The Ames Salmonella/microsome mutagenicity assay

The Ames Salmonella/microsome mutagenicity assay (Salmonella test; Ames test) is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his(+)) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner. The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used for submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories to ensure uniformity of testing procedures. This review provides historical aspects of how the Ames was developed and detailed procedures for performing the test, including the design and interpretation of results.     

Dose finding in the Ames Salmonella assay

Threshold dose/concentration values, such as the lowest effective dose, minimum effective dose or the lowest effective concentration (LED, MED or LEC, respectively) are in use as an alternative to the mutagen potency measures based on the 'rate' measurements (e.g., the slope of the initial part of the dose-response curve). In this respect, several statistical procedures for the corresponding so-called 'dose finding' were proposed during the last decades. However, most of them disregard the discrete nature of responses such as the plate colony count in the Ames Salmonella assay. When the plate counts agree with the Poisson assumption, two procedures considered here seem to be appropriate for the dose finding. One is based on the stepwise collapsing of the homogeneous control and dose counts; another consists of constructing the confidence limits for the mutation induction factor (MIF). When the dose and control counts are non-overlapping, the simple 'visual' non-parametric estimation of LED is possible. Applicability and validity of the methods is demonstrated with the two data sets on the mutagenicity of the beta-carboline alkaloid, harmine, and one of the oxidation products of apomorphine.     

Effects of pH on weak and positive control mutagens in the Ames Salmonella plate assay

The effects of pH on the mutagenic activity of several chemicals were evaluated in the standard Ames Salmonella typhimurium plate-incorporation assay. The pH of the base agar was varied between 6.0 and 8.0. The positive control compounds routinely used in this laboratory, 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and nitrofurantoin, showed increasing mutagenic activity as the pH was decreased to 6.0. However, the activity of two weakly mutagenic cosmetic ingredients, 2,2',4,4'-tetrahydroxybenzophenone and trans-4-phenyl-3-buten-2-one, was completely eliminated at pH levels near 6.0. It is concluded that plates poured with agar with pH levels below 7.0 can result in strong responses for the positive control chemicals but give negative results for some mutagens.     

Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

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)     

Structure-activity relationships of aromatic amines in the Ames Salmonella typhimurium assay

The author tried in a somewhat limited work to quantitatively correlate the electronic and steric intramolecular interactions of substituents on the amino group (influencing the enzymatic reactions of aromatic amines) and the mutagenic event. It was assumed that there is a correlation between these biotransformations and the electronic state of aromatic amines at the ionic dissociation equilibrium. The approach is rather empirical and arbitrary but the overall agreement between experimental mutagenic potencies and the values calculated was encouraging and led the author to further developments. It is hoped that the concepts used in this work may be applied to other aromatic molecules bearing an amino group.     

Mutagenicity studies of p-substituted benzyl derivatives in the Ames Salmonella plate-incorporation assay

The mutagenicity of 24 benzyl derivatives, containing a variety of substituents and leaving groups, were assayed in strain TA100 using the Ames plate-incorporation assay. p-Nitrobenzyl chloride (12 000 revertants/mumole), p-nitrobenzyl tosylate (6100 revertants/mumole), and p-acetoxybenzyl chloride (100 revertants/mumole) were mutagenic; none of the remaining 21 compounds were mutagenic. p-Nitrobenzyl chloride was also found to be mutagenic in strain TA98 (700 revertants/mumole), but not in strain TA98NR (a strain deficient in nitro reductase activity). p-Acetoxybenzyl chloride was nonenzymatically hydrolyzed to p-hydroxybenzyl alcohol and p-acetoxybenzyl alcohol. These findings suggest that nitrobenzyl derivatives were mutagenic due to nitro reductive metabolism and that p-acetoxybenzyl chloride was mutagenic due to the intermediate formation of p-hydroxybenzyl chloride during the hydrolysis of p-acetoxybenzyl chloride.     

Structure-activity relationships of aromatic diamines in the Ames Salmonella typhimurium assay. Part II.

Structure-activity relationships in the case of aromatic monoamines, diversely substituted on the ring, using the mutagenic activity in the Ames test were studied in part I. This part II is based on the same general principles but applied to phenylene diamines (ortho, para and meta) diversely substituted on the ring.     

The assessment of the mutagenic potential of vehicle engine exhaust in the Ames Salmonella assay using a direct exposure method

A method for the assessment of the mutagenic activity of vehicle engine exhaust in the Ames assay is described in which the bacterial strains used (TA98 and TA98/DNP) are exposed to the freshly produced engine exhaust using a "Cassella' slit sampler. The method is found to be effective both in the presence and absence of metabolic activation, using Aroclor-1254-induced rat liver S9 fractions. A comparison is made between the direct exposure method and the standard methods involving the collection of particulate samples on glass fibre filters and the testing of various extracts of these samples. Possible uses of the direct exposure testing method are suggested and the effect of sampling techniques on the results obtained in the Ames assay is also discussed.     

The relationship between growth and allometry

The asymptotic allometric equations are derived by considering the relative growth of different physiological and anatomical quantities. The growth rate is assumed to be proportional to the value of the quantity, where the proportion coefficient depends on time. The allometric exponents are calculated for some organs of man and compared with the experimental values for primates. The exponents are generally time-dependent during the growth. The effect of the choice of the origin of time axis on the asymptotic allometric exponents is studied.     

The anti-mutagenic and antioxidant effects of bile pigments in the Ames Salmonella test

The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.     

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.     

Peroxyacetyl nitrate: measurement of its mutagenic activity using the Salmonella/mammalian microsome reversion assay

Exposures of Salmonella typhimurium strain TA100 with and without S9 metabolic activation to low ppm levels of pure peroxyacetyl nitrate (PAN) in the gas phase were conducted. Measurements of the gas-phase PAN exposure concentration and the concentration of its decomposition products in surrogate test media led to a measured mutagenic activity of 34 +/- 5 revertants/mumole. The data indicate that PAN is a relatively weak direct-acting mutagen with TA100.     

The relationship between cell and population growth

A steady-state model of cell volume frequency distribution using the method of Williams (1971) is derived. Results are compared to a Monte Carlo simulation of cell growth and division. It is suggested that the Monte Carlo method might be of value for investigating cell and population properties for which analytic methods are not currently available.     

A procedure for the statistical evaluation of Ames Salmonella assay results. Comparison of results among 4 laboratories

Ames Salmonella test data collected in our laboratory and 3 National Cancer Institute contract laboratories were analyzed to study the distribution of experimental errors associated with the test. It is shown that the Poisson distribution is not appropriate, and that the power transformation model Y = (revertants/plate)lambda, with lambda = 0.2 as estimated by the methods of Box and Cox, produced a measurement scale on which the experimental errors could be adequately described by a normal (Gaussian) distribution with a constant variance. The modeling procedure enables one to properly use analysis of variance, regression analysis, and Student's t test to analyze Ames Salmonella test results, and well-known statistical quality control procedures to monitor laboratory performance. The method detects weak mutagenic activity and measures the amount and uncertainty of the increase in revertants/plate. The development of the power transformation model is discussed and examples of its use in the interpretation of Ames Salmonella assay results are included.     

Mutagenicity of mono-nitrobenzene derivatives in the Ames test and rec assay

The mutagenicities of 37 mono-nitrobenzene derivatives, i.e. the ortho, meta, and para isomers of nitrotoluene, nitrophenol, nitroaniline, nitroanisole, nitrobenzaldehyde, nitrobenzyl chloride, nitrobenzonitrile, nitroacetophenone, nitrophenetol, nitrobenzoic acid, nitrophenylacetic acid, and nitrocinnamic acid and p-nitrothiophenol, were tested in the Ames test and rec assay. The rec assay gave more positive results than the Ames test. The para-substituted nitrobenzene derivatives were always positive in the rec assay as were all but 2 in the Ames test, while 5 out of 12 ortho and meta derivatives were mutagenic in the Ames test. The results of the present study, combined with those of the previous study, are discussed with special reference to the structure-mutagenicity relationships.     

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.