The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.     

Comparative mutagenicity of aliphatic epoxides in Salmonella

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.     

Detoxication of aliphatic epoxides by diol formation and glutathione conjugation

The Ames procedure with Salmonella typhimurium strain TA100 was used to follow the detoxication by rat liver fractions of two series of aliphatic epoxides. The epoxides employed were 3-chloro-, 3,3-dichloro- and 3,3,3-trichloropropylene oxides and also p-methoxyphenyl-, phenyl- and p-nitrophenylglycidyl ethers. In our procedure with preincubation of the epoxides with rat liver fractions prior to the Ames tests, there was more detoxication of both systems by glutathione conjugation (non-enzymatic and transferase promoted) than by the hydrolase pathways. Non-enzymatic reaction with glutathione was more pronounced for the chloro series than for the glycidyl ethers. An HPLC system was developed which was capable of quantitative measurements of the phenylglycidyl ethers together with their diol and glutathione conjugate products. A comparison of the HPLC and Ames test results indicates that the glutathione transferase reported to be present in Salmonella could be playing a role in detoxication by the Ames test. Diols were measured more readily by HPLC than by use of the Ames test in the microsomal fraction and were detected in the cytosol with the glycidyl ethers while they were not by the Ames procedure. However, all three epoxides were converted to a greater extent to their glutathione conjugates than to their diols. Thus, while literature references question the availability of the glutathione detoxication system for epoxides produced by membrane-bound enzymes, such detoxication would be of primary importance where direct-acting environmental epoxides come into contact with the cytosolic enzymes prior to possible reaction with bionucleophiles.     

Mutagenicity of aromatic glycidyl ethers with Salmonella

6 aromatic glycidyl ethers containing naphthyl, biphenyl or benzylphenyl substituents were synthesized. These epoxides together with the commercially available compounds 2-biphenylyl glycidyl ether were examined for dose-mutagenicity relationships using the plate incorporation Ames test with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined for these compounds and 3 phenyl glycidyl ethers by concurrent testing at a single dose with strain TA100. Meaningful correlations could not be established for the mutagenicity of these epoxides to their molecular volumes, partition values, nor to their reactivities with the model nucleophile, 4-(4-nitrobenzyl) pyridine. However, it was noted that increased conjugated aromatic unsaturation with its resulting planarity led to increased mutagenicity and that this effect decreased when it was further removed from the epoxide moiety.     

Specific DNA adducts induced by some mono-substituted epoxides in vitro and in vivo

Alkyl epoxides are important intermediates in the chemical industry. They are also formed in vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in many toxicology assays which has been associated with their covalent binding to DNA. Here aspects of the formation and properties of DNA adducts, induced by some industrially important alkenes and mono-substituted epoxides are discussed. These include propylene oxide, epichlorohydrin, allyl glycidyl ether and the epoxy metabolites of styrene and butadiene. The major DNA adducts formed by epoxides are 7-substituted guanines, 1- and 3-substituted adenines and 3-substituted cytosines. In addition, styrene oxide and butadiene monoepoxide are able to modify exocyclic sites in the DNA bases, the sites being in the case of styrene oxide N(2)- and O(6)-positions of guanine, N(6)-adenine as well as N(4)-and O(2)-cytosine. In vivo the main adduct is the 7-substituted guanines. The 1-substituted adenines have also shown marked levels, and these adducts should also be targets in biomonitoring of human exposures. Due to its low mutagenicity, 7-substituted guanines are considered as a surrogate marker for other mutagenic lesions, e.g. those of 1-adenine or 3-uracil adducts.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate structure-activity relationships of epoxides in mammalian cells. For this purpose the SCE-inducing potency of 58 epoxides was determined. Of these, 16 failed to induce SCE in V79 cells. According to the substitution of the oxirane ring the results show general agreement with results obtained in the Ames test. Mono-substituted epoxides had the highest genotoxic potency compared to di- and tri-substituted epoxides. In detail, there are differences in genotoxic potency between bacteria and mammalian cells which can be explained by differences in the cellular uptake of the compounds and by detoxification reactions.     

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.     

In vitro genotoxic evaluation of conventional bleached and biobleached softwood pulp mill effluents

The effluents of pulp and paper mills contain about 300 different chemical compounds; many of them are mutagens and clastogens. Genotoxic studies have shown that chlorination stage liquors are significantly more genotoxic, in the Ames Salmonella assay, than the other process of lignin extraction, and that lyophilized effluents are genotoxic in cultured mammalian cells. Since these effluents from conventional bleaching stages are genotoxic, Chilean industries are interested in changing this process to a less toxic one, such as biobleaching using enzymes. In this study, we tested the in vitro genotoxicity of two types of effluents: an effluent obtained from a conventional radiata pine kraft-bleaching process (effluent D) and one derived from a biobleaching process with hemicellulase (effluent B). Both effluents were tested without any concentration or purification steps in the Ames Salmonella assay (TA100) and in the micronuclei (MN) and sister chromatid exchange (SCE) tests in CHO cells. The results showed that neither effluent induced base pair substitution mutations in the Ames Salmonella assay, and neither increased the micronucleus frequency in CHO cells. But, both increased the SCE frequencies in CHO cells, showing that this assay is more sensitive than the other ones, and that the two effluents contained chemical compounds in amounts enough to induce in vitro genotoxicity measured by the SCE induction.     

Sister-chromatid exchange and chromosome aberrations induced by paracetamol in vivo in bone-marrow cells of mice.

Sister-chromatid exchange (SCE) and chromosome aberrations (CA) induced by paracetamol (PC), a common analgesic, were studied in vivo on bone-marrow cells of mice. The trend tests for the evidence of dose-response effects for both SCE and CA were significant. The significant increase in SCE as well as CA induced by PC may be attributed to the fact that PC can induce genotoxicity through DNA damage. Thus, the present study indicates that PC was genotoxic in vivo in bone-marrow cells of mice.     

Biodegradation of Glycidol and Glycidyl Nitrate

When calcium hydroxide is used to desensitize glycerol trinitrate (nitroglycerine)-containing waste streams, the epoxides glycidol and glycidyl nitrate are formed. The epoxide rings of both compounds are unstable to heat in aqueous solutions, and they open to form glycerol 1-mononitrate and presumably glycerol. These transformations were accelerated by microbial activity. Glycerol 1-mononitrate was slowly denitrated to form glycerol. Glycidol and glycidyl nitrate caused base-pair substitutions in the Ames test for mutagenicity, whereas glycerol 1-mononitrate tests were negative.     

Chromosomal aberrations in mouse lymphocytes exposed in vitro and in vivo to benzidine and 5 related aromatic amines

Mouse lymphocytes were exposed in vitro for 2 h or in vivo for 24 h to benzidine and related aromatic amines to test for chromosome aberrations (CA) and mitotic indices. Uninduced mouse S9 was used to activate the amines for the in vitro tests to be consistent with the in vivo tests. Contrary to a previous report, no difference could be established in the genotoxicity of benzidine following activation with uninduced S9 compared to induced S9. There were concentration related increases in CA for benzidine and all the amines in vitro except for 4,4′-diaminostilbene which exhibited the greatest cellular toxicity towards cultured lymphocytes. Benzidine and its derivatives showed significant increases in CA in vivo compared to its negative control. The CA values for 4-aminostilbene were significantly higher than the other amines in both in vivo and in vitro studies. These genotoxicity results for 4-aminostilbene are consistent with our previous report of the pronounced CA effects in murine bone-marrow cells but would not be predicted from Salmonella mutagenicity tests.     

Mutagenicities of glycidyl ethers for Salmonella typhimurium: relationship between mutagenic potencies and chemical reactivity

The lethal and mutagenic effects of ethyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-naphthylethyl, 2-naphthylethyl and 9-anthrylmethyl glycidyl ethers on Salmonella typhimurium (TA100, TA1535, TA98 and TA1538) were investigated. LD30-value became smaller with an increase in compound hydrophobicity. The mutagenicities of these compounds in TA100 increased in the order: 1-naphthylethyl glycidyl ether less than 2-naphthylethyl glycidyl ether less than benzyl glycidyl ether less than 2-naphthylmethyl glycidyl ether less than 1-naphthylmethyl glycidyl ether less than 9-anthrylmethyl glycidyl ether. 1-Naphthylmethyl and 2-naphthylmethyl glycidyl ethers were mutagenic toward TA1535. In TA98, 1-naphthylmethyl and 9-anthrylmethyl glycidyl ethers showed mutagenic activity and 9-anthrylmethyl glycidyl ether was more mutagenic than 1-naphthylmethyl glycidyl ether. 9-Anthrylmethyl glycidyl ether was also active in TA1538. In the reaction of glycidyl ethers with deoxyguanosine and related compounds, glycidyl ethers attacked at only N-7 of guanine. The alkylation rates of glycidyl ethers toward guanine residues in DNA were determined and the exciplex-formation ability of 7-substituted guanines was studied. The reactivity of glycidyl ethers with guanine residues in DNA has not provided a sufficient explanation for the variation in mutagenic potencies of glycidyl ethers.     

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine.

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and pi orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenicities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.     

Cloning of an epoxide hydrolase-encoding gene from Aspergillus niger M200, overexpression in E. coli, and modification of activity and enantioselectivity of the enzyme by protein engineering

The gene encoding an epoxide hydrolase from Aspergillus niger M200 has been cloned and its sequence determined. The gene is interrupted by seven introns, one exon being only nine nucleotides long. The non-coding 5'- and 3'-regions of the mRNA are composed of 47 and 76 nucleotides, respectively. Overexpression of the fungal epoxide hydrolase in E. coli TOP10 has led to a 15-fold increase in specific activity (compared to the wild-type strain). Saturation mutagenesis at codon 217 resulted in the discovery of nine enzyme variants showing in several cases profound differences in activity and enantioselectivity towards various epoxides when compared to the data of the wild-type enzyme. The site 217 is located at the entrance of the tunnel that provides the substrate with access to the active site. The exchange of Ala at this position for Cys has led to a doubled enantioselectivity (E-value of 5.0) towards benzyl glycidyl ether. The same substitution resulted in a threefold-enhanced activity of the enzyme towards allyl glycidyl ether and styrene oxide without affecting enantioselectivity. The variant A217L showed an enhanced enantioselectivity towards tert-butyl glycidyl ether reaching an E-value of 100 (from 60 for the wild-type enzyme). Replacement of A217 by Val has led to higher activity towards allyl glycidyl ether by a factor of six. The substitutions Ala-->Glu and Ala-->Gln increased the enantioselectivity towards allyl glycidyl ether and styrene oxide by over 50% to E-values of 10 and 16, respectively. The study underlines that single amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity of the epoxide hydrolase from A. niger M200.     

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds.

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

Genotoxicity of aerosol extracts. Some methodological aspects and the contribution of urban and industrial locations

The genotoxicity of aerosol extracts was investigated with the Ames test and an SCE test in vitro. In addition to experiments to evaluate current techniques, a so-called gradient study was carried out in an industrialized part of The Netherlands to obtain insight into the contribution of urban and industrial locations to the genotoxicity of aerosol extracts. The influence of variations in the sampling was investigated. The results indicate that moderate variations in the volume of air sampled per unit of time and of the duration of the sampling period do not have a great influence on the effect measured. Experiments with filters impregnated with 14C-labelled benzo[a]pyrene showed that this compound is converted during sampling into directly active mutagens; no evaporation occurred. The results of experiments to evaluate the extraction procedure indicated that an 8-h Soxhlet extraction with methanol is better than, or as good as, Soxhlet extraction with other solvents. The gradient study showed that the aerosol downwind from industrial/urban areas exerted a reproducibly stronger effect in the Ames test and the SCE test when compared with aerosol from sea air.     

Genotoxicity of aerosol extracts: Some methodological aspects and the contribution of urban and industrial locations

The genotoxicity of aerosol extracts was investigated with the Ames test and an SCE test in vitro. In addition to experiments to evaluate current techniques, a so-called gradient study was carried out in an industrialized part of The Netherlands to obtain insight into the contribution of urban and industrial locations to the genotoxicity of aerosol extracts. The influence of variations in the sampling was investigated. The results indicate that moderate variations in the volume of air sampled per unit of time and of the duration of the sampling period do not have a great influence on the effect measured.Experiments with filters impregnated with ¹⁴C-labelled benzo[a]pyrene showed that this compound is converted during sampling into directly active mutagens; no evaporation occurred. The results of experiments to evaluate the extraction procedure indicated that an 8-h Soxhlet extraction with methanol is better than, or as good as, Soxhlet extraction with other solvents.The gradient study showed that the aerosol downwind from industrial/urban areas exerted a reproducibly stronger effect in the Ames test and the SCE test when compared with aerosol from sea air.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.