Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella spiral and plate assays

A factorial experimental design was used within this study to evaluate the influence of multiple metabolic activation system concentrations on the dose-response exhibited by promutagens (indirect-acting mutagens) in the Salmonella spiral and plate assays. The mutagenic activity of the three compounds used spanned three orders of magnitude. The mutagenic activity of the compounds ranged from 10 to 100 revertants/micrograms for acetylaminofluorene (2AAF) to more than 1000 revertants/micrograms for 2-aminoanthracene (2AA). Benzo [a] pyrene (BaP) activity was within an intermediate range (100-1000 revertants/micrograms). During a single experiment, a mutagen was tested in TA100 at 13 doses plus a negative control dose. Each dose was tested at 10 S9 concentrations. The S9 concentrations ranged from 0.1 mg protein/plate to 4 mg protein/plate in the standard plate assay and from 0.25 to 4.90 mg-equivalents in the spiral assay. The spiral Salmonella assay, an automated version of the standard assay, generates dose-response data from a concentration gradient on a single agar plate, thereby providing a straightforward approach to this type of study. This study demonstrates not only that even small differences in S9 concentrations can affect the measurement of mutagenic potency but that S9/compound interactions cannot be generalized through the use of interaction studies. This study also shows that spiral assay data and plate assay data for promutagens cannot be compared directly unless the S9 concentrations for all chemical doses are also comparable.     

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.     

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.     

A “spiral test” applied to bacterial mutagenesis assays

A new procedure (the spiral test) has been set up and validated for the distribution of chemicals in bacterial multagenesis asssays. This metzhod involves the use of a special instrument (spiral plater), which dispenses, along a spiral track, decreasing volumes of liquid samples, from the near centre to the periphery of a rotating agar plate. A gradient of concentration of a compound up to about 1500:1 is thus formed on a single plate. The activity of 18 mutagens of various potencies and chemical classes was checked in the Salmonella/microsome test by dispensing their solutions either on the surface of top agar (method A) or of the minimal-glucose agar medium, before the addition of molten top agar incorporating bacteria and eventually S9 mix (method B).Compared with the spot test, the gradient of concentration of a compound produced by the spiral diluter was much wider and more gradual. Even non-diffusible chemicals (e.g. benzo[a]pyrene and benz[a]anthracene) were efficiently detected in the spiral test, as well as very weak (e.g. mebanazine and trimethylphosphate) or borderline (e.g. perylene, 1,1-dimethylhydrazine and procarbazine) mutagens, which were negative in the spot test. Method B was at least as sensitive as the plate-incorporation test, such a goal being achieved in a single plate instead of in serial plates. Technical problems made method A less sensitive, but it was more efficient in detecting unstable mutagens (e.g. β-propiolactone). Like the plate test, the spiral test appeared to be suitable for a semi-quantitative assessment of mutagenicity data, and was efficient in demonstrating both the activation of promutagens and the deactivation of some directly acting mutagens. Preliminary assays were also carried out with repair-proficient (WP2) or -deficient (TM1080: lex A^?/polA^?/R391 and CM871: lexA^?/uvrA^?/recA^?) trp^? strains of E. coli.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.     

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.     

Mutagenic action of a series of epoxides.

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the additon of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinly cyclohexene diepoxide, glycidol, glycidal-dehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose-reponse curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20-50 times more potent in producing mutation that were the other 3 epoxides in the dose-response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Methods for the spiral Salmonella mutagenicity assay including specialized applications

An automated approach to bacterial mutagenicity testing - the spiral Salmonella assay - was developed to simplify testing and to reduce the labor and materials required to generate dose-responsive mutagenicity information. This document provides the reader with an overview of the spiral assay and a discussion of its application for examining the mutagenic potential of pure compounds, complex environmental mixtures, and interactive effects. Guidelines for performing a routine spiral assay are presented, and alternative test methods intended to overcome a variety of technical difficulties (such as restricted sample availability, sample viscosity or volatility, etc.) are recommended. Methods for the computerized analysis of data and the interpretation of results are discussed.     

Automated agar plate streaker: a linear plater on Society for Biomolecular Sciences standard plates

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.     

Mutagenic and alkylating activities of organophosphate impurities of commercial malathion

The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.     

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bj?rseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Glutathione activation of chloropicrin in the Salmonella mutagenicity test

Chloropicrin (CCl3NO2) is a major soil fumigant for control of fungi, insects and nematodes and may by formed by chlorination of drinking water. It is also a strong lacrimator and induces sister chromatid exchanges in cultured human lymphocytes. Mutagenicity assays of CCl3NO2 in Salmonella typhimurium TA100 establish that it is toxic but not mutagenic at 500 nmol/plate but becomes mutagenic but not toxic on addition of S9 (previous work) or 1-2 molar equivalents of glutathione (GSH) (this study). The preincubation assay is superior to the plate incorporation test giving 2-4-fold higher revertants/nmol. Using the preincubation assay with GSH at 5 mM (a biomimetic level) in the top agar gives linear dose-response relationships for CCl3NO2 and its dechlorination products CHCl2NO2 and CH2ClNO2 with 0.56, 0.56 and 1.8 revertants/nmol, respectively. The mutagenicity values for CHCl2NO2 and CH2ClNO2 are the same in the presence and the absence of GSH, which only improves the linearity at high levels by reducing toxicity to the bacteria. GSH activation of CCl3NO2 mutagenicity may be due to reductive dechlorination of the trichloro compound to the more active CHCl2NO2 and CH2ClNO2. Alternatively, the mutagenicity may result from an intermediate GSH conjugate such as GSCCl2NO2 or GSCHClNO2. In comparison, the mutagenicity of CH2Br2 and CH2I2 is affected little if any by addition of GSH and these dihalomethanes are much less active than the halonitromethane series. It therefore appears that CCl3NO2 is not mutagenic in the absence of activation and that the dechlorinated metabolites CHCl2NO2 and CH2ClNO2 are moderately potent bacterial mutagens, consistent with the possible genotoxicity of CCl3NO2 in mammals.     

Mutagenicity test of dyes used in cosmetics with the Salmonella/mammalian-microsome test.

37 dyes including 3 anthraquinone, 22 azo; 5 xanthene, 5 fluorandiol, and 2 thioindigo dyes, were tested for mutagenic potential with the Salmonella/mammalian-microsome test. Two frame-shift histidine mutants (TA1537 and TA98) and two base-pair substituted histidine mutants (TA1535 and TA100) of Salmonella typhimurium were employed. Both the spot test and the plate-incorporation assay indicated that one azo dye, D&C Orange No. 17, was mutagenic with three of the bacterial test strains. The mutagenic response of D&C Orange No. 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No; 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No. 17, beta-naphthol was not mutagenic but 2,4-dinitroaniline was mutagenic to the same Salmonella strains as D&C Orange No. 17 . Dimethyl sulfoxide extracts of lipsticks of similar formula but without D&C Orange No. 17 were tested in the plate incorporation assay. Only those containing D&C Orange No. 17 were mutagenic and the dye was mutagenic at concentrations consumed in normal daily use.     

Mutagenicity and DNA- modifying activity: A comparison of two microbial assays

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1^?) assay procedures for detecting environmental mutagens are discussed.The chief limitation of the pol A1^?1- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described.Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1^?1 system.For optimal testing, it is recommended that the two microbial assays be used in tandem.The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.     

Mutagenicity of aryl propylene and butylene oxides with salmonella

10 aryl propylene oxides and 6 aryl butylene oxides were synthesized. Dose-mutagenicity relationships were studied for these compounds and for 1,2-epoxybutane, using both the preincubation and plate incorporation Ames tests with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined by concurrent testing at single doses with the plate incorporation assay in strain TA100. In both series of compounds, mutagenicity showed very correlation to chemical reactivity, molar volume and partition values. However, all compounds were mutagenic in at least one system with the propylene oxides being more mutagenic than the corresponding butylene oxide derivatives. The naphthyl derivatives in each series were the most mutagenic.     

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.     

Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of nifurtimox (nfx) and 8 nfx analogues has been investigated with the L-arabinose forward-mutation assay of Salmonella typhimurium. The nfx analogues tested were obtained by replacing the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1); pyrazol-1-yl (2); benzimidazol-1-yl (3); 1,2,4-triazol-4-yl (4); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (5); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (6); 1-adamantyl (7); 4,6-diphenylpyridin-1-yl-2-one (8). The mutagenic activity of each chemical was determined by the standard plate-incorporation test, in the presence or absence of the S9 activation mixture. The 9 compounds were mutagenic and exhibited linear dose-mutagenic response relationships. They were direct-acting mutagens and showed a nearly 1000-fold range in mutagenic potency from chemical 1 to nfx. In most cases, the addition of S9 mixture to the test plates decreased the mutagenicity of compounds. This effect was particularly noticeable in the case of chemicals 1-3, 5 and 7 where a more than 70% decrease in mutagenic activity was observed in the presence of the S9 mixture. The mutagenic potency of compounds in the Ara test showed a negative linear correlation with previously reported antitrypanosomal activity. Thus, chemicals 6 and 8 with in vitro activities against Trypanosoma cruzi clearly superior to that of nfx showed 2 of the lowest mutagenic potencies in the Ara test and these were only somewhat higher than the mutagenicity of the reference drug.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.     

Mutagenicity of 7H-dibenzo[c,g]carbazole and metabolites in Salmonella typhimurium

7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen of environmental import. Reverse-mutation plate-incorporation assays for mutagenicity were undertaken in Salmonella typhimurium strains TA98 and TA100. Results were negative when no exogenous activation system was used, as well as when assays incorporated liver homogenates (S9) from rats, mice and rabbits. By contrast DBC was mutagenic in a forward mutation assay in Salmonella strain TM677 using resistance to 8-azaguanine for selection. Metabolites of DBC were generated by incubation with rat-liver microsomes and separated by HPLC. Two of these metabolites were directly mutagenic for Salmonella strain TM 677 while two others were mutagenic upon addition of S9. Synthetic phenolic derivatives of DBC were also mutagenic in this assay when further metabolized. It is likely that metabolites of DBC phenols constitute the biologically active forms.