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Scientific
Publications - Work Done by Microbiology Reader
Nylund, L. and Einistö, P., Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test, Mutation Research, 1992, vol. 272, pp. 205-214 ABSTRACT Mutagenicity testing of biological samples and proteins is
complicated by the presence of histidine and histidine-related growth factors
which may produce a false positive result in the Ames/Salmonella plate
incorporation test. A bioassay method, utilizing an automated
dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator
bacteria, was used to estimate the presence of histidine-related growth factors
in three enzyme solutions submitted for mutagenicity testing. One of the
solutions was clearly positive in the Ames/Salmonella test and also contained
the highest amount of L-histidine-HCl-equivalents. The two other solutions, with
low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and
negative results, respectively, in the Ames/Salmonella test. Studies were also
performed with strains TA98, TA100 and TA1535 to determine the amount of added
L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella
test. Because the minimum amount of L-histidine-HCl required to double the
number of revertant colonies was 150 nmol/plate, and the maximum amount of
L-histidine-HCl-equivalents supplied by the enzyme preparations was 40
nmol/plate at the highest tested dose, the mutagenicity test results of the
enzyme solutions cannot be explained solely by histidine or related compounds.
Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3)
and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal
differences in efficiencies to extract histidine and histidine-related compounds
in the urines. Amounts of 'histidine' in concentrates of urine were measured
using the bioassay method and a chemical method employing derivatization with
fluorescamine. The fluorescamine method also efficiently detected
3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which
was found to be unable to support auxotrophic growth in TA1535, leading to
exaggerated estimations of the auxotrophic growth enhancing properties of urine
extracts. The urine extracts, and pure L-histidine-HCl, were tested using a
two-step fluctuation test to estimate auxotrophic growth factor effects in this
type of test. Because of a strong dilution effect when adding the histidine-free
selection medium, the fluctuation test employed in this study was not found to
be particularly sensitive to growth factors. The results of this study indicate
that use of a bioassay, employing the same indicator bacteria as the
mutagenicity test themselves, is a reliable way to measure histidine-related
growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)
(order Full Text from publisher)
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