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.     

Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague–Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.     

Absence of mutagenic action of 5 beta-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5 beta-cholanoic acids including 2 of the bile acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5 beta-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.     

Absence of mutagenic action of 5β-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5β-cholanoic acids including 2 of the biie acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5β-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.     

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

Testing threshold and hormesis in a random effects dose-response model applied to developmental toxicity data

Here we describe a random effects threshold dose-response model for clustered binary-response data from developmental toxicity studies. For our model we assume that a hormetic effect occurs in addition to a threshold effect. Therefore, the dose-response curve is based on two components: relationships below the threshold (hormetic u-shaped model) and those above the threshold (logistic model). In the absence of hormesis and threshold effects, the estimation procedure is straightforward. We introduce score tests that are derived from a random effects hormetic-threshold dose-response model. The model and tests are applied to clustered binary data from developmental toxicity studies of animals to test for hormesis and threshold effects. We also compare the score test and likelihood ratio test to test for hormesis and threshold effects in a simulated study.     

Detecting and Estimating Hormesis Using a Model-Based Approach

Hormesis is defined as a dose-response relationship that is stimulatory at low doses, but is inhibitory at higher doses. In a given experiment, it is not unusual to observe enhanced responses at low doses, however, such enhanced responses may not imply hormesis, but the random fluctuation of the data. Statistical tests can be developed to detect hormesis when enhanced responses at low concentrations are observed. We propose the use of a model-based approach to detect the presence of, and estimate the extent of, hormesis. This approach includes two steps: detection and estimation. In the detection step, we compare the full and the reduced models. The full model describes the dose-response relationship incorporating the hormetic effect; the reduced model describes the dose-response relationship without the hormetic effect. The full model is an extension of the reduced model and has an extra parameter that measures the amount of increase in response at low doses. A test of statistical significance of this extra parameter can essentially be a test for detecting hormesis. In the estimation step, we obtain the area under the best-fitted dose-response curve falling within the hormetic zone. Considering both the number of concentrations within the hormetic zone and the magnitude of the stimulatory response, we propose using the ratio of the area under the hormetic zone (AUC_H) and the area under the best-fitted curve from zero to zero equivalent point (AUC_ZEP) as an estimate of magnitude of the hormetic effect. Two numerical examples are used to illustrate the use of this model-based approach.     

Bacterial mutagenicity testing of 49 food ingredients gives very few positive results

49 substances permitted for use in food in the United States was tested for mutagenicity in the Ames Salmonella typhimurium assay and in Escherichia coli strain WP2. Four of these substances caused increases in revertant counts in S. typhimurium. Two of these four (papain and pepsin) were found to contain histidine, and therefore the results of the tests on these two substances could not be taken as demonstrating mutagenicity. The other two substances causing increases in revertant counts (hydrogen peroxide and potassium nitrite) were mutagenic. The results on one chemical, beta-carotene, were evaluated as inconclusive or questionable. The remaining 44 substances were nonmutagenic in the test systems used. It is concluded that, for those generally physiologically innocuous chemicals tested, there are very few 'false positives' in the bacterial test systems used.     

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2

The Escherichia coli WP2 tryptophan reverse mutation assay detects trp(-) to trp(+) reversion at a site blocking a step in the biosynthesis of tryptophan prior to the formation of anthranilic acid. The different WP2 strains all carry the same AT base pair at the critical mutation site within the trpE gene. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity. In general the WP2 strains are used as a substitute for, or as an addition to Salmonella strain TA102 which also carries an AT base pair at the mutation site. The assay is also recommended together with the Ames assay for data submission to regulatory agencies. National and international guidelines have been established for performing these mutagenicity assays.The E. coli WP2 assay procedures are the same as those described elsewhere in this volume for the Ames Salmonella assay (Mortelmans and Zeiger, 2000) with the exception that limited tryptophan instead of limited histidine is used. This chapter is an addendum to the previous chapter and the reader should refer to the previous chapter for details regarding experimental procedures and assay design.     

Mutagenic activity of acrinol in Salmonella typhimurium

Acrinol, which is used as a disinfectant and an abortifacient in several countries, was tested for mutagenicity by the Ames test system. After incubation with a rat-liver S9 microsomal preparation, acrinol showed potent mutagenicity for Salmonella typhimurium strains TA98 and TA100, although it had no direct mutagenicity for the microorganisms.     

Statistical modeling and analyses of a base-specific Salmonella mutagenicity assay

Statistical features of a base-specific Salmonella mutagenicity assay are considered in detail, following up on a previous report comparing responses of base-specific Salmonella (Ames II) strains with those of traditional tester strains. In addition to using different Salmonella strains, the new procedure also differs in that it is performed as a microwell fluctuation test, as opposed to the standard plate or preincubation test. This report describes the statistical modeling of data obtained from the use of these new strains in the microwell test procedure. We emphasize how to assess any significant interactions between replicate cultures and exposure doses, and how to identify a significant increase in the mutagenic response to a series of concentrations of a test substance.     

Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane- and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n- and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These compounds were generally found to be more potent with regard to micronucleus induction when tested without metabolic activation (20 h treatment). In conclusion, the iso-propyl esters of the three sulfonic acids under study were found to be the strongest mutagens, either when tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-butyl ester was the only compound shown to be devoid of a genotoxic potential in both tests.     

The involvement of dimethyl sulfoxide in a bacteriotoxic response of the Ames assay tester strains TA98 and TA100

Dimethyl sulfoxide is a widely accepted and recommended solvent in which to dissolve compounds to be tested for mutagenicity via the Ames Salmonella/mammalian microsome assay. Using tester strains TA98 and TA100, we observed a bacteriotoxic response with various fractions isolated from beer when dissolved in DMSO but not when dissolved in water. Further characterization of the role of solvent in simple model systems consisting of butanol, DMSO and bacteria strongly suggests a chemical reaction occurs between dimethyl sulfoxide and specific chemical constituents of the test substance, nutrient broth, or the Ames bacterial strains. The result of such an interaction could be misinterpreted as a toxic response to the test substance when, in fact, the bacteriotoxicity could be due to another compound, chemically distinct from the test substance.     

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.     

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)     

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.     

The Salmonella mutagenicity test: evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.     

Antimutagenic effect of amino acids on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC50) of amino acids that inhibit 50% of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.     

Reduction in Ames Salmonella mutagenicity of mainstream cigarette smoke condensate by tobacco protein removal

The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay.     

What is the role of histidine in studies of faecal mutagenicity?

Mutagenicity testing can be used to assay faeces for genotoxic substances and the results are reported to correlate with population risk for colorectal cancer (Ehrich et al., 1979). It has been suggested that histidine in faeces may cause false positive results (Venitt and Bosworth, 1983). To determine the relationship between histidine and false positive mutagenicity assays aliquots of non-mutagenic faecal extract and saline were supplemented with histidine and subjected to the Ames Salmonella/mammalian microsome mutagenicity assay (Ames et al., 1975). Using high-pressure liquid chromatography the analytical recovery of histidine from water and faecal extract supplemented with histidine was equivalent (r = 0.998, p less than 0.001). Histidine was measured in faecal extracts (1 in 10 dilutions) from 35 volunteers, 10 patients with inflammatory bowel disease and 4 with rectal cancer. These extracts were also assayed for mutagens using the Salmonella/mammalian microsome mutagenicity assay. None of the faecal extracts gave mutagenicity ratios above 2. Faecal extracts from volunteers were free of detectable histidine. Although 9 of those from inflammatory bowel disease patients contained histidine (mean +/- SEM 255 +/- 34 mumoles l-1) as did 1 extract from a rectal cancer patient (50 mumoles l-1), none contained sufficient histidine to give a false positive Salmonella/mammalian microsome mutagenicity assay result (800 mumoles l-1 in test solution). Our results do not implicate histidine as a cause of error in faecal mutagenicity testing by the Salmonella/mammalian microsome mutagenicity assay.