . An audit of the experimental data was conducted for these 2-year carcinogenesis studies of oxytetracycline hydrochloride . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year feed studies of oxytetracycline hydrochloride, there was equivocal evidence of carcinogenicity for male F344/N rats, as indicated by increased incidences of pheochromocytomas of the adrenal gland . There was equivocal evidence of carcinogenicity for female F344/N rats fed diets containing oxytetracycline hydrochloride, as indicated by increased incidences of adenomas of the pituitary gland . There was no evidence of carcinogenicity for male or female B6C3F1 mice fed diets containing 6,300 or 12,500 ppm oxytetracycline hydrochloride for 2 years . Synonyms: 2-naphthacenecarboxamide,4(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6-10,2,12a-pentahydroxy-6-methyl-1,11-dioxo-monohydrochloride; Biosolvmycin; Hydrocyclin; Liquamycin; Otetryn; Oxlopar; 5-hydroxytetracycline hydrochloride; Terramycin Hydrochloride; Tetramine; Tetran Hydrochloride Natl Toxicol Program Tech Rep Ser, 1988 Jan, 320, 1 - 158 NTP Toxicology and Carcinogenesis Studies of Rotenone (CAS No . 83-79-4) in F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of rotenone (more than 98% pure), a pesticide, were conducted in B6C3F1 mice and F344/N rats for 14 days, 13 weeks, and 2 years . Results of the Fourteen-Day Studies: In the 14-day studies (dietary rotenone concentrations of 0-600 ppm in the first 14-day studies and 0-4,800 ppm in the second 14-day studies), rough hair coats and dose-related decreases in mean body weight gain were observed in rats . Rats fed diets containing rotenone at concentrations of 1,200 ppm or higher lost weight . No compound-related toxic effects were observed in mice . Results of the Thirteen-Week Studies: In the 13-week studies (concentrations of 0-1,200 ppm rotenone in feed for rats and 0-50,000 ppm for mice), compound-related effects included lower body weight gain in rats at 150 ppm or more; and bone marrow atrophy and inflammation and hyperplasia of the forestomach in male rats at 300 ppm or more and in female rats at 150 ppm or more . These findings were used to establish the dietary concentrations of rotenone for the 2-year studies . Experimental Design for the Two-Year Studies: Two-year studies of rotenone were conducted by administering diets containing 0, 38, or 75 ppm rotenone to groups of 50 F344/N rats of each sex for 103 weeks . Groups of 50 B6C3F1 mice of each sex were administered diets containing 0, 600, or 1,200 ppm rotenone on the same schedule . The estimated average amount of rotenone consumed per day was 1.7 mg/kg or 3.5 mg/kg for low dose or high dose rats and 115 mg/kg or 250 mg/kg for low dose and high dose mice . Survival and Mean Body Weight in the Two-Year Studies: Survival of control and dosed rats was similar (male: control, 22/50; low dose . 31/50; high dose, 30/50; female: control, 27/50; low dose, 32/50; high dose, 31/50) . Mean body weights of dosed and control male rats were comparable . Mean body weights of high dose female rats were 5%-9% lower than those of the controls between weeks 58 and 88 . Survival of high dose male mice was significantly greater than that of the controls (male: 29/50; 36/50; 47/50; female: 37/50; 42/50; 45/50) . Final mean body weights of dosed mice were lower than those of the controls by 8%-13% for males and 17%-24% for females . Neoplastic Effects in the Two-Year Studies: Parathyroid gland adenomas were observed in 1/41 control, 0/44 low dose, and 4/44 high dose male rats . The historicalincidence of this uncommon tumor in untreated control male rats in NTP studies is 4/1,314 (0.3%) . Because these tumors are rare and because the highest incidence ever seen in a control group is 1/50, the increase in these tumors may have been related to rotenone administration . The incidence of subcutaneous tissue fibromas, fibrosarcomas, sarcomas, myxosarcomas, or neurofibrosarcomas (combined) in low dose female rats was greater (P<0.05) than that in the controls (0/50; 5/50; 3/50) . These tumors were combined because of their possible common histiogenic origin from fibroblasts or undifferentiated mesenchymal cells . The incidence of those tumors in the low dose females was greater than the historical rats at this laboratory (9/337, 3% +/- 1%) and throughout the Program (50/2,021, 2% +/- 2%) . Because of the lack of a significant dose-related trend and because statistical significance was attained only by combining tumors of differing morphology, the subcutaneous tissue tumors in female rats were not considered to be chemically related . The incidences of these tumors in dosed male rats were not significantly different from that in the controls . Hepatocellular adenomas or carcinomas (combined) occurred in male mice with a negative (P<0.02) trend, and the incidence in the high dose group was lower than that in the controls (12/47; 12/49; 1/50) . Because this low rate of combined liver tumors is unusual, this decrease may have been related to rotenone administration . Subcutaneous tissue fibromas, sarcomas, fibrosarcomas, or neurofibrosarcomas (combined) in male mice occurred with a significant (P<0.05) negative trend (8/49; 4/50; 2/50) . The incidence in the high dose group was signignificant (P&lt;0.05) negative trend (8/49; 4/50; 2/50) . The incidence in the high dose group was significantly lower than that in the controls by the life table test (P=0.01) . Genotoxicity: Rotenone was not mutagenic when tested according to a preincubational protocol with Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 with or without metabolic activation by rat or hamster liver S9 . Rotenone induced forward mutations in the mouse L5178Y/TK&plusmn; lymphoma assay without activation; it was not tested in the presence of S9 . Results of tests with rotenone in Chinese hamster ovary cells were negative for induction of sister chromatid exchanges (SCEs) in the absence of exogenous metabolic activation (at concentrations at which the chemical was very toxic), equivocal for SCEs in the presence of rat liver S9 (due to a nonrepeatable positive response when tests were conducted up to toxic concentrations), and negative for chromosomal aberrationsin both the presence and absence of metabolic activation . Data Audit: An audit of the experimental data was conducted for the 2-year studies of rotenone . No data discrepancies were found that influenced the final interpretations . Conclusions: Under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity of rotenone for male F344/N rats, as indicated by an increased incidence of parathyroid gland adenomas (uncommon tumors) . There was no evidence of carcinogenic activity in female F344/N rats fed diets containing 38 or 75 ppm rotenone . There was no evidence of carcinogenic activity for male or female B6C3F1 mice fed diets containing 600 or 1,200 ppm rotenone for 2 years . The decreased incidence of liver neoplasms in male mice may have been related to the administration of rotenone . Synonym: 1,2,12,12a-tetrahydro-8,9-dimethoxy-2-1-methylethenyl)-{1}benzopyrano{3,4-b}furo2,3-h}{1}benzopyran-6(6H)-one Trade Names of Formulations: Derrin; Derris; Tubatoxin; Nicouline; Prentox; Noxfish; Rotocide; Barbasco; Cube Root; Haiari; Dactinol Natl Toxicol Program Tech Rep Ser, 1987 Oct, 321, 1 - 182 NTP Toxicology and Carcinogenesis Studies of Bromodichloromethane (CAS No . 75-27-4) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Bromodichloromethane (99% pure), one of several trihalomethanes commonly formed after chlorination of water, was selected for study because no carcinogenicity data were available for this compound and because chloroform, a related trihalomethane, had been found to cause tumors in rodents . The general population might be exposed to bromodichloromethane in drinking water supplies, in swimming pools, and in a variety of food substances . Single-administration, 14-day, 13-week, and 2-year studies were conducted in F344/N rats and B6C3F1 mice . The chemical was administered by gavage in corn oil because human exposure is primarily oral . Additional studies were performed to evaluate the potential for genetic damage in bacteria and mammalian cells . Results of the Short-Term Studies: In the single-administration studies, the chemical was administered at doses of 150-2,500 mg/kg per day . All rats and female mice at 1,250 and 2,500 mg/kg and all male mice at 600, 1,250, and 2,500 mg/kg died; 2/5 male rats, 1/5 female rats, and 2/5 female mice at 600 mg/kg died; all animals at lower dose levels survived . In the 14-day studies, rats received doses of 38-600 mg/kg, and mice received doses of 19-300 mg/kg per day . One female rat at 38 mg/kg and one female rat at 600 mg/kg died . Weight loss or decreased weight gain was seen at 300 and 600 mg/kg in male and female rats . All male mice at 150 and 300 mg/kg died, and one female mouse at 300 mg/kg died; no weight effects were observed in surviving mice . Dose-related necropsy findings included reddened renal medullae in male rats at 600 mg/kg and in male mice at 150 and 300 mg/kg . Clinical signs seen in high dose groups after dosing were hyperactivity in rats and lethargy in mice . In the 13-week studies, male and female rats received doses of 19-300 mg/kg per day, male mice received doses of 6.25-100 mg/kg per day, and female mice received doses of 25-400 mg/kg per day . Five of 10 male rats and 2/10 female rats at 300 mg/kg died . None of the mice died . Final body weights of male and female rats at 150 and 300 mg/kg were lower than those of vehicle controls (45%-88% of vehicle control weights); final body weights of male mice at 100 mg/kg and female mice at 400 mg/kg were 92% and 94% of those of the vehicle controls . Centrilobular degeneration in the liver and degeneration and necrosis of the kidney were seen in male rats at 300 mg/kg; centrilobular degeneration was seen in female rats at 300 mg/kg; degeneration andnecrosis of the kidney were seen in male mice at 100 mg/kg, and centrilobular degeneration of the liver was seen in female mice at 200 and 400 mg/kg . Experimental Design of the Two-Year Studies: The 2-year toxicology and carcinogenesis studies of bromodichloromethane were conducted by administering the chemical in corn oil by gavage, 5 days per week for 102 weeks, to groups of 50 male and female rats at doses of 0, 50, or 100 mg/kg per day; to groups of 50 male mice at doses of 0, 25, or 50 mg/kg per day; and to groups of 50 female mice at doses of 0, 75, or 150 mg/kg per day . The study in male rats was restarted because at 10.5 months into the original study, a temperature elevation killed 45/50 vehicle control male rats . Survival and Body Weight in the Two-Year Studies: Final survival of dosed rats was comparable to that of vehicle controls (male: vehicle control, 28/50; low dose, 36/50; high dose, 28/50; female: 34/50; 27/50; 41/50) . Mean body weights of high dose male and female rats were decreased during the last 1.5 years of the study; final mean body weights of high dose male and female rats were 88% and 79% of the vehicle control mean weights . Final mean body weights of low dose male and female rats were comparable to those of the vehicle controls . Final survival of dosed male mice was comparable to that of the vehicle controls (34/50; 32/50; 42/50) . At week 84, survival of female mice was greater than 50% in all dose groups . After week 84, survival of dosed and vehicle control female mice was reduced (final survival: 26/50; 13/50; 15/50), and this decreased survival was ass6/50; 13/50; 15/50), and this decreased survival was associated with ovarian abscesses (8/50; 19/47; 18/49) . The final mean body weight of high dose male mice was 95&percnt; that of the vehicle controls; the final mean body weight of low dose male mice was comparable to that of the vehicle controls . Mean body weights of the high dose female mice were decreased during the last 1.5 years of the study; the final mean body weight was 75&percnt; that of the vehicle controls . The final mean body weight of the low dose female mice was 91&percnt; that of the vehicle controls . Nonneoplastic Effects in the Two-Year Studies: Compound-related nonneoplastic lesions included cytomegaly and tubular cell hyperplasia of the kidney and necrosis and fatty metamorphosis of the liver in male rats; eosinophilic cytoplasmic change, clear cell change, focal cellular change, and fatty metamorphosis of the liver and tubular cell hyperplasia of the kidney in female rats; fatty metamorphosisof the liver, renal cytomegaly, and follicular cell hyperplasia of the thyroid gland in male mice; and follicular cell hyperplasia of the thyroid gland in female mice . Neoplastic Effects in the Two-Year Studies: Bromodichloromethane caused compound-related increases in the incidences of neoplasms of the large intestine and kidney in male and female rats, the kidney in male mice, and the liver in female mice, as shown in the table (see page 5 of the Technical Report) . The neoplasms of the large intestine and kidney are uncommon tumors in F344/N rats and B6C3F1 mice . Administration of bromodichloromethane was also associated with a decrease in the tumors of the adrenal glands in male rats, the pituitary and mammary glands in female rats, and the pituitary gland in female mice . Genetic Toxicology: Bromodichloromethane was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 when tested by the preincubational protocol at concentrations up to 1,000 ug/plate with or without metabolic activation . The compound was not mutagenic in the mouse lymphoma L5178Y/TK&plusmn; assay in the presence of S9 but did induce forward mutations in the system in the presence of metabolic activation from rat liver S9 . Cytogenetic tests with Chinese hamster ovary cells demonstrated no induction of chromosomal aberrations orsister chromatid exchanges following treatment with bromodichloromethane in either the presence or absence of metabolic activation . Data Audit: An audit of the experimental data was conducted for the 2-year toxicology and carcinogenesis studies of bromodichloromethane . No discrepancies were found that influenced the final interpretations of the results of these studies . Conclusions: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity for male and female F344/N rats and B6C3F1 mice as shown by increased incidences of tubular cell adenomas and adenocarcinomas in the kidney and adenocarcinomas and adenomatous polyps in the large intestine in male and female rats, increased incidences of tubular cell adenomas and adenocarcinomas in the kidney of male mice, and increased incidences of hepatocellular adenomas and carcinomas in female mice . Synonym: dichlorobromoethane Natl Toxicol Program Tech Rep Ser, 1987 Jan, 322, 1 - 172 NTP Toxicology and Carcinogenesis Studies of Phenylephrine Hydrochloride (CAS No . 61-76-7) in F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Phenylephrine hydrochloride is a sympathomimetic amine recommended for use as a nasal decongestant and as a mydriatic in ophthalmic applications . In 1977, total U.S . human exposure was estimated at 1.9 x 107 g per year . Phenylephrine hydrochloride was nominated for toxicology and carcinogenesis studies because of a lack of previous long-term studies and because two other sympathomimetic agents (soterenol hydrochloride and mesuprine hydrochloride) produced mesovarial leiomyomas in Sprague-Dawley rats . Toxicology and carcinogenesis studies of USP-grade phenylephrine hydrochloride were conducted by administering diets containing the chemical (99% pure) to F344/N rats and B6C3F1 mice of each sex in studies of 14 days, 12 weeks, and 2 years . In the 14-day studies, no toxic effects were seen in rats or mice fed diets containing up to 2,000 ppm phenylephrine hydrochloride . Doses were increased in the 12-week studies, and deaths of male rats and male mice were observed in groups fed diets containing 10,000 or 20,000 ppm; 1/10 male rats in the 5,000-ppm group died . Other than inflammatory eye lesions (considered secondary to the pharmacologic drying action of the chemical), no specific organ toxicity was noted . Body weights decreased as concentrations of phenylephrine hydrochloride in the diet were increased, and feed consumption was lower in dosed rats . Doses of 0, 620, and 1,250 ppm for rats and 0, 1,250, and 2,500 ppm for mice were selected for the 2-year studies because of the decreased body weight gains in animals given higher doses in the 12-week studies . In the 2-year studies, the approximate amount of phenylephrine hydrochloride consumed per day was 24 mg/kg for low dose rats, 50 mg/kg for high dose rats, 133 mg/kg for low dose mice, and 270 mg/kg for high dose mice . Body weight differences in rats appeared to be dose related, and dosed animals were 3%-15% lighter than controls . Body weights of dosed mice averaged 3%-14% lower than those of controls throughout the 2-year studies . Survival of high dose male rats was greater than that of the controls (control, 30/50; low dose, 33/50; high dose, 42/50); differences in survival were not significant for female rats (42/50; 34/50; 36/50), male mice (35/50; 38/50; 43/50), or female mice (37/50; 34/50; 34/50) . Few nonneoplastic lesions were related to phenylephrine hydrochloride dosing in rats or mice . Chronic focal inflammation of the liver was observed at increased incidences in dosed rats (male: 2/50; 13/50; 17/50; female:17/50; 28/50; 35/50) . Inflammation of the prostate was seen more frequently in dosed than in control males (10/50; 24/50; 24/50) . The incidence of focal cellular change in the liver was increased slightly in high dose male mice (0/50; 2/50; 7/50) . In male rats, mononuclear cell leukemia (24/50; 9/50; 5/50) and pheochromocytomas of the adrenal gland (14/49; 11/50; 2/50) occurred with negative trends, and the incidences in the high dose group were lower than those in the controls . No increases in neoplasia were seen in dosed male or female rats or mice . Phenylephrine hydrochloride was not mutagenic in four strains of Salmonella typhimurium (TA100, TA1535, TA1537, and TA98) with or without Aroclor 1254-induced liver S9 from male Sprague-Dawley rats or male Syrian hamsters . The results of mutagenicity studies of phenylephrine hydrochloride were equivocal in the mouse lymphoma L5178Y/TK+/- assay in the absence of S9; it was not tested in the presence of S9 . Phenylephrine hydrochloride induced sister-chromatid exchanges (SCEs) but not chromosomal aberrations in Chinese hamster ovary cells . The increase in SCEs was seen only in the absence of metabolic activation with S9 . An audit of the experimental data was conducted for the 2-year studies of phenylephrine hydrochloride . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year studies, there was no evidence of carcinogenicity of phenylephrine hydrochloride for male B6C3F1 mice given 1,250 or 2,500 ppm in feed . Survival of high dose male rats was greater than of high dose male rats was greater than that of controls, and the incidences of mononuclear cell leukemia and pheochromocytomas were lower in dosed than in control male rats . Inflammation was observed more frequently in the liver and prostate gland of dosed male rats than in controls . Synonyms: benzene methanol,3-hydroxy-a{(methylamino)methyl}hydrochloride (R)-; (-)-meta-hydroxy-a-{(methylamino)methyl}benzyl alcohol hydrochloride; meta-Synephrine hydrochloride; Neo-synephrinereg. Natl Toxicol Program Tech Rep Ser, 1987 Nov, 323, 1 - 172 NTP Toxicology and Carcinogenesis Studies of Dimethyl Methylphosphonate (CAS No . 756-79-6) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Dimethyl methylphosphonate (98% pure) is one of four chemicals nominated by the U.S . Army for toxicology and carcinogenesis studies because it was being considered for use to simulate the physical and spectroscopic (but not the biologic) properties of anticholinesterase (nerve) agents . Dimethyl methylphosphonate is also used as a flame retardant, a preignition additive for gasoline, an antifoam agent, a plasticizer and stabilizer, a textile conditioner and antistatic agent, and an additive for solvents and low-temperature hydraulic fluids . The United States produces 0.2-2 million pounds (91,000-910,000 kg) of per year . Gavage was chosen as the route of administration for all four candidate "simulants" to mimic potential exposure . Experimental Design: Dimethyl methylphosphonate was administered in corn oil by gavage to male and female F344/N rats and B6C3F1 mice in single-administration, 15-day, and 13-week studies to obtain toxicity data, to establish dose levels for the 2-year studies, and to identify target tissues . Additional studies were also performed to determine toxicity to the reproductive system of male F344/N rats and B6C3F1 mice and to study the potential for genetic damage in bacteria, mammalian cells, and Drosophila . Single-Administration Studies: In the single-administration studies, dimethyl methylphosphonate was given to rats and mice at doses up to 6,810 mg/kg body weight . No compound-related deaths were seen in male or female rats or male mice; two high dose female mice died . Rats exhibited inactivity, unsteady gait, and prostration after dosing; mice were inactive after dosing . Fifteen-Day Studies: Rats and mice received doses of 0, 1,250, 2,500, 5,000, 10,000, or 15,000 mg/kg dimethyl methylphosphonate per day . Compound-related deaths occurred in the three highest dose groups of rats and the two highest dose groups of mice . Rats receiving doses of 2,500 mg/kg or higher were inactive and at 5,000 or 10,000 mg/kg had an unsteady gait after dosing; mice exhibited inactivity, shallow breathing, and prostration at doses of 10,000 mg/kg or higher . No lesions were reported in rats . Nonneoplastic lesions of the stomach were seen in some male mice at doses of 1,250 mg/kg and higher and in some female mice at doses of 5,000 mg/kg and higher . Thirteen-Week Studies: Dimethyl methylphosphonate was given at doses up to 8,000 mg/kg per day . Compound-related deaths occurred at 2,000, 4,000, and 8,000 mg/kg in rats and at 4,000 and 8,000 mg/kg in mice . Mean body weights of rats at 1,000 mg/kg and mice at 2,000 mg/kg were similar to those of the vehicle controls; decreased weight gain was seen at higher doses . No compound-related clinical signs were reported . Minimal to mild renal and testicular lesions were seen at all doses in male rats, but the severity of these lesions did not increase with increasing dose of the chemical . No apparent target tissues were identified in female rats or male and female mice . Doses selected for the 2-year studies were based on body weight effects and mortality seen in the 13-week studies; the lesions seen in the kidney of male rats at the end of the 13-week studies were judged not to be life threatening . In the 2-year studies, dimethyl methylphosphonate was administered in corn oil by gavage at doses of 0, 500, or 1,000 mg/kg per day to groups of 50 F344/N rats of each sex and at 0, 1,000, or 2,000 mg/kg per day to groups of 50 B6C3F1 mice of each sex . All animals were dosed 5 days per week for 103 weeks . Body Weight and Survival in the Two-Year Studies: Mean body weights of high dose male rats were 5%-10% lower than those of the vehicle controls between weeks 28 and 76 and were 10%-24% lower between weeks 80 and 104 . Mean body weights of high dose female rats were 8%-12% lower than those of the vehicle controls after week 80 . Survival of male rats was greater than 50% in all groups until week 80, and after this time, survival decreased in both groups, with the survival at the end of the study being 27/50 in vehicle control, 17/50 in low dose, and 4/50 in high dose groups . Survival of in low dose, and 4/50 in high dose groups . Survival of low dose female rats was comparable to that of the vehicle controls, but the final survival of high dose female rats was decreased (vehicle control, 30/50; low dose, 33/50; high dose, 23/50) . No other compound-related clinical signs were observed . Mean body weights of high dose male mice were 7&percnt;-16&percnt; lower than those of the vehicle control males between weeks 36 and 76, and those of high dose female mice were 6&percnt;-12&percnt; lower between weeks 88 and 103 . Decreased survival between weeks 23 and 45 in high dose male mice was associated with fighting . Seventeen high dose male and 22 high dose female mice died during week 45; these deaths were associated with the accidental administration of a dose mixture that had a concentration 34&percnt; greater than the targeted amount . Eleven low dose male mice died on the same day during week 77 . By the end of the study, 29/50 vehicle control, 12/50 low dose, and 0/50 high dose male mice were alive; 41/50, 30/50, and 2/50 female mice survived to the end of the study . Renal Effects in the Two-Year Studies: Administration of dimethyl methylphosphonate to male rats increased the average severity of nephropathy and caused mineralization (calcification) of the collecting tubules in the renal papilla (12/50; 41/50; 36/49), hyperplasia of the transitional epithelium lining the renal pelvis and overlying the renal papilla (0/50; 23/50; 21/49), and focal hyperplasia of the renal tubular epithelium (0/50; 8/50; 9/49) . Administration of dimethyl methylphosphonate to male rats was also associated with the occurrence of rare renal tubular cell adenocarcinomas (0/50; 2/50; 3/49) and papillomas of the transitional epithelium lining of the renal pelvis (0/50; 2/50; 3/49); a transitional cell carcinoma occurred in a low dose male rat . There were no tubular cell or transitional cell neoplasms of the kidney in female rats . Hematopoietic System Effects in the Two-Year Studies: The incidence of mononuclear cell leukemia was increased in high dose male rats (10/50; 11/50; 17/50) . Genetic Toxicity: Dimethyl methylphosphonate was not mutagenic when tested in the Salmonella typhimurium/microsome assay by the preincubational protocol with strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation . The chemical did induce forward mutations in the mouse lymphoma L5178Y/TK&plusmn; assay system in the absence of metabolic activation . Treatment of cultured Chinese hamster ovary cells with dimethyl methylphosphonate did not induce chromosomal aberrations; however, sister chromatid exchanges were induced after exposure to this chemical in both the presence and absence of metabolic activation . When fed to Drosophila, dimethyl methylphosphonate induced a significant increase in the frequency of sex-linked recessive lethal mutations but did not induce reciprocal translocations . Dimethyl methylphosphonate caused a dominant lethal effect in male rats and mice . Studies of Reproductive Effects: Dimethyl methylphosphonate caused a dose-related increase in the number of fetal resorptions in undosed female rats and mice mated with males that received the chemical by gavage in water 5 days per week for 13 weeks at doses of 0-2,000 mg/kg per day . After the 13-week dosing period, histopathologic changes were seen in the kidney and testis of male rats but not in male mice; dosed male rats sired fewer litters and fewer pups per litter . Dose-related decreases in sperm count and sperm motility occurred in male rats but not in male mice . Toxic effects to the reproductive system of male rats and mice were reversible after a 13-to 14-week recovery period . Data Audit: An audit of the experimental data was conducted for the 2-year studies on dimethyl methylphosphonate . No data discrepancies were found that influenced the final interpretations . Conclusions: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of dimethyl methylphosphonate for male F344/N rats as shown by increased incidences of tubular cell hyperplasia, tubular cell adenocarcinomas, hyperplasia of the transitional cell epithelium, and transitional cell papillomas of the kidney . There was an increased incidence of mononuclear cell leukemia in male rats at 1,000 mg/kg . Renal toxicity and decreased survival occurred in dosed male rats . There was no evidence of carcinogenic activity of dimethyl methylphosphonate for female F344/N rats given doses of 500 or 1,000 mg/kg . The study in male B6C3F1 mice was an inadequate study of carcinogenic activity because of decreased survival in both dosed groups . There was no evidence of carcinogenic activity for female B6C3F1 mice receiving dimethyl methylphosphonate at 1,000 mg/kg; decreased survival of female mice at 2,000 mg/kg made this group inadequate for determination of carcinogenic activity . Synonyms: fyrol DMMP; methyl phosphonic acid, dimethyl ester; DMMP; methanephosphonic acid dimethyl ester; dimethyl methanephosphonate Natl Toxicol Program Tech Rep Ser, 1987 Oct, 324, 1 - 126 NTP Toxicology and Carcinogenesis Studies of Boric Acid (CAS No . 10043-35-3) in B6C3F1 Mice (Feed Studies); National Toxicology Program ; Boric acid is a component of cosmetics and pharmaceuticals and is also used in numerous industrial processes . Earlier long-term studies did not demonstrate a carcinogenic effect in Sprague-Dawley rats . Because of potential widespread human exposure, corroborative evidence was sought in a second species . Toxicology and carcinogenesis studies were conducted by feeding technical-grade boric acid (99.7% pure) to groups of male and female B6C3F1 mice for 14 days, 13 weeks, and 2 years . In the 14-day studies (five mice per group), mortality occurred in mice fed 25,000 ppm, 50,000 ppm, or 100,000 ppm boric acid; hyperplasia and/or dysplasia of the forestomach was also seen in these dose groups . No compound-related gross pathologic or histopathologic effects were seen in male or female mice exposed at concentrations up to 12,500 ppm in feed . In the 13-week studies, groups of 10 male and 10 female mice were fed boric acid at concentrations up to 20,000 ppm; 8 male mice and 1 female mouse receiving 20,000 ppm and 1 male receiving 10,000 ppm boric acid died before the end of the studies . Male and female mice receiving 20,000 ppm boric acid weighed 23% and 18% less, respectively, than did the controls at the end of the studies . Testicular atrophy in 8/10 male mice, hyperkeratosis and acanthosis of the stomach in 8/10 male and female mice, and extramedullary hematopoiesis of the spleen in all male and female mice receiving 20,000 ppm boric acid indicated that the testis, stomach, and spleen were potential target organs in the 2-year studies . Based on these results, 2-year toxicology and carcinogenesis studies were conducted by feeding diets containing boric acid at concentrations of 0, 2,500, or 5,000 ppm to groups of 50 male and 50 female mice . Survival of high dose male mice after week 63 and of low dose mice after week 84 was lower than that of the controls (final survival: control, 41; low dose, 30; high dose, 22), which may have reduced the sensitivity of the carcinogenicity study; the numbers of female mice (33; 33; 37) that survived to the end of the studies were considered adequate for toxicologic evaluation . Body weight gain was reduced in each sex after week 30; mean final body weights were 7% and 13% below control values for exposed male mice and 7% and 20% below those of controls for exposed female mice . No chemically related clinical signs were reported . At the top dose, boric acid caused an increased incidence of testicular atrophy (control, 3/49; low dose, 6/50; high dose, 27/47) and interstitial cell hyperplasia (0/49; 0/50; 7/47) inmale mice . The testicular atrophy was characterized by variable loss of spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa from the seminiferous tubules . The seminiferous tubules contained primarily Sertoli cells and variable numbers of spermatogonia . In some mice, there were accumulations of interstitial cells, indicating hyperplasia . In low dose male mice, there were increased incidences of hepatocellular carcinomas (5/50; 12/50; 8/49) and hepatocellular adenomas or carcinomas (combined) (14/50; 19/50; 15/49) and an increased incidence of subcutaneous tissue fibromas, sarcomas, fibrosarcomas, or neurofibrosarcomas (combined) (2/50; 10/50; 2/50) . No increased incidence of subcutaneous tissue neoplasms was seen in male mice receiving 5,000 ppm . Because the incidence of subcutaneous tissue tumors is variable in historical controls, because there was no corresponding increase in the high dose male mice, and because the incidence of hepatocellular tumors was not significant by the incidental tumor test and was within the historical control range, neither of these tumors was considered to be related to the administration of boric acid . Boric acid was not mutagenic in the Salmonella/microsome assay with Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 . Boric acid was negative in the mouse lymphoma L5178Y/TK+/- assay and did not induce sister-chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells . All assays were preformed with anhamster ovary cells . All assays were preformed with and without metabolic activation . The data, documents, and pathology materials from the 2-year studies of boric acid were audited at the NTP Archives . The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report . Under the conditions of these 2--year feed studies, there was no evidence of carcinogenicity of boric acid at doses of 2,500 or 5,000 ppm for male or female B6C3F1 mice . Testicular atrophy and interstitial cell hyperplasia were observed in high dose male mice . The decrease in survival of dosed male mice may have reduced the sensitivity of this study . Synonyms: orthoboric acid; boracic acid Natl Toxicol Program Tech Rep Ser, 1987 Jan, 325, 1 - 122 NTP Toxicology and Carcinogenesis Studies of Pentachloronitrobenzene (CAS No . 82-68-8) in B6C3F1 Mice (Feed Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of pentachloronitrobenzene (99% pure), a fungicide, were conducted by administering diets containing 0, 2,500, or 5,000 ppm pentachloronitrobenzene to groups of 50 B6C3F1 mice of each sex for 103 weeks . These doses were selected because, in 13-week studies in which the chemical was administered in feed at doses up to 20,000 ppm in male mice and up to 40,000 ppm in female mice, body weight gain depression was observed at 10,000 ppm and above in males and female and deaths occurred at 40,000 ppm in females . The National Cancer Institute had conducted 2-year (diet) studies in B6C3F1 mice and Osborne-Mendel rats (See TR-61 reported in 1978) . Survival among male mice was low, not all livers were examined from dosed female mice, and the size of the control group was considered to be small . For these reasons, the NCI decided to conduct additional 13-week and 2-year studies in B6C3F1 mice . Under the conditions of the NCI studies, pentachloronitrobenzene was not carcinogenic in either Osborne-Mendel rats or B6C3F1 mice . In the studies reported in this Technical Report, the survival of male mice was comparable among control and dosed groups (control, 35/50; low dose, 31/50; high dose, 32/50) . Final mean body weights of low dose and high dose male mice were 96% and 90% that of the controls . All groups of female mice showed evidence of bacterial infection . At week 84, survival in dosed and control female mice was 38/50; 34/50; 30/50; after week 84, survival in dosed groups decreased, with the final survival being 30/50; 20/50; 15/50 . The mean body weight of high dose female mice was more than 10% lower than that of the control group after week 20 and was 21% lower than controls at week 104 . The mean body weight of low dose female mice was within 10% that of the control group until week 88 and was 18% lower than controls at week 104 . No compound-related neoplastic lesions were seen in either male or female mice . The nonneoplastic lesions observed in female mice were considered to be secondary to bacterial infection (primarily Klebsiella) and included hematopoiesis of the liver (9/50; 21/50; 23/50) and spleen (14/50; 23/48; 27/50), plasma cell hyperplasia of the mediastinal lymph nodes (1/44; 4/47; 9/45), and ovarian abscesses (12/49; 22/50; 29/50) . Pentachloronitrobenzene was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence or absence of Aroclor 1254-induced male Syrian hamster or male Sprague-Dawley rat liver S9 when tested according to the preincubational protocol . Pentachloronitrobenzene was not mutagenic at the TK+/- locus of L5178Y mouse lymphoma cells in the presence or absence of Aroclor 1254-induced F344/N rat liver S9 . In cultured Chinese hamster ovary cells, pentachloronitrobenzene did not induce sister-chromatid exchanges but did induce chromosomal aberrations both with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 . An audit of the experimental data was conducted for the 2-year studies of pentachloronitrobenzene . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year feed studies, there was no evidence of carcinogenicity for either male or female B6C3F1 mice receiving 2,500 or 5,000 ppm of pentachloronitrobenzene . Infection is considered to have decreased survival of the female mice and thus reduced the sensitivity for determining the presence or absence of a carcinogenic response . Synonyms or Trade Names: Avicolreg.; PCNB; quintozene; Botrilexreg.; Brassicolreg.;Folosanreg.; PKhNB; Tilcarexreg.; Terraclorreg.; Tritosanreg. Natl Toxicol Program Tech Rep Ser, 1986 Aug, 303, 1 - 190 NTP Toxicology and Carcinogenesis Studies of 4-Vinylcyclohexene (CAS No . 100-40-3) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of 4-vinylcyclohexene (greater than 98% pure), a dimer of 1,3-butadiene present in the off-gasses from tire curing, were conducted by administering the chemical in corn oil by gavage 5 days per week at doses of 0, 200, or 400 mg/kg body weight to groups of 50 F344/N rats and B6C3F1 mice of each sex for 103 weeks . Doses selected for the 2-year studies were based on survival, body weight gains, and histopathologic effects observed during the 14-day and 13 week studies . All rats and most mice in the 14-day studies died when administered doses greater than or equal to 1,250 mg/kg, although no compound-related gross or histopathologic effects were observed . Final body weights were reduced in the 13-week studies in male rats receiving doses greater than or equal to 400 mg/kg of 4-vinylcyclohexene, in female rats receiving 800 mg/kg, and in female mice receiving 600 mg/kg . Extensive mortality was observed only in mice dosed at 1,200 mg/kg . Compound-related histopathologic effects in the 13-week studies included hyaline droplet degeneration of the proximal convoluted tubules of the kidney in dosed male rats, the severity of which was dose related, and a reduction in the number of primary follicles and mature graafian follicles in the ovaries of female mice receiving 1,200 mg/kg of 4-vinylcyclohexene . No compound-related gross or histopathologic effects were evident in dosed female rats or male mice in the 13-week studies . Many dosed rats died early in the 2-year studies (male: vehicle control, 17/50; low dose, 37/50; high dose, 45/50; female: vehicle control, 10/50; low dose, 22/50; high dose, 36/50; P<0.001 for all groups except low dose female rats, for which P=0.022) . The poor survival of dosed male and female rats reduced the sensitivity of the studies for detecting the possible carcinogenic effects of 4-vinylcyclohexene . Mean body weights of dosed rats were comparable to those of their respective vehicle controls, except for high dose males late in the study . Survival of high dose mice of each sex was lower (P<0.001) than that of the vehicle controls, whereas survival of low dose mice of each sex was comparable to that of the vehicle controls . Mean body weights of high dose mice of each sex were generally lower than those of the vehicle controls throughout most of the 2-year studies . Administration of 4-vinylcyclohexene to F344/N rats by gavage for 2 years was associated with a slightly increased incidence of epithelial hyperplasia of the forestomach (1/50; 3/50; 5/47) and squamous cell papillomas or carcinomas (combined) of the skin in high dose males (0/50; 1/50; 4/50) . Low dose female rats, whose survival was more similar to that of the vehicle controls, had a marginally increased incidence of adenomas or squamous cell carcinomas (combined) of the clitoral gland (1/50; 5/50; 0/49) . In B6C3F1 mice, administration of 4-vinylcyclohexene for two years by gavage was associated with mild, acute inflammatory lesions and epithelial hyperplasia of theforestomach, especially in males (0/47; 7/50; 7/46), and with an increased incidence of a number of other nonneoplastic lesions, including lung congestion in high dose males and females, splenic red pulp atrophy in high dose males, congestion of the adrenal gland in high dose females, and cytologic alteration of the adrenal cortex in low dose and high dose females . The incidences of uncommon ovarian neoplasms were markedly increased (P<0.01) in both groups of dosed female mice (mixed tumor, benign: 0/49; 25/48, 52%; 11/47, 23%; granulosa cell tumor or carcinoma {combined}: 1/49, 2%; 10/48, 21%; 13/47, 28%) . In addition, a slight increase in the incidence of adrenal gland adenomas in high dose females was observed (0/50; 3/49, 6%; 4/48, 8%) . The extensive mortality seen in the high dose male mice confounded interpretation of the increased incidences of malignant lymphomas and alveolar/bronchiolar adenomas or carcinomas (combined) of the lung seen in these animals surviving to the end of the study (malignant lymphomas: 3/37, 8%; 5/39, 13%; 4/7, 57d of the study (malignant lymphomas: 3/37, 8&percnt;; 5/39, 13&percnt;; 4/7, 57&percnt;; alveolar/bronchiolar adenomas or carcinomas {combined}: 3/37, 8&percnt;; 9/39, 23&percnt;; 3/7, 43&percnt;) . 4-Vinylcyclohexene was not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, or TA98 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 when tested according to the preincubational protocol . However, several of its metabolites, including 4-vinylcyclohexene diepoxide, have been shown to be mutagenic in Salmonella and/or induce chromosomal damage in vitro . An audit of the experimental data was conducted for these 2-year carcinogenesis studies on 4-vinylcyclohexene . No data discrepancies were found that influenced the final interpretations . 4-Vinylcyclohexene was administered by gavage in corn oil to F344/N rats and B6C3F1 mice of each sex at doses of 200 or 400 mg/kg for 103 weeks . Under these conditions, the 2-year gavage studies of 4-vinylcyclohexene in male and female rats and male mice were considered inadequate studies of carcinogenicity because of extensive and early mortality at the high dose or at both doses and the lack of conclusive evidence of a carcinogenic effect . There was clear evidence of carcinogenicity of 4-vinylcyclohexene for female mice, as shown by markedly increased incidences of uncommon ovarian neoplasms at both doses . In addition, the increased incidence of adrenal gland adenomas in high dose female mice may have been related to the administration of 4-vinylcyclohexene . Synonym: 4-ethenylcyclohexene Natl Toxicol Program Tech Rep Ser, 1987 Apr, 304, 1 - 225 NTP Toxicology and Carcinogenesis Studies of Chlorendic Acid (CAS No . 115-28-6) in F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Chlorendic acid is a chemical intermediate used in the preparation of fire-retardant polyester resins and plasticizers . Toxicology and carcinogenesis studies of chlorendic acid (greater than 98% pure) were conducted by administering the chemical in feed to groups of 50 male and 50 female F344/N rats and B6C3F1 mice at concentrations of 0, 620, or 1,250 ppm for 103 weeks . The estimated mean daily consumption of chlorendic acid was 27 and 56 mg/kg body weight for low dose and high dose male rats and 39 and 66 mg/kg for low dose and high dose female rats . In mice, the estimated daily consumption was 89 and 185 mg/kg for low dose and high dose males and 100 and 207 mg/kg for low dose and high dose females . These concentrations were selected because higher levels in the 14-day and 13-week studies caused decreased mean body weights, more deaths, and increased incidences of liver lesions (rats: centrilobular cytomegaly, mitotic alterations, bile duct hyperplasia; mice: centrilobular cytomegaly, mitotic alterations, coagulative necrosis) relative to control groups . Survival and feed consumption of dosed male and female rats and mice in the 2-year studies were similar to those of controls . Mean body weights of high dose male and female rats and mice were lower than those of controls . Mean body weights of high dose female rats were 16%-24% lower than those of controls during the second half of the study . In the 2-year chlorendic acid feed studies, incidences of nonneoplastic lesions of the liver in dosed male rats (cystic degeneration) and dosed female rats (granulomatous inflammation, pigmentation, and bile duct hyperplasia) were increased . The incidences of neoplastic nodules of the liver were significantly increased in dosed male rats (control, 2/50; low dose, 21/50; high dose, 23/50) and high dose female rats (1/50; 3/39; 11/50) . The incidence of hepatocellular carcinomas was also increased in high dose female rats (0/50; 3/49; 5/50) . In mice, the incidences of nonneoplastic lesions of the liver were increased in dosed males (coagulative necrosis) and high dose females (mitotic alterations) . The incidences of hepatocellular adenomas (5/50; 9/49; 10/50), hepatocellular carcinomas (9/50; 17/50; 20/50), and hepatocellular adenomas or carcinomas (combined) (13/50; 23/49; 27/50) were increased in dosed male mice . Hepatocellular carcinomas metastasized to the lung in 2/50 control, 4/49 low dose, and 7/50 high dose male mice . Hepatocellular adenomas or carcinomas (combined) were not significantly increased in female mice (3/50; 7/49; 7/50) . The incidences of acinar cell hyperplasia (0/49; 4/50; 4/50) and acinar cell adenomas (0/49; 4/50; 6/50) of the pancreas were increased in dosed male rats relative to those of controls . Pancreatic acinar cell adenoma is an uncommonneoplasm in untreated control F344/N rats in NTP studies (3/1,667) . In dosed male rats, incidences of alveolar/bronchiolar adenomas of the lung (0/50; 3/50; 5/50) were increased . The incidences of alveolar/bronchiolar adenomas or carcinomas (combined) in dosed female mice were also increased (1/50; 5/50; 6/50) . Preputial gland carcinomas occurred at a greater incidence in low dose male rats (1/50; 8/50; 4/50) than in controls . An adenoma and a squamous cell papilloma were observed in two low dose male rats . The incidences of sarcomas, fibrosarcomas, or neurofibrosarcomas (combined) of the salivary gland (1/50; 2/49; 4/50) were increased in dosed male rats . The incidences in the dosed groups were not significantly different from that in the controls, but these tumors are uncommon in F344/N rats receiving no treatment (3/1,689) . Chlorendic acid was not mutagenic in strains TA100, TA98, TA1535, or TA 1537 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver activation when tested according to the preincubational protocol . Chlorendic acid was mutagenic in the L5178Y/TK+/- mouse lymphoma cell forward assay (in the absence of activation) at a dose resulting in toxicity . An audit of the experimental dudit of the experimental data was conducted for the 2-year studies of chlorendic acid . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenicity of chlorendic acid for male F344/N rats as shown by increased incidences of neoplastic nodules of the liver and acinar cell adenomas of the pancreas . Increased incidences of alveolar/bronchiolar adenomas and preputial gland carcinomas may also have been related to the administration of chlorendic acid . There was clear evidence of carcinogenicity of chlorendic acid for female F344/N rats as shown by increased incidences of neoplastic nodules and of carcinomas of the liver . There was clear evidence of carcinogenicity of chlorendic acid for male B6C3F1 mice as shown by increased incidences of hepatocellular adenomas and of hepatocellular carcinomas . There was no evidence of carcinogenicity of chlorendic acid for female B6C3F1 mice given chlorendic acid in the diet at concentrations of 620 or 1,250 ppm for 103 weeks. Natl Toxicol Program Tech Rep Ser, 1986 May, 305, 1 - 202 NTP Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C23, 43% Chlorine) (CAS No . 108171-27-3) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of chlorinated paraffins (C23, 43% chlorine), an extreme-pressure lubricant and flame retardant, were conducted by administering the chemical in corn oil by gavage to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days per week for 103 weeks . Additional groups of 10 rats per sex and dose were examined at 6 and at 12 months . Male rats received doses of 0, 1,875, or 3,750 mg/kg body weight; female rats were given 0, 100, 300, or 900 mg/kg . Male and female mice received 0, 2,500, or 5,000 mg/kg . Doses selected for the 2-year studies were based on the results from 13-week studies in which rats of each sex received 0 to 3,750 mg/kg, and mice of each sex, 0 to 7,500 mg/kg . No toxicity of chlorinated paraffins (C23, 43% chlorine) was observed in male rats or in male or female mice in the 13-week studies . A dose-related inflammation of the liver was observed in female rats in the 13-week studies and in male and female rats in the 13-week studies and in male and female rats at 6 and 12 months in the 2-year studies . Chlorinated paraffins (C23, 43% chlorine) administration did not influence mean body weights of rats during the 2-year studies, but both male and female low dose mice gained less weight than did vehicle controls or the high dose groups . Survival of dosed and vehicle control groups was similar for each sex and species (male rats: vehicle control, 30/50; low dose, 32/50; high dose, 27/50; female rats: 34/50; 30/50; 33/50; 31/50; male mice: 29/50; 36/50; 28/50; female mice: 21/50; 22/50; 20/50) . For female mice, 60%-70% of the early deaths in each group were attributed to utero-ovarian infection . The lower survival for female mice may have decreased the sensitivity of this study to detect a carcinogenic effect . Pheochromocytomas of the adrenal gland medulla occurred with an increased incidence in female rats exposed to chlorinated paraffins (C23, 43% chlorine) (vehicle control, 1/50; low dose, 4/50; mid dose, 6/50; high dose, 7/50) . However, adrenal gland medullary hyperplasia was not increased (6/50; 3/50; 1/50; 6/50) . Malignant lymphomas were increased in dosed male mice (6/50; 12/50; 16/50) . High dose female mice showed a marginal increase in the incidence of hepatocellular carcinomas (1/50; 1/49; 6/50) and in the incidence of adenomas or carcinomas (combined) (4/50; 3/49; 10/50) . The primary nonneoplastic lesion associated with chlorinated paraffins (C23, 43% chlorine) administration was a diffuse lymphohistiocytic inflammation in the liver and in the pancreatic and mesenteric lymph nodes of male and female rats . Splenic congestion was a secondary effect . These lesions occurred earlier and at lower doses in female rats than in male rats . No significant nonneoplastic lesions were considered compound related in mice . Chlorinated paraffins (C23, 43% chlorine) was not mutagenic in strains TA100, TA1535, TA97, or TA98 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 when assayed according to the preincubation protocol . An audit of the experimental data was conducted for these 2-year studies of chlorinated paraffins (C23, 43% chlorine) . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for male F344/N rats given 1,875 or 3,750 mg/kg per day . There was equivocal evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for female F344/N rats as shown by an increased incidence of adrenal gland medullary pheochromocytomas . There was clear evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for male B6C3F1 mice as shown by an increase in the incidence of malignant lymphomas . There was equivocal evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for female B6C3F1 mice as shown by a marginal increase in the incidence of hepatocellular neoplasms . *The Chemical Abstract Service Seal increase in the incidence of hepatocellular neoplasms . *The Chemical Abstract Service Service (CAS) number that appeared on this technical report at the time of publication (63449-39-8) reflects the generic CAS number for chlorinated paraffins . This number has been replaced in the NTP Chemtrack chemical tracking system with the more appropriate number. Natl Toxicol Program Tech Rep Ser, 1986 May, 307, 1 - 186 NTP Toxicology and Carcinogenesis Studies of Ephedrine Sulfate (CAS No . 134-72-5) in F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Ephedrine sulfate is a sympathomimetic amine that affects both the central and peripheral nervous systems . An effective bronchiodilator and weak vasoconstrictor, ephedrine sulfate is used extensively in nonprescription pharmaceutical preparations such as nose drops, cold tablets, cough syrups, and, in particular, asthma relief medicines . Ephedrine sulfate was nominated for carcinogenesis studies by the National Cancer Institute because of its widespread and long-term use for the relief of symptoms associated with asthma . In 14-day repeated-exposure studies, F344/N rats of each sex received diets containing 0-1,500 ppm ephedrine sulfate or drinking water containing 0-1,200 ephedrine sulfate; B6C3F1 mice received diets or drinking water containing 0-5,000 ppm ephedrine sulfate . In the feed studies, the average feed consumption by dosed rats and mice was comparable to that of their respective controls . The average water consumption by rats and mice decreased with increasing concentration of ephedrine sulfate in the drinking water . Thus, subsequent studies used the feed route of administration . Doses for the 2-year studies were selected on the basis of results from 13-week studies in which F344/N rats of each sex were given diets containing 0, 125, 250, 500, 1,000, or 2,000 ppm ephedrine sulfate and B6C3F1 mice of each sex were given diets containing 0, 310, 630, 1,250, 2,500, or 5,000 ppm ephedrine sulfate . The major response that occurred during the 13-week studies was compound-associated reduction in weight gain . Toxicology and carcinogenesis studies of ephedrine sulfate were conducted by administering diets containing 0, 125, or 250 ppm ephedrine sulfate to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex for 103 weeks . The estimated average amount of ephedrine sulfate consumed per day during the 2-year study was 4 mg/kg and 9 mg/kg for low dose and high dose male rats, 5 mg/kg and 11 mg/kg for female rats, 14 mg/kg and 29 mg/kg for male mice, and 12 mg/kg and 25 mg/kg for female mice . Survival of chemically exposed female rats during the 2-year study was greater than that of the controls (control, 27/50; low dose, 39/50; high dose, 39/50); survival of exposed male rats and male and female mice was comparable to that of controls . Throughout most of the 2-year studies, mean body weights of rats and mice of each sex receiving diets containing ephedrine sulfate were lower than those of controls . Neoplasms that occurred in these studies were not considered to be related to administration of ephedrine sulfate . Two high dose female mice had ovarian granulosa cell tumors, and luteomas were found in one low dose and one high dose female mouse . Because of the low incidence, these uncommon, benign tumors could not be clearly related to ephedrine sulfate administration . Ephedrine sulfate was not mutagenic in four strains of Salmonella typhimurium (TA100, TA1535, TA97, or TA98) with or without Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9 activation . Ephedrine sulfate did not induce sister-chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells . An audit of the experimental data was conducted for these 2-year studies of ephedrine sulfate . No data discrepancies were found that influenced the final interpretations . Under the conditions of these studies, there was no evidence of carcinogenicity for F344/N rats or B6C3F1 mice of either sex receiving 125 or 250 ppm ephedrine sulfate in the diet for 2 years. Natl Toxicol Program Tech Rep Ser, 1986 May, 308, 1 - 206 NTP Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C12, 60% Chlorine) (CAS No . 108171-26-2*) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis assessments of chlorinated paraffins (C12, 60% chlorine), a material widely used as a flame retardant and extreme-pressure lubricant, were conducted in male and female F344/N rats and male and female B6C3F1 mice in single-administration, 16-day, 13-week, and 2-year studies . Doses used in the 2-year studies were 0, 312, or 625 mg/kg body weight per day administered by gavage in corn oil five times per week to groups of 70 male and female rats and 0, 125, or 250 mg/kg administered to groups of 50 male and female mice . Ten male and 10 female rats were killed after 6 and 12 months of dosing and examined for toxicity . No chemically related toxicity was observed in single-administration studies in which male and female rats received doses of chlorinated paraffins (C12, 60% chlorine) up to 13,600 mg/kg body weight and male and female up to 27,200 mg/kg . In 16-day studies, deaths did occur in groups of male and female rats given 7,500 mg/kg and in groups of male and female mice given doses of 1,875 mg/kg or higher . In 13-week studies, no chemically related deaths occurred among male and female rats given up to 5,000 mg/kg or mice given up to 2,000 mg/kg . Increased liver weights were noted in dosed rats and mice of each sex in the short-term studies, and dosed male rats showed more severe nephropathy than did vehicle controls . Doses selected for the 2-year studies were those that caused a minimal increase in liver weight in the short-term studies . Liver and kidney weights were increased in dosed rats killed at 6 and 12 months . Morphometric measurements demonstrated hepatocyte hypertrophy in the livers of dosed rats . Lesions of the kidney tubules and interstitial inflammation increased with dose in male and female rats . During the 2-year studies, body weights of high dose male rats were 8%-12% lower than those of vehicle controls after week 20, and body weights of dosed female mice were about 10% lower than those of vehicle controls during the second year . Survival of dosed male rats was lower than that of vehicle controls after about week 85, perhaps due to toxicity to the kidney (final survival: vehicle control, 27/50; low dose, 6/50; high dose, 3/50) . Survival of low dose female rats was lower than that of vehicle controls (34/50; 24/50; 29/50) . Survival of dosed male mice was not significantly different from that of vehicle controls (34/50; 31/50; 31/50) . Survival of high dose female mice was lower than that of vehicle controls after about week 75 (final survival: 36/50; 31/50; 25/50) . Chemically related nonneoplastic lesions consisted of hypertrophy and minimal focal necrosis of the liver in rats; erosion, inflammation, and ulceration of the glandular stomach and forestomach a in male rats; and formation ofmultiple cysts in the kidney tubules of male rats . The incidence of nephropathy was also increased in dosed female rats and mice . The maximum tolerated dose may have been exceeded in male and female rats . Neoplastic lesions associated with chlorinated paraffins (C12, 60% chlorine) administration were found in the liver of rats and mice of each sex (see table p . 12 of Technical Report) Dosed male rats showed increased incidences of kidney tubular cell hyperplasia (1/50; 9/50; 12/49) and of tubular cell adenomas (0/50; 7/50; 3/49); two low dose males had tubular cell adenocarcinomas . The incidences of mononuclear cell leukemia were increased in dosed male rats (7/50; 12/50; 14/50) and in low dose female rats (11/50; 22/50; 16/50) . Pancreatic acinar cell tumors occurred at increased incidences in low dose male rats (11/50; 22/50; 17/50) . Follicular cell adenomas or carcinomas (combined) of the thyroid gland were found at increased incidences in both female rats (0/50; 6/50; 6/50) and female mice (8/50; 12/49; 15/49) . Chlorinated paraffins (C12, 60% chlorine) was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley or male Syrian hamster liver S9 when tested according to the preincubational protocol . An ed according to the preincubational protocol . An audit of the experimental data was conducted for these 2-year studies on chlorinated paraffins (C12, 60&percnt; chlorine) . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenicity of chlorinated paraffins (C12, 60&percnt; chlorine) for F344/N rats based on increased incidences of hepatocellular neoplasms (primarily neoplastic nodules) in male and female rats, of adenomas or adenocarcinomas (combined) of the kidney tubular cells in male rats, and of follicular cell adenomas or carcinomas (combined) of the thyroid gland in female rats . Mononuclear cell leukemia in dosed male rats may have been related to administration of chlorinated paraffins (C12, 60&percnt; chlorine) . There was clear evidence of carcinogenicity of chlorinated paraffins (C12, 60&percnt; chlorine) for B6C3F1 mice as shown by increased incidences of hepatocellular adenomas and of adenomas or carcinomas (combined) in dosed male and female mice and increased incidences of adenomas and of adenomas or carcinomas (combined) of thyroid gland follicular cells in dosed female mice. Natl Toxicol Program Tech Rep Ser, 1986 May, 309, 1 - 242 NTP Toxicology and Carcinogenesis Studies of Decabromodiphenyl Oxide (CAS No . 1163-19-5) In F344/N Rats and B6C3F1 Mice (Feed Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of decabromodiphenyl oxide, a flame retardant for plastics and other materials, were conducted by exposing groups of 50 male and 50 female F344/N rats and B6C3F1 mice at 0, 25,000, and 50,000 ppm in the diet for 103 weeks . These concentrations were selected because no toxicity was observed at any dose in the 14-day or 13-week studies and 50,000 ppm chemical in the diet is considered to be the highest dose to which rats and mice can be exposed for extended periods of time without reducing the nutritional value of the diet . No compound-related gross or microscopic pathologic effects were observed in the 14-day or 13-week studies . Body weights of dosed male and female rats and mice in the 2-year studies were comparable to those of the controls . Decreased survival of low dose male rats was not believed to be compound related . No other effects on survival were observed in the 2-year studies . Loss of control male mice (presumably due to fighting) was significant during the first part of the study . In the 2-year studies, nonneoplastic lesions were observed at increased incidences in rats and mice of each sex . Thrombosis and degeneration of the liver, fibrosis of the spleen, and lymphoid hyperplasia were observed in high dose male rats . Degeneration of the eye was observed in low dose female rats . Nonneoplastic lesions observed in dosed mice were granulomas in the liver of low dose males and hypertrophy in the liver of low dose and high dose males . Follicular cell hyperplasia was observed in thyroid glands of dosed male mice (control, 2/50; low dose, 10/50; high dose, 19/50) . The incidences of neoplastic nodules in the liver of low and high dose male rats (1/50; 7/50; 15/49) and high dose female rats (1/50; 3/49; 9/50) were significantly greater than those in the controls . Mononuclear cell leukemia occurred in dosed male rats with a positive trend (30/50; 33/50; 35/50); this marginal increase was not considered biologically significant . Acinar cell adenomas were observed in the pancreas of four high dose male rats, and a sarcoma was observed in the spleen of one low dose and one high dose male rat . Hepatocellular adenomas or carcinomas (combined) occurred at marginally increased incidences in dosed male mice (8/50; 22/50; 18/50) . The incidences of thyroid gland follicular cell adenomas or carcinomas (combined) were increased in dose male mice (0/50; 4/50; 3/50) . A study of decabromodiphenyl oxide absorption from the gastrointestinal tract indicated that absorption was minimal, possibly less than 1%, at the doses administered in the 2-year studies . Additional chemical analysis indicated than the decabromodiphenyl oxide used in these studies contained several less brominated diphenyl oxides . Therefore, since absorption and toxicity of minor impurities are unknown, effects observed in these studies must be attributed to the approximately 95% pure preparation used rather than to pure decabromodiphenyl oxide . Decabromodiphenyl oxide was not mutagenic in strains TA1535, TA1537, TA98, or TA100 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced Sprague-Dawley male rat or Syrian hamster liver S9 when tested according to the preincubational protocol . Decabromodiphenyl oxide was not mutagenic in the mouse lymphoma L5178Y/TK+/- assay in the presence or absence of Aroclor 1254-induced F344/N male rat liver S9 . Decabromodiphenyl oxide did not induce sister-chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells in vitro in the presence or absence of S9 prepared from livers of Aroclor 1254-induced male Sprague-Dawley rats . An audit of experimental data was conducted for these 2-year studies on decabromodiphenyl oxide . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year feed studies of decabromodiphenyl oxide, there was some evidence of carcinogenicity for male and female F344/N rats as shown by increased incidences of neoplastic nodules of the liver in low dose (25,000 ppm) males and high dose (50, and high dose (50,000 ppm) groups of each sex . There was equivocal evidence of carcinogenicity for male B6C3F1 mice as shown by increased incidences of hepatocellular adenomas or carcinomas (combined) in the low dose group and of thyroid gland follicular cell adenomas or carcinomas (combined) in both dosed groups . There was no evidence of carcinogenicity for female B6C3F1 mice receiving 25,000 or 50,000 ppm in the diet . Several nonneoplastic lesions were observed at increased incidences, the most notable being thyroid gland follicular cell hyperplasia in male mice . Synonyms: decabromodiphenyl ether; bis(pentabromophenyl)ether; DBDPO Natl Toxicol Program Tech Rep Ser, 1986 Sep, 310, 1 - 206 NTP Toxicology and Carcinogenesis Studies of Marine Diesel Fuel (NO CAS) and JP-5 Navy Fuel (CAS No . 8008-20-6) in B6C3F1 Mice (Dermal Studies); National Toxicology Program ; Toxicology and carcinogenesis studies were conducted by applying marine diesel fuel or JP-5 navy fuel to clipped dorsal interscapular skin of male and female B6C3F1 mice to determine both systemic and dermal effects . Doses for the 2-year studies were set by conducting 14-day and 13-week studies . Doses of 2,000-40,000 mg/kg marine diesel fuel were applied neat in the 14-day studies; in the 13-week studies, doses of 250-4,000 mg/kg marine diesel fuel in acetone were applied with a dose volume of 0.1 ml . Doses of 5,000-40,000 mg/kg JP-5 navy fuel in ethanol were applied in the 14-day studies with a dose volume of 0.5 ml; in the 13-week studies, doses of 500-8,000 mg/kg JP-5 navy fuel in acetone were applied with a dose volume of 0.2 ml . For the 2-year studies, doses were selected which did not cause deaths, decrease body weight gain, or produce excessive dermatitis in the 14-day or 13-week studies . Two-year studies were conducted by administering marine diesel fuel or JP-5 navy fuel by dermal application to groups of 49 or 50 male and 50 female B6C3F1 mice at doses of 0, 250, or 500 mg/kg in an acetone vehicle with a dose volume of 0.1 ml . Both sexes of mice dosed with 500 mg/kg marine diesel fuel (84-week exposure) and female mice dosed with 500 mg/kg JP-5 navy fuel (90-week exposure) were killed early because of excessive irritation and ulceration at the site of application and to prevent the spread of infection . Survival rates at those times were 26/50 males and 29/50 females dosed with marine diesel fuel and 17/50 females dosed with JP-5 navy fuel . Survival rates at the end of the studies (104 weeks) were reduced (P<0.01) in low dose female mice receiving marine diesel fuel (40/50 in vehicle controls compared with 12/50 in the low dose group) or with JP-5 navy fuel (44/50 in vehicle controls compared with 33/50 in the low dose group) . Body weight gain was decreased below that of the vehicle controls after week 30 in all groups of mice receiving marine diesel fuel and in both sexes of mice receiving the high dose of JP-5 navy fuel . There was a marked increase in the incidence of chronic dermatitis in mice receiving marine diesel fuel or JP-5 navy fuel . Chronic dermatitis was defined as a composite lesion of epidermal histopathologic changes generally consisting of acanthosis, hyperkeratosis, and in some instances necrosis and ulceration of the overlying epidermis . Dermal changes frequently included fibrosis, increased amounts of melanin, and the presence of acute and chronic inflammatory cell infiltrates . A dose-related, proportional increase in the severity of the lesions was twofold to threefold greater in the dosed groups than in the vehicle controls . The averagedegree of severity of the lesions was judged to be minimal in the vehicle controls, mild in the low dose groups, and moderate in the high dose groups of mice dosed with marine diesel fuel or JP-5 navy fuel . There were similar responses at the site of inguinal skin to which the chemicals had migrated after application, but the degree of severity of the lesions was judged to be minimal to mild in the vehicle control and dosed groups of mice . Squamous cell papillomas or carcinomas (combined) occurred with a positive trend (P<0.05) at the site of application in male mice administered marine diesel fuel (vehicle control, 0/49; low dose, 0/49; high dose, 3/49) . The total numbers of mice with squamous cell papillomas or carcinomas (combined) both for the site of application and the adjacent inguinal skin were 1/50, 2/49, and 3/50 for the vehicle control, low dose, and high dose groups of male mice and 0/50, 1/45, and 2/48 for female mice . There are no NTP historical data for B6C3F1 mice that received acetone by dermal application . The NTP historical incidence of squamous cell papillomas or carcinomas (combined) in untreated male and female B6C3F1 mice is 0.3%-0.4% in over 3,500 observations . Marine diesel fuel was not mutagenic in Salmonella typhimurium TA98, TA100, TA1535, or TA1537, and JP-5 navy fuel was not mutagenic in strains TA97, TA98, TA100, or TA1535 in th100, or TA1535 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster S9 when tested according to the preincubation protocol . Audits of the experimental data were conducted for these 2-year studies on marine diesel fuel and JP-5 navy fuel . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year dermal studies, marine diesel fuel at doses of 250 and 500 mg/kg resulted in dose-related increased incidences of squamous cell neoplasms of the skin (primarily carcinomas), providing equivocal evidence of carcinogenicity for male and female B6C3F1 mice . The sensitivity for detecting systemic carcinogenicity in female mice dosed with marine diesel fuel was reduced by poor survival . Under the conditions of these 2-year dermal studies, JP-5 navy fuel at doses of 250 and 500 mg/kg provided no evidence of carcinogenicity for male and female B6C3F1 mice. Natl Toxicol Program Tech Rep Ser, 1986 Aug, 311, 1 - 197 NTP Toxicology and Carcinogenesis Studies of Tetrachloroethylene (Perchloroethylene) (CAS No . 127-18-4) in F344/N Rats and B6C3F1 Mice (Inhalation Studies); National Toxicology Program ; Tetrachloroethylene is used primarily as a dry cleaning agent, an industrial solvent for fats, oils, tars, rubber, and gums, and a metal degreasing agent . Tetrachloroethylene had antihelminthic uses, particularly for hookworms (1.6-8 g/60 kg), and was formerly used in combination with some grain protectants and fumigants . Toxicology and carcinogenesis studies of tetrachloroethylene (99.9% pure) were conducted by inhalation exposure of groups of 50 male and 50 female F344/N rats and B6C3F1 mice 6 hours per day, 5 days per week, for 103 weeks . The exposure concentrations used (0, 200, or 400 ppm for rats and 0, 100, or 200 ppm for mice) were selected on the basis of results from 13-week inhalation studies in which groups of 10 rats and 10 mice of each sex were exposed to tetrachloroethylene at 100-1,600 ppm for 6 hours per day, 5 days per week . During the 13-week studies, 1,600 ppm tetrachloroethylene was lethal to 20%-70% of the rats and mice and reduced the final body weights of survivors . In rats, tetrachloroethylene at 200-800 ppm caused minimal to mild hepatic congestion . In dosed male and female mice, minimal to mild hepatic leukocytic infiltration, centrilobular necrosis, bile stasis (400-1,600 ppm), and mitotic alteration (200-1,600 ppm) were produced . Tetrachloroethylene exposure also caused minimal renal tubular cell karyomegaly in mice at concentrations as low as 200 ppm . During the 2-year studies, exposure to tetrachloroethylene did not consistently affect body weight gains in either rats or mice . Exposure at 400 ppm tetrachloroethylene reduced the survival of male rats (control, 23/50; low dose, 20/50; high dose, 12/50) . This reduced survival may have been related to an increased incidence of mononuclear cell leukemia . Tetrachloroethylene at both exposure concentrations reduced the survival of male mice (46/50; 25/50; 32/50), whereas exposure at 200 ppm reduced female mouse survival (36/50; 31/50; 19/50) . Early deaths in mice may have been related to the development of hepatocellular carcinomas . Both concentrations of tetrachloroethylene were associated with increased incidences of mononuclear cell leukemia in male rats (28/50; 37/50; 37/50) . In female rats, tetrachloroethylene increased the incidence of leukemia (18/50; 30/50; 29/50) and decreased the time to occurrence of the disease . Tetrachloroethylene produced renal tubular cell karyomegaly in male and female rats, renal tubular cell hyperplasia in male rats, and renal tubular cell adenomas or adenocarcinomas (combined) in male rats (1/49; 3/49; 4/50) . The incidence of the renal tubular cell tumors was not statistically significant; these uncommon tumors have been consistently found at low incidences in male rats in other 2-year studies of chlorinated ethanes and ethylenes . One low dose male rat had a kidney lipoma, and another had a nephroblastoma . Four high dose male and two high dose female rats had gliomas of the brain, whereas one control male and one control female had this tumor . In male and female mice, tetrachloroethylene caused dose-related increases in the incidences of hepatocellular neoplasms . In males, tetrachloroethylene at 200 ppm increased the incidence of hepatocellular adenomas (11/49; 8/49; 18/50) and at both concentrations increased the incidence of hepatocellular carcinomas (7/49; 25/49; 26/50) . In female mice, tetrachloroethylene at both concentrations increased the incidences of hepatocellular carcinomas (1/48; 13/50; 36/50) . Tetrachloroethylene also produced renal tubular cell karyomegaly in both sexes of mice, and one low dose male mouse had a tubular cell adenocarcinoma . In these inhalation studies, there were no neoplastic changes in the respiratory tracts of either species, but there was an increase in the incidence of squamous metaplasia in the nasal cavities in dosed male rats (0/50; 5/50; 5/50) . Tetrachloroethylene was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence or absence of male Syrian hamster or male Sprague-Dawley rat liver S9 . Tetrachloroethylene was not mutagenic in was not mutagenic in L5178Y/TK&plusmn; mouse lymphoma cells with or without metabolic activation and did not induce sex-linked recessive lethal mutations in Drosophila melanogaster . Tetrachloroethylene did not induce sister-chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of metabolic activation . An audit of the experimental data was conducted for these 2-year studies on tetrachloroethylene . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenicity of tetrachloroethylene for male F344/N rats as shown by an increased incidence of mononuclear cell leukemia and uncommon renal tubular cell neoplasms . There was some evidence of carcinogenicity of tetrachloroethylene for female F344/N rats as shown by increased incidences of mononuclear cell leukemia . There was clear evidence of carcinogenicity for B6C3F1 mice as shown by increased incidences of both hepatocellular adenomas and carcinomas in males and of hepatocellular carcinomas in females . Synonyms: carbon bichloride; carbon dichloride; ethylene tetrachloride; per; perc; perchlor; perchlorethylene; perchloroethylene; perk; tetrachlorethylene; 1,1,2,2-tetrachloroethylene Trade names: Ankilostin; Antisal 1; Dee-Solv; Didakene; Dow-Per; ENT 1860; Fedel-Un; Nema; Perclene; Percosolv; Perklone; PerSec; Tetlen; Tetracap; Tetraleno; Tetravec; Tetroguer; Tetropil Natl Toxicol Program Tech Rep Ser, 1986 Apr, 312, 1 - 198 NTP Toxicology and Carcinogenesis Studies of n-Butyl Chloride (CAS No . 109-69-3) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of n-butyl chloride (greater than 99.5% pure), a solvent as well as an alkylating agent, were conducted by exposing groups of F344/N rats and B6C3F1 mice to n-butyl chloride in corn oil by gavage for 14 days, 13 weeks, and 2 years . In the 14-day studies, no compound-related gross pathologic effects were observed in groups of five male or female rats or mice administered doses of up to 3,000 mg/kg body weight . However, deaths occurred in the groups administered 750, 1,500, or 3,000 mg/kg . Tremors and convulsions following gavage administration were observed . In the 13-week studies, groups of 10 male and 10 female rats were administered up to 500 mg/kg n-butyl chloride, and similar groups of mice received up to 1,000 mg/kg, Three of 10 male rats in the 500 mg/kg dose group and one female mouse in the 120 mg/kg dose group died before the end of the studies . Mild to moderate extramedullary hematopoiesis was observed in 3/10 male rats receiving 500 mg/kg . Mean body weights of male and female rats receiving 250 or 500 mg/kg were lower than those of the vehicle controls . Convulsions were observed in male and female rats receiving 250 mg/kg or higher and in 2/10 female mice receiving 1,000 mg/kg . Based on these results, 2-year toxicology and carcinogenesis studies of n-butyl chloride were conducted by administering doses of 0, 60, or 120 mg/kg in corn oil by gavage to groups of 50 male and 50 female rats and doses of 0, 500, or 1,000 mg/kg to groups of 50 male and 50 female mice . In the 2-year studies, survival relative to that of vehicle controls was significantly lower in high dose male rats (40/50 vs 17/50) and high dose female rats (35/50 vs 11/50) and in male mice receiving 1,000 mg/kg (33/50 vs 10/50) . Due to excessive mortality in the 1,000 mg/kg female mice, the group was terminated in the 45th week and a second series of 2-year studies in mice of each sex was started at concentrations of 0 and 250 mg/kg . Male mice in the 1,000 mg/kg group had 10% lower mean body weights than the vehicle control group . No adverse effects on survival or body weights in other dosed groups of rats and mice were observed . Convulsions were observed before or after gavage administration on several occasions during the rat studies . These observations were noted primarily in the high dose groups (male: vehicle control, 1/50; low dose, 3/50; high dose, 27/50; female: vehicle control, 0/50; low dose, 7/50; high dose, 45/50) . Hemorrhage of the brain and alveoli were observed primarily in high dose male and female rats dying from convulsions . Lymphoid depletion of the spleen and splenic hemosiderosis were also observed inthese animals . In mice, convulsions were observed only in the first studies (in the high dose female mice that were terminated early and in 6/50 high dose male mice) . Pheochromocytomas of the adrenal gland occurred at marginally increased incidence in low dose female rats (1/50; 6/50; 1/49) . Hyperplasia was observed in 3/50 vehicle controls, 7/50 low dose females, and 4/49 high dose females . The incidence of pheochromocytomas was low, not dose related, and not seen in males, and thus it was not considered to be compound related . Cytoplasmic vacuolization of the adrenal cortex occurred at increased incidences in males (5/50; 10/50; 20/50) but not in female rats . Nephropathy of the kidney occurred at increased incidences in female rats (13/50; 25/50; 20/50) but not in male rats . Additional nonneoplastic lesions such as congestion, inflammation, or nephrosis were not present to any degree in either vehicle control or dosed female rats . An increased incidence of alveolar/bronchiolar adenomas or carcinomas (combined) was observed in the 500 mg/kg group of female mice (3/50 vs 9/50), but little effect was seen in the 250 mg/kg group (6/50 vs 8/50) . The incidences of adenomas or carcinomas (combined) in dosed female mice were not significantly different from that in the pooled vehicle control group from the first and second studies (pooled controls, 9/100; 250 mg/kg, 8/50; 500 mg/kg, 9/50) . The lack o0) . The lack of hyperplasia in female mice and the negative trend in male mice suggest that these marginal effects were probably not related to the administration of n-butyl chloride . An increased incidence of hepatocellular adenomas or carcinomas (combined) was observed in the 500 mg/kg dose group of female mice (3/50 vs 8/50) but not in the 250 mg/kg dose group (9/50 vs 7/50) . An increased incidence of hemangiosarcomas was observed in male mice in the first study (1/50; 3/50; 4/50) but not in the second study (4/50 vs 2/50) . Neither of these marginal effects was regarded as compound related . n-Butyl chloride was not mutagenic in Salmonella typhimurium strains TA98, TA1535, or TA1537 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 or in the presence of male Syrian hamster liver S9 . n-Butyl chloride was mutagenic in the mouse lymphoma L5178Y/TK&plusmn; assay in the absence of Aroclor-induced male rat liver S9 and was not tested in the presence of S9 . n-Butyl chloride did not induce sister-chromatid exchanges or chromosomal aberrations in Chinesehamster ovary cells in the presence or absence of Aroclor-induced male rat liver S9 . An audit of the experimental data was conducted for the 2-year studies of n-butyl chloride . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity of n-butyl chloride for male and female F344/N rats at daily doses of 60 or 120mg/kg, for male B6C3F1 mice at doses of 250, 500, or 1,000 mg/kg, or for female B6C3F1 mice at doses of 250 or 500 mg/kg . Chemical-induced toxicity in high dose rats (primarily females) reduced the sensitivity of the study for determining carcinogenicity . Synonyms: 1-chlorobutane; butyl chloride; n-propylcarbinyl chloride Natl Toxicol Program Tech Rep Ser, 1985 Nov, 287, 1 - 180 NTP Toxicology and Carcinogenesis Studies of Dimethyl Hydrogen Phosphite (CAS No . 868-85-9) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Dimethyl hydrogen phosphite (DMHP) is used as an intermediate in the production of insecticides and herbicides, as an additive to lubricants, and as a stabilizer in oil and plaster and was considered for use as a chemical to simulate the physical (but not the biologic) properties of anticholinesterase agents . Results of 13-week gavage studies in F344/N rats (0-400 mg DMHP/kg body weight) and in B6C3F1 mice (0-1,500 mg DMHP/kg body weight) were used to identify short-term toxicity and to establish doses for the 2-year toxicology and carcinogenesis studies . In these studies, dimethyl hydrogen phosphite (greater than 97% pure) was administered for 103 weeks in corn oil by gavage to groups of 50 male F344/N rats and to groups of 50 male and female B6C3F1 mice at doses of 0, 100, or 200 mg/kg and to groups of 50 female F344/N rats at doses of 0, 50, or 100 mg/kg . In the 2-year studies, survival of high dose male rats and high dose male mice was lower (P<0.05) than that of the vehicle controls (male rats: vehicle control, 39/50; low dose, 29/50; high dose, 23/50; male mice: 42/50; 34/50; 32/50) . At the end of the studies, mean body weights were lower than those of the corresponding vehicle controls for high dose male rats (-15%), for high dose female rats (-5%), and for high dose male mice (-5%) . Dimethyl hydrogen phosphite caused dose-related increases in nonneoplastic and neoplastic lesions of the lung in male and female rats . In high dose male rats, there were increased incidences of lung neoplasms, including squamous cell carcinomas (0/50; 0/50; 5/50), alveolar/bronchiolar adenomas (0/50; 0/50; 5/50), and alveolar/bronchiolar carcinomas (0/50; 1/50; 20/50) . In high dose female rats, there was a marginal increase in the incidence of alveolar/bronchiolar carcinomas of the lung (0/50; 1/49; 3/50) . Hyperplasia of the lung and chronic interstitial pneumonia were increased in dosed male rats and in high dose female rats . Dimethyl hydrogen phosphite caused increases in forestomach lesions in male and female rats . In male rats, there was an increased incidence of forestomach neoplasms, including squamous cell papillomas (0/50; 1/50; 3/50) and squamous cell carcinomas (0/50; 0/50; 3/50) . High dose male rats had increased incidences of hyperkeratosis and hyperplasia of the forestomach . In high dose female rats, the incidence of forestomach hyperplasia was increased . Neoplastic lesions of the forestomach (a squamous cell papilloma and a squamous cell carcinoma) were found in two high dose female rats . Mineralization of the cerebellum was seen in high dose male rats (12/49) and in no other group . Focal calcification of the testis occurred at increased incidence in dosed male mice in the 2-year studies (2/50; 9/47; 24/50) . Compound-related testicular atrophy was seen in male mice in the 13-week study . Dimethyl hydrogen phosphite did not induce any neoplasms in male or female mice . Dimethyl hydrogen phosphite was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9 . This chemical did not induce sex-linked recessive lethal mutations in Drosophila melanogaster . An audit of the experimental data was conducted for these carcinogenic studies on dimethyl hydrogen phosphite . No data discrepancies were found that influenced the final interpretations . Under the conditions of these gavage studies, there was clear evidence of carcinogenicity in male rats receiving dimethyl hydrogen phosphite, as shown by increased incidences of alveolar/bronchiolar adenomas, alveolar/bronchiolar carcinomas, and squamous cell carcinomas of the lung and of neoplasms of the forestomach . There was equivocal evidence of carcinogenicity in female F344/N rats receiving dimethyl hydrogen phosphite, as shown by marginally increased incidences of alveolar/bronchiolar carcinomas of the lung and of neoplasms of the stomach . There was no evidence of carcinogenicity in male or female B6C3F1 mice receiving dimethyl hydrogen phosphite at doses of 100 ogen phosphite at doses of 100 or 200 mg/kg for 103 weeks . Synonyms: phosphonic acid, dimethyl ester (9CI); dimethyl phosphite; dimethyl phosphorus acid; methyl phosphonate; dimethyl phosphonate; dimethoxyphosphine oxide; TL 585; DMHP; phosphorous acid, dimethyl ester; dimethylphosphite; dimethyl phosphonate; dimethylphosphorous acid; bis(hydroxymethyl) phosphine oxide Natl Toxicol Program Tech Rep Ser, 1986 Jan, 291, 1 - 198 NTP Toxicology and Carcinogenesis Studies of Isophorone (CAS No . 78-59-1) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of isophorone (greater than 94% pure), a widely used solvent and chemical intermediate, were conducted by administering 0, 250, or 500 mg isophorone/kg body weight per day by gavage in corn oil to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days per week for 103 weeks . Doses selected for the 2-year studies were based on the 16-day studies in which rats and mice of each sex received doses of 0-2,000 mg/kg per day and on 13-week studies in which rats and mice of each sex received doses ranging from 0 to 1,000 mg/kg per day by gavage in corn oil . No chemically related gross or histopathologic effects were observed in the 16-day or 13-week studies, but 1/5 high dose male rats, 4/5 high dose female rats, and all high dose male and female mice died during the 16-day studies . During the 13-week studies, 1/10 high dose female rats and 3/10 high dose female mice died . The high dose for the 2-year studies was set at 500 mg/kg per day for each sex of rats and mice, based mainly on the deaths in the 13-week studies . Throughout the 2-year study, the mean body weights of the high dose male rats averaged 5% lower than those of the vehicle controls . During the second year, the mean body weights of the female high dose rats averaged 8% lower than those of the vehicle controls, and the high dose female mice averaged 5% lower . The survival of high dose male rats was significantly lower than that of the vehicle controls after week 96 (final survival: vehicle control, 33/50; low dose, 33/50; high dose, 14/50) . The survival of dosed female rats was poor (30/50; 23/50; 20/50), due in part to 20 gavage-related accidental deaths of dosed animals . The survival of male mice was also low (16/50; 16/50; 19/50), but there was a significant trend toward increased survival of dosed female mice relative to that of the vehicle controls (26/50; 35/50; 34/50) . Dosed male rats showed a variety of proliferative lesions of the kidney (tubular cell hyperplasia: 0/50; 1/50; 4/50; tubular cell adenoma: 0/50; 0/50; 2/50; tubular cell adenocarcinoma: 0/50; 3/50; 1/50; epithelial hyperplasia of the renal pelvis: 0/50; 5/50; 5/50) . Dosed male rats also exhibited increased mineralization of the medullary collecting ducts (1/50; 31/50; 20/50), and low dose male rats showed a more severe nephropathy than is commonly seen in aging F344/N rats . Carcinomas of the preputial gland were increased in high dose male rats (0/50; 5/50; 5/50) . With the exception of a moderate increase in nephropathy (21/50; 39/50; 32/50), female rats did not show chemically related increased incidences of neoplastic or nonneoplastic lesions . In high dose male mice, isophorone exposure was associated with increased incidences of hepatocellular adenomas and carcinomas (18/48; 18/50; 29/50) and of mesenchymal tumors of the integumentary system (fibroma, fibrosarcoma, neurofibrosarcoma, or sarcoma: 6/48; 8/50; 14/50) . An increased incidence of lymphomas or leukemias was noted in low dose male mice (8/48; 18/50; 5/50) . Coagulative necrosis (3/48; 10/50; 11/50) and hepatocytomegaly (23/48; 39/50; 37/50) were observed more frequently in the livers of dosed male mice than in vehicle controls . No compound-related neoplastic or nonneoplastic lesions associated with isophorone exposure were seen in female mice . Isophorone was not mutagenic in strains TA100, TA1535, TA1537, or TA98 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 . Isophorone was weakly mutagenic in the mouse L5178Y/TK+/- assay in the absence of S9; it was not tested in the presence of S9 . Isophorone induced sister-chromatid exchanges in the absence of S9 in Chinese hamster ovary cells; it did not induce sister-chromatid exchanges in the presence of Aroclor 1254-induced male rat liver S9, and it did not induce chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of S9 . An audit of the experimental data was conducted for the 2-year toxicology and carcinogenesis studies of isophorcarcinogenesis studies of isophorone . No data discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenicity of isophorone in male F344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas in animals given 250 or 500 mg/kg per day; carcinomas of the preputial gland were also observed at increased incidence in male rats given 500 mg/kg . There was no evidence of carcinogenicity in female F344/N rats given 250 or 500 mg/kg per day . For male B6C3F1 mice, there was equivocal evidence of carcinogenicity of isophorone as shown by an increased incidence of hepatocellular adenomas or carcinomas (combined) and of mesenchymal tumors in the integumentary system in animals given 500 mg/kg per day and by an increase in malignant lymphomas in animals given 250 mg/kg per day . There was no evidence of carcinogenicity of isophorone in female B6C3F1 mice given 250 or 500 mg/kg per day . Synonym: 3,5,5-trimethyl-2-cyclohexen-1-one Natl Toxicol Program Tech Rep Ser, 1985 Aug, 293, 1 - 192 NTP Toxicology and Carcinogenesis Studies of HC Blue No . 2 {2,2'-((4-((2-Hydroxyethyl)amino)-3-nitrophenyl)imino)bis(ethanol)} (CAS No . 33229-34-4) in F344/N Rats and B6C3F 1 Mice (Feed Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of HC Blue No . 2 (approximately 98% pure), a semipermanent hair dye, were conducted by administering the test chemical in feed for 103 weeks to groups of 50 F344/N rats of each sex and for 104 weeks to groups of 50 B6C3F 1 mice of each sex . The dietary concentrations used were 0, 5,000, or 10,000 ppm for male rats and male mice and 0, 10,000, or 20,000 ppm for female rats and female mice . These concentrations were selected on the basis of results from single-administration gavage and 14-day and 13-week feed studies . For the 2-year studies, the average daily doses were approximately 195 and 390 mg/kg in male rats, 465 and 1,000 mg/kg in female rats, 1,320 and 2,240 mg/kg in male mice, and 2,330 and 5,600 mg/kg in female mice . The survival of high dose male rats and male mice was better than that for controls, and the survival of dosed female rats was comparable to that of the controls . The survival of high dose female mice was reduced (P<0.05) relative to that of controls (control, 35/50; low dose, 27/50; high dose 19/50); this reduced survival was attributed to a reproductive tract infection . Final mean body weights relative to those of controls were depressed less than 10% in dosed male rats, whereas depressions of 13% and 22% were observed in the low dose and high dose groups of female rats . Final mean body weights for dosed male mice were within 5% of control values, but final mean body weights for dosed females were 15% (low dose) and 22% (high dose) lower than that of controls . A dose-related increase in the incidence of hyperostosis of the skull was detected in rats (male, 5/50, 8/50, 25/49; female, 2/50, 19/50, 49/50) and in 1/49 high dose male and 4/50 high dose female mice . Mixed mesenchymal neoplasms of the kidney were detected in 2/50 high dose female rats; none was observed in any other group of female or male rats . This tumor is considered uncommon and has not been found in 1,863 historical control female F344/N rats . A negative trend in fibroadenomas of the mammary gland was seen in female rats (20/50, 10/50, 4/50) . A marginal (P=0.05) positive trend occurred in the incidence of lymphomas in male mice (1/50; 5/48; 8/49); the incidences in the dosed groups were not significantly greater than that in the controls when survival differences were taken into account . HC Blue No . 2 was mutagenic for strains TA97 and TA98 but not for strains TA100 or TA1535 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9 . HC Blue No . 2 was mutagenic in the mouse lymphoma L5178Y/TK+/- assay in the presence of Aroclor 1254-induced male F344/N rat liver S9 . An audit of the experimental data was conducted for these carcinogenic studies on HC Blue No . 2 . No data discrepancies were found that influenced the final interpretations . Under the conditions of these studies, there was no evidence of carcinogenicity in male and female F344/N rats or in male and female B6C3F 1 mice receiving HC Blue No . 2 in the diet at concentrations of 0.5% and 1.0% for males and 1.0% and 2.0% for females for 2 years . HC Blue No . 2 administration caused a dose-related increase in the incidence of hyperostosis of the skull in male and female rats . Synonym: 2,2'-((4-((-hydroxyethyl)amino)-3-nitrophenyl)imino)bis(ethanol) Natl Toxicol Program Tech Rep Ser, 1986 Dec, 294, 1 - 109 NTP Toxicology and Carcinogenesis Studies of Chlorinated Trisodium Phosphate (CAS No . 56802-99-4)* in B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Two-year toxicology and carcinogenesis studies of chlorinated trisodium phosphate, an inclusion complex of trisodium phosphate and sodium hypochlorite used in various cleaning compounds, were conducted by administering 0, 500, or 1,000 mg/kg (dose volume: 10 ml/kg) of the chemical in water by gavage, 5 days per week for 103 weeks, to groups of 50 male and 50 female B6C3F1 mice . Groups of mice receiving 250 mg/kg were included in these studies but were removed after 6 months because of a lack of toxicity in the 500 and 1,000 mg/kg groups . Two-year studies were begun in male and female F344/N rats at doses of 0, 500, 1,000, or 2,000 mg/kg of chlorinated trisodium phosphate in water by gavage (10 ml/kg) . The 2,000 mg/kg groups were killed at 15 weeks because of poor survival, and the other groups were killed at 35 weeks because of toxicity in the 1,000 mg/kg group . The doses selected for the 2-year studies were based on the general lack of adverse effects seen in the 14-day and 13-week studies in which rats received 0-1,000 mg/kg and mice received 0-2,000 mg/kg by gavage in water . No compound-related histopathologic effects were observed in the 14-day or the 13-week studies in mice . In the 2-year studies, survival and mean body weights of dosed and vehicle control male mice groups were comparable (survival-- vehicle control, 39/50; low dose, 35/50; high dose, 32/50) . Survival of the dosed female mice was lower than that of the vehicle controls (30/50; 16/50; 21/50), although at week 80 survival of female mice was 42/50, 39/50, and 36/50 . The mean body weights of the high dose female mice were lower than those of the vehicle control mice, primarily after week 32; final body weights were 11% lower in the high dose group compared with that in the vehicle controls . The lower survival and mean body weights of the dosed female mice may have been due to a greater incidence of uterine/ovarian infections in these mice rather than to a direct toxic effect of chlorinated trisodium phosphate . Nine of 20 vehicle control, 20/34 low dose, and 21/29 high dose female mice that died before the end of the studies had such infections . This reduced survival decreased the sensitivity of the study of female mice for detecting the presence or absence of carcinogenic effects . At no site was the incidence of neoplasms considered to be related to the administration of chlorinated trisodium phosphate . Minimal necrosis and fatty changes were observed in the livers of male mice . Kidneys in male mice were characterized by small, multifocal areas of mineralization, primarily in the cortex but not at the corticomedullary junction or in the tubes of the medulla . Neither effect was considered compound related . Five different types of ovarian neoplasms were found in six dosed female mice; because these lesions were from tissues of different embryonic origin, they were considered unrelated to administration of chlorinated trisodium phosphate . Chlorinated trisodium phosphate was weakly mutagenic in strain TA1535 of Salmonella typhimurium in the presence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 . This compound was not mutagenic in strains TA97, TA98, or TA100 . An audit of the experimental data was conducted for these 2-year studies of chlorinated trisodium phosphate . No data discrepancies were found that influenced the final interpretation of these experiments . Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity for either male or female B6C3F1 mice given chlorinated trisodium phosphate by gavage in water for 103 weeks at doses of 500 or 1,000 mg . Survival of dosed female mice was 78% and 72% after 80 weeks and 32% and 42% at the termination of the study . The studies in male and female F344/N rats were considered to be inadequate studies of carcinogenicity because the experiments were terminated at 35 weeks due to poor survival . Synonym: sodium hypochlorite phosphate Natl Toxicol Program Tech Rep Ser, 1987 Feb, 296, 1 - 290 NTP Toxicology and Carcinogenesis Studies of Tetrakis(hydroxymethyl)phosphonium sulfate (THPS) (CAS No . 55566-30-8) and Tetrakis(hydroxymethyl)phosphonium chloride (THPC) (CAS No . 124-64-1) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Toxicology and carcinogenesis studies of tetrakis(hydroxymethyl)phosphonium sulfate (THPS) and tetrakis(hydroxymethyl)phosphonium chloride (THPC) were conducted because of the widespread use of these chemicals as flame retardants in cotton fabrics . THPS was available as a 72% aqueous solution and THPC as a 75% aqueous solution . Short-term gavage studies with a range of doses were conducted first to identify toxic effects and affected sites and to determine doses for the 2-year studies . The doses selected for the 14-day studies ranged from 12.5 to 200 mg/kg THPS for rats and mice, 9.4 to 150 mg/kg THPC for rats, and 18.8 to 300 mg/kg THPC for mice . Mortality and reduction in body weight gain occurred at the two highest doses in the 14-day studies . There was hind limb paralysis in some rats and mice dosed at the highest concentrations of THPS and THPC . In the 13-week studies, doses of THPS ranged from 5 to 60 mg/kg in rats and from 2 to 180 mg/kg in mice; doses of THPC ranged from 3.75 to 60 mg/kg in rats and from 1.5 to 135 mg/kg in mice . Mortality and reduction in body weight gain occurred at the two higher doses for both sexes and species . Vascular degeneration of hepatocytes or hepatocellular necrosis was a common histopathologic finding . Hind limb paralysis was noted in rats and mice receiving the highest dose of THPC, and axonal degeneration, characterized by swollen axon sheaths, missing or fragmented axons, and some proliferation of neurolemma cells, was observed in rats . These lesions were found in the sciatic nerve, dorsal roots of the caudal spinal nerves, and the tracts of the spinal cord, particularly in the dorsal column of the lumbar cord . Two-year studies were conducted in F344/N rats by administering 0, 5, or 10 mg/kg THPS or 0, 3.75, or 7.5 mg/kg THPC in deionized water by gavage to groups of 49 or 50 animals of each sex, 5 days per week for 103 or 104 weeks . Groups of 49 or 50 B6C3F1 mice were administered 0, 5, or 10 mg/kg THPS (each sex), 0, 7.5, or 15 mg/kg THPC (males), or 0, 15, or 30 mg/kg THPC (females) . Survival of male rats was reduced for the low dose (after week 102) and the high dose (after week 67) groups given THPS compared with that of the vehicle controls; survival at terminal kill was as follows: vehicle control, 28/50; low dose, 13/50; high dose, 16/50 . Survival of the high dose group of female rats given THPC was lower after week 70 than that of the vehicle controls (survival at terminal kill: 37/50; 34/50; 21/50) . Mean body weights of rats dosed with THPS or THPC were comparable to those of the vehicle controls . There was no difference in survival or mean body weights between the vehicle controls and mice dosed with either THPS or THPC . No neurotoxicity or any other signs of clinical toxicity were observed . A nonneoplastic effect common to 13-week and 2-year exposure to THPS or THPC was an increase in the incidence of hepatocellular lesions, primarily cytoplasmic vacuolization . The incidences of this lesion in the two-year studies were dose related for all studies except for the mice receiving THPS . Other lesions observed included focal hyperplasia of the adrenal medulla in high dose male mice given THPS and follicular cell hyperplasia of the thyroid gland in high dose female mice given THPC . The increased incidences of hematopoietic system lesions observed in these studies were not considered biologically related to chemical exposure because the increases were marginal, no dose-response relationship was observed, and the incidences of these lesions are highly variable in untreated rats and mice . The incidences of mononuclear cell leukemia in low dose male rats administered THPS or THPC were somewhat greater than those in the vehicle controls (THPS: 30/50; 36/50; 20/50; THPC: 19/50; 25/50; 16/50) . Low dose male mice administered THPS had an increased incidence of malignant lymphomas when compared with vehicle controls (2/50; 9/50; 0/50) . These marginal increases in the incidences of hematopoietic system tumors were not considered related to chemical exposure, since they were signific were significant only by the life table tests and were not dose related . THPC demonstrated no mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation . Both THPS and THPC induced forward mutations in mouse lymphoma L5178Y cells without metabolic activation; neither was tested in the presence of S9 . THPC increased the frequency of sister-chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells in the presence and absence of exogenous metabolic activation . An audit of the experimental data was conducted for the 2-year studies of THPS and THPC . No discrepancies were found that influenced the final interpretations . Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity of THPS in either sex of F344/N rats or B6C3F1 mice given 5 or 10 mg/kg . There was no evidence of carcinogenicity of THPC in either sex of F344/N rats given 3.75 or 7.5 mg/kg, in male B6C3F1 mice given 7.5 or 15 mg/kg, or in female B6C3F1 mice given 15 or 30 mg/kg. Natl Toxicol Program Tech Rep Ser, 1986 Jan, 298, 1 - 186 NTP Toxicology and Carcinogenesis Studies of Dimethyl Morpholinophosphoramidate (CAS No . 597-25-1) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Dimethyl morpholinophosphoramidate (DMMPA, greater than 99% pure) was developed for use as a simulant for the physical (but not biologic) properties of anticholinesterase agents in chemical defense training . Because of the potential for human exposure, the toxicity and carcinogenicity of DMMPA were investigated . Fourteen-day and 13-week studies were conducted to determine short-term toxicity, to identify target organs, and to establish doses for the 2-year toxicology and carcinogenesis studies . In the 14-day studies, groups of five male and five female F344/N rats were administered DMMPA in corn oil by gavage daily at 0, 313,