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Quantitative metabolism of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) by liver microsomes of uninduced mice and rats was studied. Both enzymatic systems metabolize 7-Cl-BA preferentially at the C-8 and C-9 aromatic double bond region, approximately 42 and approximately 56% respectively, of the total metabolites. 7-Cl-BA and 7-Br-BA were metabolized considerably at C-3 and C-4, C-5 and C-6, C-8 and C-9, and C-10 and C-11. While 7-Cl-BA trans-3,4-dihydrodiol was formed in a 7-8% yield of the total metabolites in both enzymatic systems, 7-Br-BA trans-3,4-dihydrodiol was formed 16.0 and 9.9% respectively, from the mouse and rat liver microsomal metabolism. In mutagenicity assays with the Salmonella typhimurium tester strain TA100 in the presence of S9 activation enzymes, both of these trans-3,4-dihydrodiols exhibited higher mutagenicity than 7-Cl-BA and 7-Br-BA, while the other trans-dihydrodiol metabolites were either essentially inactive or weaker than the parent compounds. These results suggest that 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol are the proximate metabolites of 7-Cl-BA and 7-Br-BA. Metabolism of 7-Cl-BA and 7-Br-BA by mouse liver microsomes was also in a stereoselective manner, preferentially giving trans-dihydrodiol metabolites an R, R stereochemistry.

2-Nitropyrene, the geometric isomer of the most studied nitropolycyclic aromatic hydrocarbon (nitro-PAH), 1-nitropyrene, is an environmental contaminant detected in ambient air and a potent direct-acting mutagen. Its metabolic activation leading to the formation of DNA adducts was studied. The activated metabolite, N-hydroxy-2-aminopyrene, was prepared and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA, the resulting nucleosides were separated by HPLC, and the adducts were characterized by mass and proton NMR spectral analysis. Both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene, in a 5:2 ratio, were identified. These adducts were then utilized as standards to identify the DNA adducts formed from reaction of [3H]2-nitropyrene with DNA mediated by liver microsomes and cytosols of mouse and rat. In all cases, both adducts were formed. The quantities of the two adducts formed in each system were: mouse liver microsomes (11.3 pmol [3H]2-nitropyrene/mg DNA), rat liver microsomes (23), mouse liver cytosol (11.4) and rat liver cytosol (5.1). Thus, these adducts were formed in highest yield from rat liver microsomes and the lowest from rat liver cytosol. The deoxyguanosine/deoxyadenosine adduct ratio was higher from rat and mouse liver microsomes (7.8:9.2) than from rat and mouse liver cytosols (2.5:3.1). Our results represent the first direct demonstration of a C8-deoxyadenosine adduct being formed as a major product from the reaction of a nitro-PAH metabolite with DNA.

The products formed in the metabolism of 3-methylcholanthrylene (3MCE), either in the presence or in the absence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), with an NADPH-regenerating system and liver microsomes from 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats were separated by reversed-phase and normal-phase HPLC. The metabolites were characterized by UV-visible absorption spectral analysis, and by comparing their retention times on reversed-phase and normal-phase HPLC with authentic 3MC derivatives whenever available. In addition to 3MC trans-1,2-diol, 3MC-1-one, and 3MC-2-one reported earlier by other investigators, 3-hydroxymethylcholanthrylene (3-OHMCE), 3-OHMCE trans-11,12-dihydrodiol, 3MCE trans-11,12-dihydrodiol, 3MCE trans-9, 10-dihydrodiol. 9- and 10-hydroxy-3MCE. 3MC-2-one trans-9,10-dihydrodiol, and a chemically unstable 3MCE 1,2-epoxide were identified as metabolites of 3MCE. 3MC cis-1,2-diol, a previously reported metabolite of 3MCE, was not detectable. In the presence of TCPO, metabolites that have been identified include 3-OHMCE, 3-OHMCE 11,12-epoxide. 3MCE 11,12-epoxide, 3MC-2-one, 3MC-1-one, 9-hydroxy-3MCE, 10-hydroxy-3MCE, and an unstable metabolic intermediate 3MCE 1,2-epoxide. The results suggest that 3MCE 1,2-epoxide, 3MCE 9,10-diol-7,8-epoxide, and 3MC-2-one 9,10-diol-7,8-epoxide may be involved in the metabolic activation of 3MCE to carcinogenic form.

A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N(2)-yl)-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, approximately 1.5 adducts/10(9) nucleotides using 20 micro g DNA) and a high signal-to-noise ratio (> or =100). The CIA BPDE-DNA standard curve gave 50% inhibition at 0.60 +/- 0.08 fmol BPdG (mean +/- SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-(3)H]BPDE was assayed by radiolabeling, (32)P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-(3)H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose-response was obtained with all assays. The BPDE-DNA CIA was further validated in MCL-5 cells exposed to 4 micro M BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE-DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH-DNA adducts/10(8) nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH-DNA adduct values obtained were highly correlated (r(2) = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterial mutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P, prepared in situ from reduction of 3-nitro-B[a]P with calf thymus DNA, was studied. After enzymatic digestion of the DNA, the resulting modified nucleosides were analyzed by thermospray HPLC-MS and high-resolution proton NMR spectroscopy. The major adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubation of DNA with 3-nitro-B[a]P in the presence of the mammalian nitroreductase xanthine oxidase, and hypoxanthine. These data indicate that a mammalian nitroreductase can metabolize 3-nitro-B[a]P to an activated derivative that reacts with DNA to give a novel adduct distant from the site of N-hydroxylation.

Caloric restriction (CR) inhibited strongly the incidence of chemically-induced tumors in the neonatal B6C3F1 mouse tumorigenicity bioassay, when begun 3 months after treatment with the potent carcinogen 6-nitrochrysene. These data indicate that CR can have a profound inhibitory effect on tumor development even long after metabolic activation and DNA repair have occurred.

In contrast to 1-nitropyrene (1-NP), which is the most abundant nitropolycyclic aromatic hydrocarbon in numerous environmental sources, 2-nitropyrene (2-NP) has been detected only in the ambient air and not in direct emissions. Thus, 2-NP can be used as an indicator for monitoring human exposure to nitropolynuclear aromatic hydrocarbons in ambient air. Therefore, it is essential to determine the possible metabolic pathways of 2-NP. The metabolism of 2-NP by rat liver 9000 g supernatant was investigated. Under aerobic conditions, ring oxidation to 6-hydroxy-2-nitropyrene and nitroreduction to 2-aminopyrene (2-AP) were observed. When incubations were carried out in an atmosphere of nitrogen, 2-AP was the only metabolite detected. These results are consistent with those observed with 1-NP. In vitro metabolic activation of 2-NP to DNA adducts catalyzed by xanthine oxidase was also examined. Two adducts were characterized as N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene. The presence of deoxyadenosine adduct, which is derived from the nitroreduction pathway, may contribute to the powerful direct-acting mutagenicity of 2-NP.

Stereoselective metabolism of 9-methylanthracene (9-M-A), 9-hydroxymethylanthracene (9-OHM-A) and 9,10-dimethylanthracene (9,10-DM-A) by liver microsomes from untreated rats, and from rats pretreated with either 3-methylcholanthrene or phenobarbital was studied. The metabolites were separated by h.p.l.c. and characterized by analysis of the u.v.-visible, mass and n.m.r. spectral data and compared to published spectral data. The identified trans-dihydrodiol metabolites were 9-M-A trans-1,2- and 3,4-dihydrodiols, 9-OHM-A trans-1,2- and 3,4-dihydrodiols, and 9,10-DM-A trans-1,2-dihydrodiol. The absolute configuration and optical (enantiomeric) purity of the trans-dihydrodiol metabolites were determined. In order to assist in determining the absolute configuration of these trans-dihydrodiols, metabolism of 9-bromoanthracene by liver microsomes of rats pretreated with 3-methylcholanthrene was performed and its trans-1,2- and 3,4-dihydrodiol metabolites were determined to both possess an R,R absolute configuration. Absolute configurations of the trans-dihydrodiol metabolites of 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene were then determined by comparison of their CD spectra with those of anthracene 1R,2R-dihydrodiol or 9-bromoanthracene 1R,2R-dihydrodiol. The optical purities of the trans-dihydrodiol metabolites were determined by analysis of the chiral stationary phase h.p.l.c. profiles of the dihydrodiols or their corresponding tetrahydrodiol derivatives. The results indicated that the R,R enantiomers were the predominant products and that liver microsomes of rats pretreated with 3-methylcholanthrene exhibited much higher stereoselectivity than the liver microsomes of untreated rats or rats pretreated with phenobarbital in the formation of trans-dihydrodiols from anthracene, 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene. The methyl and hydroxymethyl substituents slightly decreased the stereoselectivity of the trans-dihydrodiol formation.

Rat liver microsomal metabolism of the potent mutagen 3-nitrobenzo[a]pyrene (3-nitro-BaP) under aerobic conditions yielded 3-nitro-BaP trans-7,8-dihydrodiol and 3-nitro-BaP trans-9,10-dihydrodiol, and under anaerobic conditions produced 3-amino-BaP. All of these metabolites were highly mutagenic in the Salmonella typhimurium reversion assay both with and without exogenous metabolic activation (S9). None of the in vitro metabolic pathways led to detoxification of 3-nitro-BaP.

1-,3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypes of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derived from a carcinogenic parent PAH, benzo[a]pyrene, are environmental contaminants and potent bacterial mutagens. In this study, the aerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomes was studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol, while metabolism under hypoxic conditions yielded 1-amino-BaP. The metabolites formed from aerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9,10-dihydrodiol by liver microsomes of untreated rats and rats pretreated with 3-methylcholanthrene and phenobarbital were quantified. Comparison of these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-position of BaP markedly affects the regioselectivity of the P-450-containing enzymes. 1-Nitro-BaP and the three metabolites were potent mutagens in Salmonella typhimurium TA98, both in the absence and in the presence of an exogenous metabolic activation system (S9). The direct and S9-mediated mutagenicities of 1-nitro-BaP and the two dihydrodiols were decreased in the nitroreductase-deficient strain TA98NR, while TA98/1,8-DNP6, an O-acetylase-deficient strain, was less sensitive to the two dihydrodiols, both with and without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equally mutagenic in all three tester strains. These observations indicate that: the metabolism of 1-nitro-BaP involves several pathways leading to mutagenic activation; the major activation pathways of 1-nitro-BaP involve nitroreduction; nitroreduction followed by O-acetylation is the major activation pathway of 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol; and 1-amino-BaP is a potent direct-acting mutagen.

The optically pure (-) and (+)trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracenes (BA trans-1,2-dihydrodiol) were obtained through the resolution of their diastereomeric di(-)menthoxyacetates by normal-phase h.p.l.c., followed with base-catalyzed hydrolysis. The (-)-BA trans-1,2-dihydrobiol has been determined to have 1R,2R absolute stereochemistry by exciton chirality method. Each of the enantiomeric and racemic BA trans-1,2-dihydrodiol was incubated with liver microsomes from untreated, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats. The racemic and enantiomeric BA trans-1,2-dihydrodiols were each metabolized to two 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydrobenz[a]anthracenes (BA 1,2,3,4-tetrol) derived from the hydrolysis of BA trans-1,2-dihydrodiol anti-3,4-epoxide (the 3,4-epoxy oxygen is trans to the 1-hydroxyl group) and syn-3,4-epoxide (the 3,4-epoxy oxygen is cis to the 1-hydroxyl group), respectively. All the BA 1,2,3,4-tetrols were identified by comparing the reversed-phase h.p.l.c. retention times of tetrols and their vicinal acetonides with the hydrolysis products of the chemically synthesized BA trans-1,2-dihydrodiol anti-3,4-epoxide, BA trans-1,2-dihydrodiol syn-3,4-epoxide, BA trans-3,4-dihydrobiol anti-1,2-epoxide, and BA trans-3,4-dihydrodiol syn-1,2-epoxide, respectively, by their modes of forming vicinal acetonides, and by ultraviolet absorption and mass spectral analyses. From the metabolism of (-)-BA trans-1,2-dihydrodiol, a BA trans-1,2-dihydrodiol anti-3,4-epoxide was the major product formed by liver microsomes from MC-treated rats whereas a BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed by liver microsomes from either untreated or PB-treated rats. In contrast, BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed from the metabolism of (+)-BA trans-1,2-dihydrodiol by all three rat liver microsomal preparations. Liver microsomes from PB-treated rats were found to catalyze the metabolism of both the racemic and the enantiomeric BA trans-1,2-dihydrodiols at a rate higher than those by liver microsomes from untreated and MC-treated rats. All BA 1,2,3,4-tetrol metabolites were found to be optically active by circular dichroism spectral analysis. The results indicate that the 'bay-region' BA trans-1,2-dihydrodiol is metabolized by rat liver microsomes predominantly at the vicinal 3,4-double bond and that each enantiomeric BA trans-1,2-dihydrodiol is metabolized to a pair of diastereomeric BA trans-1,2-dihydrodiol 3,4-epoxides with varying degrees of stereoselectivity depending on the constitutive forms of cytochrome P-450 in the rat liver microsomal preparations.

Since 6-nitrochrysene and 6-aminochrysene have shown activity in carcinogenicity bioassays, we have begun an investigation of their metabolic activation pathways and the nature of the carcinogen-DNA adducts that may be formed. N-Hydroxy-6-aminochrysene (N-hydroxy-AC), a candidate proximate or ultimate carcinogen and the highest polycyclic N-hydroxy arylamine homolog studied thus far, was prepared by direct chemical synthesis and characterized by 1H-n.m.r. spectroscopy. Its rate and extent of reaction with DNA in vitro was 20-30 nmol bound/mg DNA/30 min, which is 2-10 times greater than has been reported for several other carcinogenic N-hydroxy arylamines. Three major aminochrysene-nucleoside adducts were detected in enzymatic hydrolysates of this N-hydroxy-AC-modified DNA, and these were isolated and identified by mass and 1H-n.m.r. spectroscopy as N-(deoxyinosin-8-yl)-6-aminochrysene, 5-(deoxyguanosin-N2-yl)-6-aminochrysene, and N-(deoxyguanosin-8-yl)-6-aminochrysene. These adducts accounted for 32%, 28%, and 22% respectively, of the total DNA adducts formed. We hypothesize that the deoxyinosine adduct is derived from spontaneous oxidation of the corresponding deoxyadenosine adduct prior to or during DNA isolation and adduct preparation. DNA isolated from Sprague-Dawley rat hepatocytes which had been treated with [3H]6-aminochrysene or [3H]6-nitrochrysene contained up to 12 pmol adducts/mg DNA (4 adducts per 10(6) nucleotides). High performance liquid chromatography (h.p.l.c.) analyses of enzymatic hydrolysates of this DNA indicated that the major products formed cochromatographed with the C8-deoxyinosine and C8-deoxyguanosine adducts. N-(Deoxyinosin-8-yl)-6-aminochrysene and N-(deoxyguanosin-8-yl)-6-aminochrysene accounted for 45% and 30% respectively, of the total DNA adducts formed in these cells. The preferential modification of deoxyadenosine by N-hydroxy-6-aminochrysene and the apparent facile oxidation of this adduct to a deoxyinosine derivative is thus far unique among the reactions of N-hydroxyarylamines with DNA and would not be predicted on the basis of reactivity alone.

Previous studies have shown that 1- and 3-nitrobenzo[a]pyrene (NBaP) were mutagenic in the Salmonella reversion assay without exogenous activation and that 1-, 3- and 6-NBaP were mutagenic in the presence of hepatocytes or liver homogenate (S9). In the present study, 1-, 3- and 6-NBaP were tested for mutagenicity in Chinese hamster ovary (CHO) cells under activation conditions similar to those used in the bacterial studies. None of the NBaPs was mutagenic without exogenous activation and none was mutagenically activated by hepatocytes from unpretreated rats or rats pretreated with Aroclor 1254 or 3-methylcholanthrene. Benzo-[a]pyrene (BaP), the parent compound, induced a strong mutagenic response under all hepatocyte mediation conditions. The NBaPs did produce positive mutagenic responses with S9 activation (3- = 1- greater than 6-NBaP), but these moderate responses were less than those of BaP. The difference between the bacterial and CHO results under the variety of activation conditions suggests the importance of the endogenous metabolism of the target cell as well as the source and the type of exogenous metabolic activation.

Rat liver microsomes metabolized the weak carcinogen 7-bromobenz[a]anthracene (7-Br-BA) to form predominantly trans-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols. The dihydrodiol metabolites were isolated by reversed-phase h.p.l.c. for structural and conformational analyses. N.m.r. spectral analysis indicated that the 3,4- and 10,11-dihydrodiols preferentially adopted quasidiequatorial conformations whereas the 5,6- and 8,9-dihydrodiols (which have a peri bromo substituent) preferred quasidiaxial conformations. Comparison of c.d. spectra with those of quasidiequatorial benz[a]anthracene (BA) 3R,4R-dihydrodiol and 10R,11R-dihydrodiol indicated that the major enantiomeric 7-Br-BA-3,4- and 10,11-dihydrodiol metabolites have R,R absolute sterochemistry. The absolute conformation of 7-Br-BA-5,6- and -8,9-dihydrodiol metabolites could not be deduced by comparing their c.d. spectra with those of BA 5R,6R- and 8R,9R-dihydrodiols. Catalytic hydrogenolysis converted the enzymatically formed 7-Br-BA trans-5,6-dihydrodiol to BA trans-5,6-dihydrodiol which had a c.d. spectrum identical to that of BA 5R,6R-dihydrodiol. Catalytic hydrogenolysis and hydrogenation converted the enzymatically formed 7-Br-BA trans-8,9-dihydrodiol to a BA 8,9,10,11-tetrahydro-trans-8,9-diol which had a retention time identical to that of BA 8,9,10,11-tetrahydro-8R,9R-diol on a chiral h.p.l.c. column. These results indicate that the major enantiomers of trans-dihydrodiol metabolites formed in rat liver microsomal metabolism of 7-Br-BA all have R,R absolute stereo-chemistries.

The tumor-initiating activity of the 3,4-dihydrodiols (diols) as well as other metabolites of 8-methylbenz[a]anthracene (8-MBA) and 8-hydroxymethylbenz[a]anthracene (8-OHMBA) were examined in the classical 2-stage initiation-promotion model on mouse skin. The 3,4-diol of 8-MBA caused 2.2 times as many papillomas/mouse as did 8-MBA. The 3,4-diol of 8-OHMBA was not more tumorigenic than either 8-MBA or 8-OHMBA. None of the other diols tested, including the 5,6- and 8,9-diol of 8-MBA and the 5,6 and 10,11-diol of 8-OHMBA were remarkably tumorigenic. These data indicate that the 3,4-diol of 8-MBA is a good candidate as a proximate carcinogen of 8-MBA and further suggest that the bay region 3,4-diol-1,2-epoxide is a likely ultimate carcinogen of this compound on mouse skin.

Cyclopenta-polycyclic aromatic hydrocarbons (cyclopenta-PAHs) are a group of compounds that have been detected as environmental pollutants. Perturbation molecular orbital (PMO) calculations on their presumed ultimate carcinogenic metabolites, the cyclopenta-PAH expoxides, predict that they may have a greater biological hazard than the classic PAHs.

The 12 possible monomethylbenz[a]anthracenes (MBAs) were examined for their tumor-initiating activity in the classical two-stage initiation-promotion experiment. Based on the average number of tumors/mouse, 7-MBA was the most tumorigenic compound of the series causing 4.9 papillomas/mouse at an initiating dose of 400 nmol/mouse. At this same dose level 8-MBA and 12-MBA were equally potent causing 1.0 papillomas/mouse. 6- an 9-MBAs caused -0.6 papillomas/mouse at this dose level. These data are in general agreement with previous experiments using other model systems. Of interest is the low tumorigenicity of the 1-, 2-, 3-and 4-MBAs which has been cited in support of the bay region theory of polycyclic aromatic hydrocarbon carcinogenesis. The tumor-initiating ability of anthracene and 9,10-dimethylanthracene was also examined. Neither of these compounds possessed tumor-initiating activity.

The aerobic and hypoxic metabolism of 2-nitroanthracene (2-NA) and 9-nitroanthracene (9-NA), two components of diesel exhaust, was studied and the mutagenicities of the parent compounds and their metabolites were compared. 2-NA was metabolized by 3-methylcholanthrene-induced rat liver microsomes under aerobic conditions to 2-NA trans-5,6-dihydrodiol, 2-NA trans-7,8-dihydrodiol, 2-NA 7-keto-5,6,7,8-tetrahydro-trans-5,6-diol, 2-NA 6-keto-5,6,7,8-tetrahydro-trans-7,8-diol, 2-nitro-9,10-anthraquinone and 2-NA 5,6,7,8-tetrahydrotetrol. When incubations were conducted under hypoxic conditions, 2-aminoanthracene was produced facily. N.m.r. spectral analysis indicated that the nitro-substituent of 2-NA and all of its ring-oxidized metabolites preferentially adopted an orientation in which the nitro group was coplanar or nearly co-planar with the aromatic ring system. 2-NA and its two trans-dihydrodiol metabolites were mutagenic in Salmonella typhimurium strain TA98, both in the presence and in the absence of S9 enzymes while the two tetrahydrodiol-ketones were much less mutagenic. When assayed in strains TA98NR and TA98/1,8-DNP6, the mutagenic activities of 2-NA and the trans-7,8-dihydrodiol were decreased. 2-Aminoanthracene was mutagenic in strain TA98 only in the presence of S9 enzymes. When 2-aminoanthracene was metabolized aerobically, the corresponding trans-5,6- and 7,8-dihydrodiols were not detected. These results suggest that 2-NA can be metabolized to mutagenic products by nitroreduction and ring-oxidation followed by nitroreduction, but not nitroreduction followed by ring-oxidation. Aerobic metabolism of 9-NA produced 9-NA trans-1,2- and 3,4-dihydrodiols, while metabolism was not detected under anaerobic conditions. Previous studies indicated that 9-NA and its two metabolites were not mutagenic in TA98. The differences in the orientation of the nitro substituents in 2-NA and its ring-oxidized metabolites and in 9-NA and its metabolites can be employed to explain the strong mutagenicity of 2-NA and weak mutagenicity of 9-NA when assayed both in the absence and in the presence of S9 activation enzymes.

Aerobic metabolism of 9-nitroanthracene by uninduced rat liver microsomes produced four metabolites identified as trans-1,2- and 3,4-dihydrodiols, 1,2,3,4-tetrahydrotetrol of 9-nitroanthracene, and anthraquinone. Further metabolism of the predominant metabolite, 9-nitroanthracene trans-3,4-dihyrodiol, yielded a 1,2,3,4-tetrahydrotetrol with a trans-cis-trans configuration, indicating that a trans-dihydrodiol anti-epoxide is formed as an intermediate. The mutagenic activities of both dihydrodiols and 9-nitroanthracene in strains TA98 and TA100, both in the presence and in the absence of S9 enzymes, were very low. When 9-nitroanthracene was metabolized under anaerobic conditions, nitroreduction did not occur. The results thus explain the weak mutagenic activity of 9-nitroanthracene.

The rat liver microsomal metabolism of 3-deutero-6-nitrobenzo(a)pyrene ([3-2H]6-NO2-BaP) was studied. The metabolites were separated by h.p.l.c. The 500 MHz 1H n.m.r. spectral analysis of the metabolites indicated that the 3-hydroxy-6-NO2-BaP and 6-NO2-BaP-3,9-hydroquinone each retained 33% of the deuterium label at the C-2 position. It is proposed that the migration of deuterium occurs via an NIH shift mechanism. These results indicate that a 2,3-epoxide is a common intermediate.

Aerobic metabolism of 1-nitrobenzo[e]pyrene (1-nitro-BeP) by rat liver microsomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP, and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichloropropylene 1,2-oxide was incorporated into the metabolism, 1-nitro-BeP 4,5-oxide was the predominant metabolite, and 1-nitro-BeP trans-4,5-dihydrodiol was not detected. All of the metabolites were purified by both reversed- and normal-phase HPLC and characterized by analysis of their mass and 500 MHz proton NMR spectral data. 1-Nitro-BeP was not metabolized under hypoxic conditions. 1-Nitro-BeP and its four metabolites were assayed in Salmonella typhimurium tester strains TA98, TA98NR and TA98/1,8-DNP6, both in the presence and absence of S9 activation. As predicted, 1-nitro-BeP was a weak mutagen without S9 (2 revertants/micrograms in TA98); the addition of S9 resulted in approximately 18, 17 and 4 revertants/micrograms in TA98, TA98NR and TA98/1,8-DNP6 respectively. The two phenolic metabolites were mutagenic both in the presence and absence of S9, producing moderate responses (19-84 revertants/micrograms). In addition, while the 1-nitro-BeP 4,5-oxide was only weakly mutagenic in TA98 (6-14 revertants/micrograms), 1-nitro-BeP trans-4,5-dihydrodiol was unexpectedly potent (approximately 300 revertants/micrograms both with and without S9). These results indicate that microsomal epoxidation of 1-nitro-BeP followed by epoxide hydrolase-catalyzed hydrolysis of the resulting epoxide to the 1-nitro-BeP trans-4,5-dihydrodiol results in the most potent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP 4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic than the corresponding parent nitro-PAHs. Also, the lower S9-mediated mutagenicity of 1-nitro-BeP in TA98/1,8-DNP6 compared with TA98 indicates that the mutagenicity of 1-nitro-BeP is dependent upon nitroreduction and transesterification. Finally, we previously hypothesized that nitrated PAHs with their nitro substituents perpendicular or nearly perpendicular to the aromatic rings are very weak or nondirect-acting mutagens in Salmonella typhimurium tester strains. The results reported in this communication demonstrate that ring-oxidized derivatives of nitro-PAHs do not always follow this structure--mutagenicity correlation.

The direct enantiomeric resolution of non-K region trans-1,2-dihydrodiol, 1,2,3,4-tetrahydro-trans-1,2-diol, trans-3,4-dihydrodiol and 1,2,3,4-tetrahydro-trans-3,4-diol, K region trans- and cis-5,6-dihydrodiols and their monomethyl ethers of chrysene was studied by chiral stationary phase high-performance liquid chromatography (CSP-h.p.l.c.). The chiral stationary phase columns were packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)-phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine was bonded either ionically or covalently. Enantiomers of all dihydrodiol derivatives were resolved by one or more, but not all, of the chiral stationary phases utilized. Enantiomeric resolutions were substantially improved when the non-K region dihydrodiols were converted to tetrahydrodiols. The absolute configurations of the K region trans- and cis-5,6-dihydrodiols were established by the exciton chirality circular dichroism method. The (R,R):(S,S) enantiomer ratios, determined by CSP-h.p.l.c., of the 1,2-, 3,4- and 5,6-trans-dihydrodiols formed in the metabolism of chrysene by liver microsomes from untreated male rats of the Sprague--Dawley strain were found to be 51:49, 99:1 and 86:14, respectively; from phenobarbital-treated rats, 41:59, 99:1 and 87:13, respectively; from 3-methylcholanthrene-treated rats, 96:4, 99:1 and 92:8, respectively. The absolute configurations of chrysene 5,6-epoxide enantiomers, resolved by CSP-h.p.l.c., were elucidated by the determination of the structures and absolute configurations of their methoxylation products. Both enantiomers of chrysene 5,6-epoxide were hydrated by microsomal epoxide hydrolase to chrysene trans-5,6-dihydrodiol enriched (67-92%) in the 5R,6R enantiomer. Chrysene 5R,6S-epoxide was hydrated to trans-5,6-dihydrodiol at a rate approximately 6-fold faster than chrysene 5S,6R-epoxide.