Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay)

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.     

A study of the mutagenicity of anesthetics and their metabolites.

The commonly used volatile anesthetics, several of their metabolites, and drugs frequently employed by the anesthesiologist were screened for mutagenicity in the Salmonella/rat-liver microsomal assay system developed by Dr. B. Ames and his colleagues. Chloral hydrate, both a sedative and metabolite of trichloroethylene, was found to be weakly mutagenic. Other compounds testing including halothane, isoflurane, methoxyflurane, diazepam and chlordiazepoxide were not mutagenic. Non-volatile compounds were tested for their ability to inhibit growth of bacterial strains with decreased capacity to repair damaged DNA. None of the compounds tested inhibited the growth of DNA-repair-deficient strains relative to a strain with normal DNA-repair. Halothane and trilene were tested for direct interaction with DNA; under the experimental conditions employed, no direct interaction of these compounds and DNA could be detected.     

Prophage induction and mutagenicity of a series of anti-tumour platinum(II) and platinum(IV) co-ordination complexes

11 platinum compounds with nitrogen donor ligands, previously tested for anti-tumour activity, were studied for induction of prophage lambda and for mutagenicity in the Ames assay, with various strains of Salmonella. The compounds included cis and trans isomers of Pt(II) and Pt(IV) complexes and were tested with and without metabolic activation. All the cis compounds elicited prophage induction, whereas the trans compounds were inactive. Mutagenicity was found only in strains containing the R factor, indicating that SOS-type repair processes are required for the conversion of initial DNA lesions into mutations. Mutation induction was also influenced by the excision-repair process. The 2 trans compounds were not, or only slightly, mutagenic; all other compounds were mutagenic in at least one strain, exhibiting a 2-20-fold increase over the spontaneous background level. Addition of liver homogenate had no significant effect on the number of mutants. One compound induced exclusively frameshift mutations. The other mutagenic compounds induced frameshift mutations as well as base-pair substitutions. 7 compounds were more mutagenic for the repair-proficient than for the repair-deficient strains; only one showed the opposite effect. This suggests that for mutagenicity testing of platinum compounds, repair-proficient strains are more sensitive indicators. The differences in response of the various strains are more sensitive indicators. The differences in response of the various strains toward the compounds suggest the formation of different DNA lesions and/or a selective action of repair processes on these lesions. In general, a good qualitative correlation was observed between prophage-inducing capacity, mutagenicity in bacterial and mammalian cells and anti-tumour activity.     

The genotoxicities of N-nitrosamines in Drosophila melanogaster in vivo: the correlation of mutagenicity in the wing spot test with the DNA damages detected by the DNA-repair test

The genotoxicities of a series of N-nitrosamines were assayed in the wing spot test and a new short-term test of Drosophila melanogaster. In the spot test, larval flies trans-heterozygous for the somatic cell markers mwh and flr3 were fed the test reagents and the wing hairs in adults were inspected for clones expressing the phenotypes of the markers. In the other test, larval stock consisting of meiotic recombination-deficient (Rec-) double mutant mei-9a and mei-41D5 males and repair-proficient Rec+ females were grown on feed containing the reagents and the DNA damages were detected with the preferential killing of the Rec- larvae as an endpoint. The carcinogenic nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-butylamine (NDBA), N-nitrosomorpholine (NMOR), N-nitro-sopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR), all showed clearly positive activities in both tests. The activities in the wing spot test were ranked in a sequence of NDMA much greater than NMOR greater than NPIP greater than NDEA greater than NPYR greater than NDBA. A similar ranking was obtained in the repair assay. The genotoxicity of N-nitrosodiphenylamine (NDPhA), carcinogenicity studies of which are inconclusive, was marginal in the spot test. The non-carcinogenic N-nitrosoproline (NPRO) and the non-mutagenic N-nitrosothioproline (NTPRO) were negative in the spot test. NDPhA and NPRO were negative in the repair test as well. The DNA-repair test is thus a convenient technique for estimating the mutagenicity of compounds because of its simplicity compared with the wing spot test. These Drosophila tests may be useful in predicting carcinogenic potentials of compounds.     

An evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity. A report of the U.S. EPA's Gene-TOX Program

The detection of DNA-damaging agents by repair-deficient bacterial assays is based on the differential inhibition of growth of repair-proficient and repair-deficient bacterial pairs. The various methodologies used are described and recommendations are made for their improved use. In a survey of the literature through April 1979, 91 of 276 papers evaluated contained usable data, resulting in an analysis of 611 compounds that had been assayed in 1 or more of 55 pairs of repair-proficient and repair-deficient strains. The results indicate that (1) a liquid suspension assay is more sensitive than a spot (diffusion) test. In a review of the Escherichia coli polA assay, 45 compounds that gave "No Test" in the spot test were clearly positive or negative in the liquid suspension assay. (2) Of the 21 compounds analyzed by the E. coli polA assay and by other E. coli repair-deficient strains (e.g., rec, uvr, hcr, and exr derivatives of WP2 and AB1157), 10 were in complete agreement in all strains except uvrA strains. This indicates that strains other than polA+/polA- are useful for detecting DNA-damaging agents. However, in selecting strains for use in these assays, care should be taken to consider repair pathway specificity for particular compounds. (3) There was a 78% correspondence between results obtained with E. coli polA and Bacillus subtilis (H17/M45, 17A/45T) rec assay and between E. coli polA and Proteus mirabilis. (4) In a comparison of test results with carcinogenicity data, 44 of 71 (62%) carcinogenic compounds assayed by the polA system were positive, 10 (14%) were negative, and 17 (24%) gave No Test or doubtful results. 7 carcinogens were assayed by other E. coli strains and all were positive. 56 carcinogens were assayed in B. subtilis: 24 (43%) were positive, 9 (16%) were negative, and 23 (41%) gave No Test or doubtful results. Of the 7 carcinogens assayed in P. mirabilis, 6 (86%) were positive and 1 (14%) was negative. (5) The results were analyzed with respect to chemical classes. E. coli polA detected the highest percentage of hydroxylamines and alkyl epoxides. The B. subtilis rec assay detected the highest percentage of nitrosamines and sulfur and nitrogen oxides. It is concluded that some of these test systems are effective tools for the detection of DNA-damaging and potentially carcinogenic compounds, especially if the assay is done in liquid suspension and if more than 1 pair of tester strains is used. Advantages and disadvantages of the assay are discussed and suggestions are made for improvements in the system.     

Mutagenicity, comutagenicity, and antimutagenicity of erythrosine (FD and C red 3), a food dye, in the Ames/Salmonella assay

Erythrosine (diNa, tetraiodofluorescein) was nonmutagenic to the Ames/Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104, to a concentration of 2 mg/plate. No mutative intermediates were detected on metabolism by rat caecal cell-free extracts or rat liver S9 mixture; or on incubation with the comutagens, harman and norharman (+/- S9). Instead, an unexpected dose-dependent suppression in spontaneous reversion frequencies was observed (maximum approximately equal to 35% decrease). Erythrosine was antimutagenic to benzo[a]pyrene, but it did not decrease the mutagenicity of the other adduct-forming mutagen, 4-nitroquinoline N-oxide. The food dye was strongly antimutagenic to the bifunctional alkylating agent, mitomycin C, though it did not exhibit a similar effect on the mutagenicity of the corresponding monofunctional agent, methyl methanesulphonate. It partially depressed the mutagenic potentials of sodium azide. The antimutagenic effect of erythrosine on an intercalating agent, ethidium bromide, was discernible only at the highest dose (2 mg/plate). These results have been interpreted in terms of a genointeractive role of erythrosine. Erythrosine produced differential toxic effects in repair-deficient (TA97a, TA98, TA100) and repair-proficient (TA102, TA104) Salmonella tester strains; survival of the repair-deficient strains was found to be decreased. Photoinduced potentiation of erythrosine toxicity was observed, although light irradiation in the presence of erythrosine did not modify the reversion frequencies of the tester strains. The evidence strongly suggests that erythrosine, which exhibits nonmutagenicity in the Ames/Salmonella test, can interact with DNA repair enzymes and/or with DNA.     

Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test

Compounds of various chemical classes were comparatively assayed in the Ames reversion test with his- S. typhimurium strains TA1535, TA157 , TA1538, TA98, TA100, and, in part, TA97 , and in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA- polA-) and CM871 (uvrA- recA- lexA-). A liquid micromethod procedure for the assessment of the minimal inhibitory concentration (MIC) of test compounds, using the same reagents as the Ames test, was set up and calibrated in its technical details. Other techniques (spot test and treat-and-plate method) were applied to a number of compounds in order to obtain more complete information on their DNA-damaging activity in E. coli. From a qualitative standpoint, the results obtained in the reversion test and in the DNA-repair test (liquid micromethod) were overlapping for 96 (59 positive and 37 negative) out of 135 compounds (71.1%). There was disagreement for 39 compounds (28.9%), 9 of which were positive only in the reversion test (8 requiring metabolic activation and 5 genotoxic in the treat-and-plate method). 30 compounds were positive only in the lethality test, showing a direct DNA-damaging activity, which in half of the cases was completely eliminated by S9 mix. Although the experimental protocol intentionally included several compounds already reported as nonmutagenic carcinogens or as noncarcinogenic mutagens, the overall accuracy was 64.5% for the reversion test and 72.4% for the DNA-repair test, as evaluated for 75 compounds classified according to their carcinogenic activity. Quantitation of results was obtained in the Ames test by relating the net number of revertants to nmoles of compound and in the DNA-repair test by means of a formula relating the difference and ratio of MICs in repair-proficient and -deficient bacteria to nmoles of compound. Following these criteria, the genotoxic potency varied over a 4.5 X 10(7)-fold range among compounds positive in the reversion test and over a 6 X 10(9)-fold range among compounds damaging E. coli DNA. The genotoxic potencies in the two bacterial systems were correlated within the majority of the chemical classes under scrutiny.     

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.     

Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.     

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.     

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.     

Mutagenicity of 43 structurally related heterocyclic compounds and its relationship to their carcinogenicity.

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.     

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.     

Direct-acting mutagenic properties of some hair dyes used in New Zealand

Mutagenicity or carcinogenicity data are not publicly available on many hair dyes or dye components commonly used within New Zealand. Representative mid- to dark-warm brown hair dyes of 12 brands supplying more than 1% of the New Zealand market were tested for direct-acting mutagenicity using the bacterial 'Ames' test. Despite recent scientific advances in the development of non-mutagenic dyes, 23 of the 40 products tested gave positive results in one or both of the tester strains used. There appeared to be differences between distributors in the proportion of their hair dyes which were mutagenic. In the case of 6 out of 10 of the above dyes which had tested positive, in vitro mutagenicity or toxicity was enhanced in the presence of verapamil, suggesting that risks from hair-dye exposure may change in the case of persons using this or similar drugs. It is recognised that there are uncertainties regarding human risks from dyes which are shown to be mutagenic in in vitro tests. However, from the above results, it seems possible to produce non-mutagenic hair dyes in this color range; and in the interests of public reassurance, it may be prudent to require that such dyes be used.     

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Induction of repairable DNA damage in Escherichia coli and interaction with DNA in vitro by the radical cation of chlorpromazine

Studies were performed to determine the DNA interactions of and the induction of cytotoxic effects by the radical cation (CPZ+.) formed enzymatically from chlorpromazine (CPZ): in the presence of native DNA the lifetime of CPZ+. is markedly increased. The decreased reactivity of CPZ+. in the presence of native DNA and the concomitant increased viscosity of CPZ+.-DNA complexes strongly support the assumption that CPZ+. does form intercalation complexes with DNA. The relative strong bacteriotoxicity of CPZ+. hindered the accurate determination of mutagenesis in various Salmonella indicator strains, but a test for repairable DNA damage in Escherichia coli using various repair-deficient strains indicated that the cytotoxic action of CPZ+. is in part due to DNA alterations which can be excised in wild-type DNA repair-proficient strains. After activation of CPZ with long wavelength UV light, genetic effects are observed in S. typhimurium strain TA98, as well as in the E. coli tester strains. The possible role of CPZ+. in the photosensitization of CPZ is discussed.     

Assessment of the genotoxicity of the "Binase" enzyme preparation]

"Binase" enzyme sample (a microbial ribonuclease) has been tested for mutagenicity in a set of tests. The set included Ames test Salmonella/microsome, Escherichia coli Rec-test, bacteriophage induction assay, DNA-repair synthesis in lymphoid cells. "Binase" is shown to possess a small genotoxic effect at high concentrations. Both animal and plant S-9 fractions eliminated the effect.     

Mutagenicity tests on anthelmintics: microsomal activation of viprynium embonate to a mutagen.

Eight anthelmintic preparations readily available in Australia were tested for mutagenicity in the Salmonella typhimurium test system. A slightly modified version of the procedure recommended by Ames et al. [2] was adopted, in that the test samples were placed in "wells" cut out of the agar of a plate previously seeded with the appropriate tester strain. Addition of a mixture of rat liver microsomal enzymes and appropriate co-factors ("S-9 mix") to one of the two wells on a single plate allowed a possible requirement for metabolic activation to be recognised. Using this procedure, viprynium embonate was found to be non-mutagenic. It was however, activated by the rat liver microsome preparation to a mutagen capable of causing both base-pair substitution (detected with strain TA100) and frameshift (detected with strain TA98) mutations. The other seven compounds tested all gave negative results in this system.     

Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.