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The direct resolution of trans and cis bay region dihydrodiol and tetrahydrodiol enantiomers of benz[a]anthracene and benzo[a]-pyrene, and trans and cis K-region dihydrodiol enantiomers of benz[a]anthracene, 4-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, and 3-methylcholanthrene was evaluated by high-performance liquid chromatography using commercially available columns packed with (R)-N-(3,5-dinitrobenzoyl)phenylglycine and (S)-N-(3,5-dinitrobenzoyl)leucine either ionically or covalently bonded to γ-aminopropylsilanized silica. Except benz[a]anthracene trans-5,6-dihydrodiol, the diol enantiomers of other hydrocarbons were all resolved by at least one of the four chiral stationary phases tested. The absolute configurations of enantiomeric diols, whose hydroxyl groups' conformations are restricted due to steric factors, have been established by the exciton chirality circular dichroism method. Based on the experimental results, the mechanisms of some chiral in...

Six strains of fungi grown on Sabouraud dextrose broth in the presence of 7,12-dimethylbenz[a]anthracene (DMBA) were surveyed for their ability to metabolize DMBA. Experiments with [14C]DMBA indicated that the extent of formation of organic-soluble metabolites ranged from 6 to 28% after 5 days of incubation, depending on the organism tested. The yields of water-soluble metabolites also varied, and ranged from 1 to 33% after 5 days.Cunninghamella elegans ATCC 36112 andSyncephalastrum racemosum UT-70 exhibited the highest DMBA-metabolizing activity among the organisms surveyed.S. racemosum metabolized DMBA primarily to 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)_ and 7,12-dihydroxymethylbenz[a]anthracene (7,12-diOHMBA). Minor metabolites included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols, and glucuronide and sulfate conjugates of phenolic derivatives of DMBA. In contrast, the major DMBA metabolites produced byC. elegans were water-soluble. The predominant organic-soluble metabolites produced byC. elegans included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols. DMBA-trans-3,4-dihydrodiol was also detected. Circular dichroism spectral analysis revealed that the major enantiomer of the 7-OHM-12-MBA-trans-8,9-dihydrodiol formed by each organism has anS,S absolute configuration, while the major enantiomers of the 5,6-, 10,11- and 3,4-dihydrodiols had anR,R configuration. The mutagenic activity of extracts fromS. racemosum exposed to DMBA were determined inSalmonella typhimurium TA98. The mutagenicity of DMBA decreased by 36% over a period of 5 days as 33% of the compound was metabolized. Comparison of these results with previously reported results in mammalian systems suggests that there are similarities and differences between the fungal and mammalian oxidation of DMBA and that the overall balance of fungal metabolism is towards a detoxification rather than a bioactivation pathway.

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.

The 7-methylbenzo[a]pyrene (7-MBaP) was incubated with liver microsomes of rats pretreated with polychlorinated biphenyls (Aroclor 1254) (PCBs). Metabolites of 7-MBaP were isolated by both reversed-phase and normal-phage high performance liquid chromatography (HPLC) and were characterized by nuclear magnetic resonance, UV-visible and mass spectral analyses. The predominant metabolite of 7-MBaP was found to be 3-hydroxy-7-methylbenzo[a]pyrene (3-hydroxy-7-MBaP). Other identified metabolites include 7-MBaP 4,5-, 7,8-, and 9,10-trans-dihydrodiols, 7-hydroxymethyl-BaP, 7-hydroxymethyl-BaP trans-9,10-dihydrodiol, 9-hydroxy-7-MBaP, 3-hydroxy-7-hydroxymethyl-BaP, 7-MBaP 1,6- and 3,6- quinones, and a hydroquinone which is also formed by further metabolism of the 3-hydroxy-7-MBaP. Comparative metabolic studies of 7-MBaP and BaP indicated that, relative to that of BaP, the methyl substituent of 7-MBaP slightly increases the formation of 3-hydroxy-7-MBaP and decreases the metabolism at other regions of the 7-MBaP molecule. The finding that a 7,8-dihydrodiol is a metabolite indicates that, like BaP, 7-MBaP may also be activated to the potentially reactive 7,8-dihydrodiol 9,10-epoxides although their formations are significantly reduced.

Synthese par action d'arenals sur le carbanion forme par lithiation de la dimethyl-4,4 o-tolyl-2 oxazoline-2 (formee a partir de methyl-2 phenyl-2 propanol-1 et du chlorure d'o-toluoyle)

Es wird die Alkylierung der im Titel genannten Li-Verbindungen (Alkyl = Isopropyl, tert.-Butyl) mit Alkylhalogeniden (Alkyl = Methyl, Athyl, Isopropyl) zu den entsprechenden Dialkylderivaten untersucht.

Summary Xanthine oxidase, a mammalian nitroreductase, catalyzed the covalent binding of a series of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) trans -dihydrodiols to DNA. Some of the trans -dihydrodiols bound to DNA to a greater extent than their parent nitro-PAHs; however, when the dihydrodiol moiety was peri to the nitro substituent low levels of binding were observed. These data illustrate that ring-oxidation and hydrolysis of nitro-PAHs to their trans -dihydrodiols followed by nitroreduction is a potential metabolic pathway leading to DNA adducts in mammals.

The direct separation of 26 bay region and non-bay region mono-ol and diol enantiomers of phenanthrene, benz[a]anthracene, and chrysene was compared by high-performance liquid chromatography on commercially available columns, packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)phenylglycine(R-DNBPG) or (S)-N-(3,5-dinitrobenzoyl)leucine(S-DNBL) was either ionically or covalently bonded. In general, enantiomers of bay region mono-ols and diols are more efficiently resolved than those of non-bay region derivatives. Elution orders of enantiomers on either chiral stationary phase are the same, regardless of whether the chiral stationary phase is ionically or covalently bonded. Except for the enantiomers of 4-hydroxy-4-methyl-1,2,3,4-tetrahydrobenz[a]anthracene, 1,2,3,4-tetrahydrobenz[a]anthracene trans-1,2-diol, and benz[a]anthracene trans-1,2-dihydrodiol, elution orders of resolved enantiomers on R-DNBPG are reversed on S-DNBL. The enantiomers are generally more efficiently resolved on R-DNBPG than on S-DNBL. With the exception of the elution order of the enantiomeric 4-hydroxy-1,2,3,4-tetrahydrochrysene, the results of this study are consistent with the chiral recognition mechanisms proposed by Pirkle and co-workers, who developed the chiral stationary phases used in this study.

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.

8-Methylbenz[a]anthracene (8-MeBaA) transdihydrodiol metabolites were isolated by reversed-phase and normal-phase HPLCs from incubations of 8-MeBaA with liver microsomes or a reconstituted system containing purified cytochrome P-448 and epoxide hydrolase. Regardless of the enzyme source, the metabolically formed 8-MeBaA trans-3,4- and -5,6-dihydrodiols were found to be enriched in one enantiomeric isomer and differed only in the degree of optical purity. The 8-MeBaA trans-8,9-dihydrodiol formed by liver microsomes from either untreated or phenobarbital-treated rats was enriched with the (+)-enantiomer. In contrast, the 8-MeBaA trans-8,9-dihydrodiol formed either by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted rat liver enzyme system containing cytochrome P-448 and epoxide hydrolase was enriched with the (-)enantiomer. These results indicate that, in catalyzing the formation of 3,4- and 5,6-epoxide intermediates, the interaction with the unsubstituted 3,4- and 5,6-double bonds of 8-MeBaA by the different forms of cytochrome P-450 occur preferentially on the same face of the aromatic plane and they differ only in the degree of stereoselectivity. However, different forms of cytochrome P-450 may interact with different faces of the aromatic plane at the methyl-substituted 8,9-double bond of 8-MeBaA, resulting in the formation of trans-8,9-dihydrodiols enriched in different enantiomeric forms. This demonstrates that different forms of cytochrome P-450 may catalyze the epoxidation reaction preferentially at different sides of the methyl-substituted double bond of a planar polycyclic hydrocarbon molecule. These properties may be used to further classify and to understand the enzyme-substrate interactions of the different forms of cytochrome P-450 in the drug-metabolizing enzyme systems.

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.

8 representative 2-substituted 5-nitrofurans were assayed for mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6. The tested compounds were: 5-nitro-2-furanacrylic N-(5-nitro-2-furfurylidene)hydrazide (1); furazolidone (2); 5-nitro-2-furanacrolein (3); 5-nitro-2-furaldehyde semicarbazone (4); 5-nitro-2-furaldehyde (5); nitrofurantoin (6); 5-nitro-2-furaldehyde diacetate (7); and 5-nitro-2-furoic acid (8). These compounds exhibited markedly different mutagenic activities in TA98, and these mutagenicities were similar both in the presence and the absence of rat-liver hepatic S9 activation enzymes. The mutagenic responses ranged from potent (90-300 revertants/nmole, compounds 1-3), to medium (about 10 revertants/nmole, compounds 4 and 6), to weak (0-4 revertants/nmole, compounds 5, 7 and 8). The mutagenicity of 3 was similar in all 3 tester strains, while compound 8 was essentially inactive. The mutagenicities of 1, 4, 5 and 7 were decreased 30-75% in TA98NR, while 2 and 6 showed an even greater depression of activity in this strain. Compound 6 with S9 was about equally mutagenic in TA98 and TA98/1,8-DNP6, while the activities of 6 without S9 and 2 and 7 both with and without S9 were 50-75% lower in TA98/1,8-DNP6. Compounds 1, 4 and 5 were only about 5-10% as mutagenic in TA98/1,8-DNP6 as in TA98. These results suggest that: (i) nitrofurans and their S9-mediated metabolites have similar mutagenic potencies; (ii) with the possible exception of No. 3, nitroreduction is the major route of mutagenic activation for these nitrofurans; and (iii) for compounds 2, 6 and 7, both the presumed N-hydroxy and N,O-ester derivatives of the corresponding aminofuran metabolites appear to lead to mutations.

Summary An optically active 8,9-diol has been identified as a rat liver microsomal metabolite of the carcinogenic 8-methylbenz[a]anthracene. This is the first example indicating that the presence of a methyl group in a polycyclic aromatic hydrocarbon does not sterically block the enzymatic formation of a diol at the methyl-substituted double bond.

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.

Nitrated polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that result from various incomplete combustion processes. We have examined the activity of hepatic microsomal enzymes in rats pretreated with a series of environmentally occurring nitrated PAHs including: 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitrofluoranthene, and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of the compounds increased the cytochrome P-450 content more than 2-fold. 1,8-Dinitropyrene, 6-nitrochrysene, and 1- and 3-nitrobenzo[a]pyrene significantly increased arylhydrocarbon hydroxylase activity 2- to 8-fold higher than solvent-treated controls. The induction of 7-ethoxycoumarin O-deethylase activity paralleled that found with arylhydrocarbon hydroxylase. The maximum induction of aminopyrine N-demethylase was only 1.5-fold, and none of the nitrated PAHs caused significant increases in epoxide hydrase or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. The greatest increase was caused by 1-nitrobenzo[a]pyrene followed by 1,3-dinitropyrene, 3-nitrobenzo[a]pyrene and 6-nitrochrysene. These data suggest that nitrated PAHs may potentiate the effects of subsequent exposures to various chemical carcinogens.

The bay region anti-diolepoxide of chrysene (8) is conveniently synthesized from 3,4,5,6-tetrahydro-chrysene obtained from chrysene in two steps.

The mutagenic environmental pollutants 1-, 3-, and 6-nitrobenzo[a]pyrene were synthesized. Nitration of 7,8,9,10-tetrahydrobenzo[a]pyrene with sodium nitrate in trifluoroacetic acid and acetic anhydride at ambient temperature gave a mixture of 1-, 3-, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, which was separated by chromatography. Dehydrogenation of the isolated nitrotetrahydrobenzo[a]pyrenes with 2,3-dichloro-4,5-dicyano-1,6-benzoquinone produced 1-, 3-, and 6-nitrobenzo[a]pyrene in high yield. Comparison of the spectral data of these compounds with those obtained from direct nitration of benzo[a]pyrene confirmed that 1- and 3-nitrobenzo[a]pyrenes are indeed the minor products of the latter reaction. This confirmation also verifies that 1- and 3-nitrobenzo[a]pyrene were the minor nitrated products of benzo[a]pyrene formed in model air atmospheres. The 1-, 3-, and 6-nitrobenzo[a]pyrene were mutagenic in Salmonella typhimurium tester strains TA98 and TA100 in the presence of a mammalian microsomal (S9) activating system. Both 1- and 3-nitrobenzo[a]pyrene, but not 6-nitrobenzo[a]pyrene, were also direct-acting mutagens in these strains. However, only 6-nitrobenzo[a]pyrene exhibited weak mutagenic activity when tested in Chinese hamster ovary cells, while only 3-nitrobenzo[a]pyrene produced a concentration-dependent decrease in cellular survival.

Metabolism of 6-nitrobenzo[a]pyrene by rat liver microsomes yielded 1- and 3-hydroxy-6-nitrobenzo[a]pyrene, 6-nitrobenzo[a]pyrene-1,9- and -3,9-hydroquinone and benzo[a]pyrene-3,6-quinone. The monohydroxylated metabolites were more mutagenic than 6-nitrobenzo[a]pyrene in a Salmonella typhimurium / microsome reversion assay. These results indicate that ring hydroxylation is involved in the metabolic activation of this nitro-polycyclic aromatic hydrocarbon.

The enantiomers of trans-7,8-dihydroxy-7,8-dihydro-7-methylbenzo[a]pyrene (7-MBaP 7,8-dihydrodiol) and of trans-7,8-dihydroxy-7,8,9,10-tetrahydro-7-methylbenzo[a]pyrene (7-MBaP . 7,8-tetrahydrodiol) were directly resolved by high-performance liquid chromatography (HPLC) using a commercially available column packed with an (R)-N-(3,5-dinitrobenzoyl)-phenylglycine derivative of γ-aminopropylsilanized silica. The absolute configurations of the resolved enantiomers were determined by the exciton chirality method. Circular dichroism (CD) spectral analysis of the quasidiequatorial benzo[a]pyrene 7R,8R-dihydrodiol enantiomer and its diacetate and dimenthoxyacetate derivatives indicated conformational changes were induced upon derivatization. However, the characteristic CD Cotton effects of the quasidiequatorial 7-MBaP 7,8-dihydrodiol and its diacetate and dimenthoxyacetate derivatives were similar indicating that the conformation of 7-MBaP trans-7,8-dihydrodiol was not altered upon derivatization. Proton nuclear magnetic resonance (NMR) spectral analyses confirmed that 7-MBaP 7,8-dihydrodiol, its diacetate and dimenthoxyacetate derivatives all have quasidiequatorial conformations. The results indicate that the methyl substituent of 7-MBaP 7,8-dihydrodiol maintains a quasiaxial position regardless of the size of the acyl derivatives linked to the hydroxyl groups.

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 direct resolution of K-region cis- and trans-dihydrodiol enantiomers of 14 unsubstituted and methyl- and bromo-substituted benz[a]anthracene (BA) derivatives was investigated by high-performance liquid chromatography with commercially available columns, packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)phenylglycine (R-DNBPG) or (S)-N-(3,5-dinitrobenzoyl)leucine (S-DNBL) is either ionically or covalently bonded. BA derivatives used in this study include: BA, 1-methyl-BA, 4-methyl-BA, 7-methyl-BA, 8-methyl-BA, 10-methyl-BA, 11-methyl-BA, 12-methyl-BA, 7,12-dimethyl-BA, 7-bromo-BA, 7-bromo-1-methyl-BA, 7-bromo-11-methyl-BA, 7-bromo-12-methyl-BA, and 3-methylcholanthrene. The enantiomers of BA trans-5,6-dihydrodiol were the only compounds not resolved by any of the four chiral stationary phases (CSPs) tested. The results indicate that conformational preference of the hydroxyl group is one of the most important factor in determining the elution order of dihydrodiol enantiomers. The presence and the location of a substituent and the molecular size and shape of the dihydrodiols can significantly affect the efficiency of enantiomeric resolution. In general, the ionically bonded R-DNBPG provides the best resolution of enantiomeric quasidiequatorial trans-dihydrodiols and the R,R enantiomers are consistently more strongly retained. In contrast, the enantiomeric pairs of quasidiaxial trans-dihydrodiols are generally better resolved by the covalently bonded R-DNBPG, and the S,S enantiomers are more strongly retained. The enantiomers of cis-dihydrodiols having hydroxyl groups that adopt quasiequatorial-quasiaxial and/or quasiaxial-quasiequatorial conformations are more consistently resolved by the ionically bonded S-DNBL and in all cases the S,R enantiomers are more strongly retained. Thus, it is possible to choose a CSP which resolves the K-region dihydrodiol enantiomers with a predictable elution order.

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.

Fluorometric studies on the binding of benz[a]anthracene and benzo[a]pyrene to human serum albumin are described. The protein molecule appears to have one binding site for the hydrocarbons, but all of the sites on the protein are not fully occupied even in relatively large hydrocarbon concentrations. Equilibrium studies showed that both hydrocarbons bind to the protein to the same extent. Evidence for the energy transfer from the tryptophan residue of the protein to bound hydrocarbons is examined. By using Forster's theory, the mean distance between the tryptophan residue and bound ligand was found to be 15,2 A for benz[a]anthracene and 19.6 A for benzo[a]pyrene. It is concluded that the two hydrocarbons may bind to the same general area on the protein molecule near the tryptophan residue but at different sites. The structural differences of the hydrocarbons, which may greatly affect their orientations on the protein molecule, affect mainly the selection of the binding site rather than the binding equilibrium.

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.

In the field of chemical carcinogenesis, amino- and acetylamino-polycyclic aromatic hydrocarbons (PAHs) are among the most studied compounds. Many of these compounds have recently been detected in the environment. Presently, knowledge permitting predictions of the high-performance liquid chromatographic (HPLC) retention order of amino- and acetylamino-PAHs, particularly among their geometric isomers is lacking. In order to obtain a better understanding of the separation of these types of compounds, we have studied the separation of a series of structurally related amino- and acetylamino-PAHs derived from naphthalene, phenanthrene, anthracene, pyrene, benz[a]anthracene, benzo[a]pyrene, and benzo[e]pyrene by using reversed-phase and normal-phase HPLC columns of different types (monomeric, polymeric, and chiral stationary phase). The results indicate: (i) Pirkle-type chiral stationary phase columns and the Zorbax SIL column can efficiently separate both the amino-PAHs and acetylamino-PAHs; (ii) in general, there was no correlation between retention time and molecular size; (iii) when acetylamino-PAHs were separated on the monomeric Zorbax ODS column, the isomer with the acetylamino group located at the carbon position of higher electron density has a shorter retention time; and (iv) separation of the parent PAHs was better than that of the amino-PAHs and acetylamino-PAHs. Our results thus may provide useful information for the analysis of amino-PAHs, particularly for distinguishing the geometric isomers of environmental samples.

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.

Metabolism of 7-nitrobenz(a)anthracene (7-NO2-BA) by rat liver microsomes yielded 7-NO2-BA trans-3,4-dihydrodiol and 7-NO2-BA trans-8,9-dihydrodiol as major metabolites. Proton NMR spectral analyses indicate that 7-NO2-BA trans-3,4-dihydrodiol preferentially adopts a quasidiequatorial conformation and that 7-NO2-BA trans-8,9-dihydrodiol adopts a mixture of quasidiequatorial and quasidiaxial conformations. Circular dichroism spectral analyses of these compounds and their diacetoxy derivatives indicated that the major enantiomers of both dihydrodiols have R,R absolute stereochemistries. The identification of 7-NO2-BA trans-8,9-dihydrodiol as a metabolite of 7-NO2-BA indicates that oxidative metabolism can occur at position peri to the nitro substituent.

Extensive metabolism studies of many non-K-region dihydro-diols of unsubstituted and methyl-substituted polycyclic aromatic hydrocarbons (PAHs) by mammalian drug-metabolizing enzymes indicate that the extent of formation of vicinal dihydrodiol-epoxides is determined by the conformations and geometric location of the adjacent double bond of the dihydrodiol precursors. Unsubstituted PAH dihydrodiols with one (in the case of cis-dihydrodiols) or both (in the case of trans-dihydrodiols) of the hydroxyl groups preferentially in quasi- equatorial conformations can be further metabolized to the vicinal di- hydrodiol-epoxides. The trans-dihydrodiols with both of the hydroxyl groups preferentially or exclusively in the quasi-diaxial. conformations can also be enzymatically epoxidized to form vicinal dihydrodiol-epox- ides, but the extent of these formations is highly dependent on the geometric location of the adjacent double bond of the dihydrodiols. Quasi-diaxial trans-dihydrodiols with an adjacent double bond at the bay-regions are not or minimally metabolized to the vicinal dihydrodiol-epoxides. A methyl substituent at the hydroxylated carbons or at the adjacent double bonds of the quasi-diequatorial trans-dihydrodiols can substantially or completely inhibit the vicinal dihydrodiol-epoxide formations. These findings, along with the bay-region theory, provide the molecular basis for the observed carcinogenic potencies of many bay-region containing unsubstituted and methyl-substituted PAHs.

While metabolism of benz[a]anthracene by rat liver microsomes produced a (+)5R,6R-dihydrodiol as the major enantiomer, metabolism of 12-methylbenz[a]anthracene under similar conditions gave a (−)5S,6S-dihydrodiol as the major enantiomer. This is the first example indicating that the methyl substituent of a polycyclic aromatic hydrocarbon can drastically alter the stereoselective preference of the microsomal drug-metabolizing enzyme systems toward a substrate molecule in the formation of a dihydrodiol metabolite at an unsubstituted aromatic double bond.

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.

The mutagenic environmental pollutants 1-, 3-, and 6-nitrobenzo[a]pyrene were synthesized. Nitration of 7,8,9,10-tetrahydrobenzo[a]pyrene with sodium nitrate in trifluoroacetic acid and acetic anhydride at ambient temperature gave a mixture of 1-, 3-, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, which was separated by chromatography. Dehydrogenation of the isolated nitrotetrahydrobenzo[a]pyrenes with 2,3-dichloro-4,5-dicyano-1,6-benzoquinone produced 1-, 3-, and 6-nitrobenzo[a]pyrene in high yield. Comparison of the spectral data of these compounds with those obtained from direct nitration of benzo[a]pyrene confirmed that 1- and 3-nitrobenzo[a]pyrenes are indeed the minor products of the latter reaction. This confirmation also verifies that 1- and 3-nitrobenzo[a]pyrene were the minor nitrated products of benzo[a]pyrene formed in model air atmospheres. The 1-, 3-, and 6-nitrobenzo[a]pyrene were mutagenic in Salmonella typhimurium tester strains TA98 and TA100 in the presence of a mammalian microsomal (S9) activating system. Both 1- and 3-nitrobenzo[a]pyrene, but not 6-nitrobenzo[a]pyrene, were also direct-acting mutagens in these strains. However, only 6-nitrobenzo[a]pyrene exhibited weak mutagenic activity when tested in Chinese hamster ovary cells, while only 3-nitrobenzo[a]pyrene produced a concentration-dependent decrease in cellular survival.

1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmental pollutants and are S9-mediated mutagens in the Chinese hamster ovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferase assay. In this study, we examined the pathways leading to the mutagenic activation of these compounds in CHO cells. The microsomal metabolites of 1- and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydroxy-7,8-dihydrodiols (trans-7,8-dihydrodiols) and the 1- and 3-nitro-BaP trans-9,10-dihydrodiols, were isolated and tested for mutagenicity. At the concentrations assayed, both trans-9,10-dihydrodiols were non-mutagenic with and without S9 activation. In contrast, the trans-7,8-dihydrodiols of 1- and 3-nitro-BaP were direct-acting mutagens and these responses were similar in magnitude to the S9-mediated mutagenicities of the parent nitro-BaPs. S9 increased the mutagenic responses of the trans-7,8-dihydrodiols approximately 20-fold. Inhibition of epoxide hydrolase decreased the S9-mediated mutagenicity of 1-nitro-BaP by half, but doubled the S9-mediated mutagenicity of 3-nitro-BaP. These results suggest that in CHO cells: (i) the major route of mutagenic activation of 1- and 3-nitro-BaP involves S9-generated derivatives of the trans-7,8-dihydrodiols, e.g. bay-region diol epoxides; (ii) reactive nitroarene oxides may contribute to mutation induction by 3-nitro-BaP; and (iii) metabolic routes involving trans-9,10-dihydrodiol formation result in detoxification.

Trans -3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols formed from the metabolism of 7-fluorobenz[a]anthracene by rat liver microsomes were isolated by reversed-phase high performance liquid chromatography. Ultraviolet absorption, mass, and NMR spectral analyses indicated that the 5,6- and 8,9-dihydrodiols were preferentially in quasi-diaxial conformations, whereas the 3,4- and 10,11-dihydrodiols were preferentially in quasi-diequatorial conformations. CPK space-filling models suggest that the quasi-diaxial conformation is primarily the result of electronic repulsion between the fluorine and the peri hydroxyl oxygen. These findings provide a structural basis in the interpretation of the carcinogenic potencies of some fluorinated polycyclic aromatic hydrocarbons.