The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene.

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.     

Metabolism of 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene by Cunninghamella elegans.

The fungal metabolism of 7-methylbenz[a]anthracene (7-MBA) and 7-hydroxymethylbenz[a]anthracene (7-OHMBA) was studied. 7-MBA was metabolized by Cunninghamella elegans to form 7-OHMBA-trans-8,9-dihydrodiol and 7-OHMBA-trans-3,4-dihydrodiol as the predominant metabolites. Other metabolites were identified as 7-OHMBA, 7-MBA-trans-8,9-dihydrodiol and 7-MBA-trans-3,4-dihydrodiol, and 7-MBA-8,9,10,11-tetraol. Incubation of 7-OHMBA with C. elegans cells indicated that 7-OHMBA-trans-8,9-dihydrodiol and 7-OHMBA-trans-3,4-dihydrodiol were major metabolites. The metabolism of 7-MBA by rat liver microsomes from 3-methylcholanthrene-treated rats showed that the metabolites were qualitatively similar to those formed by C. elegans, except additional dihydrodiol metabolites were formed at the 5,6 and 10,11 positions. The metabolites formed were isolated by high-performance liquid chromatography and identified by comparing their chromatographic, UV-visible absorption and mass spectral properties with those of reference compounds.     

Metabolism of benz[a]anthracene by the filamentous fungus Cunninghamella elegans.

The metabolism of the carcinogen benz[a]anthracene (BA), a tetracyclic aromatic hydrocarbon, by Cunninghamella elegans was investigated. C. elegans grown on Sabouraud dextrose broth transformed [14C]BA to labeled BA trans-8,9-dihydrodiol (90%), BA trans-10,11-dihydrodiol (6%), and BA trans-3,4-dihydrodiol (4%), but not to BA trans-5,6-dihydrodiol. These metabolites were separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography and were identified by UV and mass spectral techniques. A BA tetraol, 8 beta,9 alpha,10 alpha,11 beta-tetrahydroxy-8 alpha, 9 beta,10 beta,11 alpha-tetrahydro-BA, was also identified as a metabolite and may have arisen as an additional oxidation product of either BA 8,9- or 10,11-dihydrodiol. This is the first study in which a biologically produced BA tetraol has been identified. Our results suggest that the transformation of BA to trans-dihydrodiols by C. elegans is similar to the transformation of BA found in mammals, except that BA 5,6-dihydrodiol is not produced.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to 7,12-dimethylbenz[a]-anthracene

The syntheses of 7,12-dimethylbenz[a]anthracene 5,6-oxide, 7-acetoxymethyl-12-methylbenz[a]anthracene 5,6-oxide and a product that appears to be mainly 7-hydroxymethyl-12-methylbenz[a]anthracene 5,6-oxide are described. The compounds readily rearranged to phenols in the presence of mineral acid, and 7,12-dimethylbenz[a]anthracene 5,6-oxide and its 7-hydroxymethyl derivative reacted slowly with water to yield trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and trans-5,6-dihydro-5,6-dihydroxy-7-hydroxymethyl-12-methylbenz [a]anthracene respectively. Both epoxides were converted enzymically by rat liver microsomal fractions and homogenates into the related trans-dihydrodiols. The epoxides reacted chemically with GSH to form conjugates that were identical with the conjugates formed when the epoxides were incubated with rat liver homogenates. The GSH conjugates were more stable to acid than conjugates derived from other arene oxides. In the alkylation of 4-(p-nitrobenzyl)pyridine, 7,12-dimethyl-benz[a]anthracene 5,6-oxide was more active than the 5,6-oxides of 7-methylbenz[a]-anthracene and benz[a]anthracene.     

Metabolic activation of polycyclic aromatic hydrocarbon trans-dihydrodiols by ubiquitously expressed aldehyde reductase (AKR1A1)

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by CYP1A1 and epoxide hydrolase (EH). CYP1A1 or aldo-keto reductases (AKRs) from the 1C subfamily can further activate the trans-dihydrodiols by forming either anti-diol-epoxides or reactive and redox active o-quinones, respectively. To determine whether other AKR superfamily members can divert trans-dihydrodiols to o-quinones, the cDNA encoding human aldehyde reductase (AKR1A1) was isolated from hepatoma HepG2 cells using RT-PCR, subcloned into a prokaryotic expression vector, overexpressed in E. coli and purified to homogeneity in milligram amounts. Studies revealed that AKR1A1 preferentially oxidized the metabolically relevant (-)-[3R,4R]-dihydroxy-3,4-dihydrobenz[a]anthracene. AKR1A1 also displayed high utilization ratios (V(max)/K(m)) for the following PAH trans-dihydrodiols: (+/-)trans-3,4-dihydroxy-3,4-dihydro-7-methylbenz[a]anthracene, (+/-)trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene and (+/-)trans-7,8-dihydroxy-7,8-dihydro-5-methylchrysene. Multiple tissue expression (MTE) arrays were used to measure the co-expressed of CYP1A1, EH and AKR1A1. All the three enzymes co-expressed to sites of PAH activation. The high catalytic efficiency of AKR1A1 for potent proximate carcinogen trans-dihydrodiols and its presence in tissues that contain CYP1A1 and EH suggests that it plays an important role in this alternative pathway of PAH activation (supported by CA39504).     

Stereoselective fungal metabolism of 7,12-dimethylbenz[a]anthracene: identification and enantiomeric resolution of a K-region dihydrodiol.

Syncephalastrum racemosum UT-70 and Cunninghamella elegans ATCC 36112 metabolized 7,12-dimethylbenz[a]anthracene (7,12-DMBA) to hydroxymethyl metabolites as well as 7-hydroxymethyl-12-methylbenz[a]anthracene trans-3,4-, -5,6-, -8,9-, and -10,11-dihydrodiols. The 7,12-DMBA metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by their UV-visible absorption, mass, and nuclear magnetic resonance spectral characteristics. A comparison of the circular dichroism spectra of the K-region (5,6-position) dihydrodiol of both fungal strains with those of the 7,12-DMBA 5S,6S-dihydrodiol formed from 7,12-DMBA by rat liver microsomes indicated that the major enantiomer of the 7-hydroxymethyl-12-methylbenz[a]anthracene trans-5,6-dihydrodiol formed by both fungal strains had a 5R,6R absolute stereochemistry. Direct resolution of the fungal trans-5,6-dihydrodiols by chiral stationary-phase high-performance liquid chromatography indicated that the ratios of the R,R and S,S enantiomers were 88:12 and 77:23 for S. racemosum and C. elegans, respectively. These results indicate that the fungal metabolism of 7,12-DMBA at the K region (5,6-position) is highly stereoselective and different from that reported for mammalian enzyme systems.     

Stereoselective fungal metabolism of 7,12-dimethylbenz[a]anthracene: identification and enantiomeric resolution of a K-region dihydrodiol

Syncephalastrum racemosum UT-70 and Cunninghamella elegans ATCC 36112 metabolized 7,12-dimethylbenz[a]anthracene (7,12-DMBA) to hydroxymethyl metabolites as well as 7-hydroxymethyl-12-methylbenz[a]anthracene trans-3,4-, -5,6-, -8,9-, and -10,11-dihydrodiols. The 7,12-DMBA metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by their UV-visible absorption, mass, and nuclear magnetic resonance spectral characteristics. A comparison of the circular dichroism spectra of the K-region (5,6-position) dihydrodiol of both fungal strains with those of the 7,12-DMBA 5S,6S-dihydrodiol formed from 7,12-DMBA by rat liver microsomes indicated that the major enantiomer of the 7-hydroxymethyl-12-methylbenz[a]anthracene trans-5,6-dihydrodiol formed by both fungal strains had a 5R,6R absolute stereochemistry. Direct resolution of the fungal trans-5,6-dihydrodiols by chiral stationary-phase high-performance liquid chromatography indicated that the ratios of the R,R and S,S enantiomers were 88:12 and 77:23 for S. racemosum and C. elegans, respectively. These results indicate that the fungal metabolism of 7,12-DMBA at the K region (5,6-position) is highly stereoselective and different from that reported for mammalian enzyme systems.     

The metabolism of 7,12-dimethylbenz[a]anthracene and 7-hydroxymethyl-12-methylbenz[a]anthracene by rat liver and adrenal homogenates and by rat adrenocortical cells

The metabolism of 3H-labelled 7,12-dimethylbenz[a]anthracene (DMBA) and of 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) into solvent- and water-soluble and protein-bound derivatives has been examined in rat liver and adrenal homogenates and in rat adrenocortical cells in culture. Although the overall extents of metabolism of the substrates by the two types of homogenate were similar, there was twice as much binding to protein in incubations with the 7-hydroxymethyl derivative. Rat adrenal cells in culture metabolized DMBA more extensively than 7-OHM-12-MBA and converted much more of the parent hydrocarbon into water-soluble derivatives. Both hydrocarbons were metabolized to yield dihydrodiols that were separated and identified by high performance liquid chromatography (HPLC). The 8,9-dihydrodiol was the major dihydrodiol formed from DMBA but, with 7-OHM-12-MBA as substrate, metabolism was diverted to the 10,11- and 3,4-positions in adrenal and hepatic preparations respectively. The viability of rat adrenocortical cells in culture, as measured by trypan blue exclusion, did not appear to be affected by treatment with DMBA, 7-OHM-12-MBA, the sulphate ester of 7-OHM-12-MBA or by 3,4-dihydro-3,4-dihydroxy-7-hydroxymethyl-12-methylbenz[a]anthracene.     

The formation of dihydrodiols by the chemical or enzymic oxidation of 7-hydroxymethyl-12-methylbenz[alpha]anthracene and the possible role of hydroxymethyl dihydrodiols in the metabolic activation of 7,12-dimethylbenz[alpha]anthracene.

The formation of dihydrodiols from 7-hydroxymethyl-12-methylbenz[alpha]anthracene by rat-liver microsomal fractions, by mouse skin in short-term organ culture and by chemical oxidation in an ascorbic acid/ferrous sulphate/EDTA system has been studied using a combination of thin-layer chromatography and high pressure liquie chromatography. The 3,4-, 8,9- and 10,11-dihydrodiols were formed in all three systems. The 5,6-dihydrodiol was formed in rat-liver microsomal fractions and in chemical oxidation but was not detected as a metabolite of [7-3H]hydroxymethyl-12-methylbenz[alpha]anthracene when this compound was incubated with mouse skin in short-term organ culture. The possible role of hydroxymethyl dihydrodiols in the in vivo metabolic activation of 7,12-dimethylbenz[alpha]anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed following the treatment of mouse skin in vivo with [7,12-3H]dimethylbenz[alpha]anthracene are not the same as those that are formed following the treatment of mouse skin under the same conditions with either 7-hydroxymethyl-12-methylbenz[alpha]anthracene or 7-methyl-12-hydroxymethylbenz[alpha]anthracene.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Regio- and stereoselective metabolism of 7,12-dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.     

Metabolic and stereoselective formations of non-K-region benz(a)anthracene 8,9- and 10,11-epoxides

The non-K-region benz[a]anthracene (BA) 8,9- and 10,11-epoxides were isolated by normal-phase high-performance liquid chromatography as rat liver microsomal metabolites of BA. The identities of these epoxides were established by ultraviolet and mass spectral analyses and were further validated by the microsomal epoxide hydrolase catalyzed conversion to BA trans-8,9-dihydrodiol and trans-10,11-dihydrodiol, respectively. Circular dichroism spectral analyses of the metabolically formed non-K-region epoxides and dihydrodiols and mass spectral analyses of metabolically formed 18O-labeled non-K-region dihydrodiols and their acid-catalyzed dehydration products indicated that BA (8R,9S)-epoxide and (10S,11R)-epoxide were the predominant enantiomers formed in the metabolism at the 8,9- and 10,11- aromatic double bonds of BA, respectively, by rat liver microsomes. This is the first example demonstrating the direct detection and stereoselective metabolic formation of non-K-region epoxides of a polycyclic aromatic hydrocarbon.     

The metabolism of 7- and 12-methylbenz[a]-anthracene and their derivatives

1. 7- and 12-Methylbenz[a]anthracene were converted by rat-liver homogenates into the corresponding hydroxymethyl derivatives, products that are probably the 8,9-dihydro-8,9-dihydroxy and the 5,6-dihydro-5,6-dihydroxy derivatives, and a number of phenolic products. 2. Both hydrocarbons were converted into glutathione conjugates; that from 7-methylbenz[a]anthracene was also formed, together with 5,6-dihydro-5,6-dihydroxy- and 5-hydroxy-benz[a]anthracene, from 5,6-epoxy-5,6-dihydro-7-methylbenz[a]anthracene. 3. 7- and 12-Hydroxymethyl-benz[a]anthracene were converted into products that are probably 8,9-dihydro-8,9-dihydroxy derivatives, and into phenols. 4. The preparation of a number of derivatives of the hydrocarbons is described. 5. The oxidation of the hydrocarbons with lead tetra-acetate was investigated.     

Effects of a fluoro substituent on the fungal metabolism of 1-fluoronaphthalene.

The metabolism of 1-fluoronaphthalene by Cunninghamella elegans ATCC 36112 was studied. The metabolites were isolated by reverse-phase high-pressure liquid chromatography and characterized by the application of UV absorption, 1H nuclear magnetic resonance, and mass spectral techniques. C. elegans oxidized 1-fluoronaphthalene predominantly at the 3,4- and 5,6-positions to form trans-3,4-dihydroxy-3,4-dihydro-1-fluoronaphthalene and trans-5,6-dihydroxy-5,6-dihydro-1-fluoronaphthalene. In addition, 1-fluoro-8-hydroxy-5-tetralone, 5-hydroxy-1-fluoronaphthalene, and 4-hydroxy-1-fluoronaphthalene as well as glucoside, sulfate, and glucuronic acid conjugates of these phenols were formed. Circular dichroism spectra of the trans-3,4- and trans-5,6-dihydrodiols formed from 1-fluoronaphthalene indicated that the major enantiomers of the dihydrodiols have S,S absolute stereochemistries. In contrast, the trans-5,6-dihydrodiol formed from 1-fluoronaphthalene from 3-methylcholanthrene-treated rats had Cotton effects that are opposite in sign (R,R) to those formed by C. elegans. The results indicate that the fungal monooxygenase-epoxide hydrolase systems are highly stereoselective in the metabolism of 1-fluoronaphthalene and that a fluoro substituent blocks epoxidation at the fluoro-substituted double bond, decreases oxidation at the aromatic double bond that is peri to the fluoro substituent, and enhances metabolism at the 3,4- and 5,6-positions of 1-fluoronaphthalene.     

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.     

Metabolism of 7,12-dimethylbenz[a]anthracene and its methyl-hydroxylated metabolites: formation of phenolic metabolites at the 2-positions.

The 1- and 2-positions of 7,12-dimethylbenz[a]anthracene (DMBA) were thought not to be involved in biotransformation to 1,2-epoxide and 1,2-dihydrodiol because of steric hindrance from the 12-methyl group (Biochem. Biophys. Res. Commun. $\text{85}$: 357–362, 1978). However, we have identified four 2-phenols as rat liver microsomal metabolites of DMBA and its methyl-hydroxylated metabolites, 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]-anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. Our findings suggest that neither the 12-methyl group nor the 12-hydroxymethyl group blocks the microsomal oxygenations of the 1,2 positions of DMBA or its methyl-hydroxylated derivatives. The 2-phenols may be formed as nonenzymatic rearrangement products of the 1,2-epoxide intermediates, although their formations by a direct hydroxylation mechanism cannot be ruled out.     

The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system.     

Absolute stereochemistry of the trans-dihydrodiols formed from benzo[a]anthracene by liver microsomes.

Through application of the exciton chirality method, absolute stereochemistry has been assigned to the (+)-and (-)-enantiomers of four of the five metabolically possible trans-dihydrodiols of the polycyclic hydrocarbon benzo[a]anthracene (BA). The (+)- and (-)-enantiomers of each of these dihydrodiols can be separated as their diastereomeric bis-esters with (-)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid by high pressure liquid chromatography (HPLC). BA 3,4-, 5,6-, 8,9- and 10,11-dihydrodiol are formed in 38%, 36%, 78% and 66% enantiometric purity, respectively, by liver microsomes from phenobarbital-treated rats, whereas the liver microsomes from 3-methylcholanthrene(MC)-treated rats form BA 5,6-, 8,9- and 10,11-dihydrodiols with higher optical purity (62%, 96% and 96%, respectively). BA 3,4-dihydrodiol is formed from (+/-)-BA 3,4-oxide by microsomal epoxide hydrase in very high enantiometric purity (78%). The major enantiomer of the BA dihydrodiols formed by liver enzymes has R,R absolute stereochemistry in each case. In parallel with previous studies on the metabolism of benzo[a]pyrene, the more tumorigenic (-)-enantiomer is the predominant isomer of BA 3,4-dihydrodiol formed by liver microsomes from BA.     

The formation of dihydrodiols in the chemical or enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,h]-anthracene and chrysene.

The formation of trans-dihydrodiols from dibenz[a,c]anthracene, dibenz[a,h]anthracene and chrysene by chemical oxidation in an ascorbic acid-ferrous sulphate-EDTA system and by rat-liver microsomal fractions has been studied using a combination of thin-layer (TLC) and high pressure liquid chromatography (HPLC) to separate the mixtures of isomeric dihydrodiols. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene, the 1,2-,3,4- and 5,6-dihydrodiols of dibenz[a,h]anthracene and the 1,2-, 3,4- and 5,6-dihydrodiols of chrysene were formed in chemical oxidations. These dihydrodiols were also formed when the three parent hydrocarbons were metabolized by rat-liver microsomal fractions and, in addition, dibenz[a,c]anthracene yielded the 10,11-dihydrodiol. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene have not been reported previously either as metabolites of the hydrocarbon or as products of chemical syntheses and the 5,6-dihydrodiol of chrysene was not detected in earlier metabolic studies.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The synthesis of dibenz[a,c]anthracene 10,11-oxide and its metabolism by rat liver preparations

The synthesis of dibenz[a,c]anthracene 10,11-oxide is described. The oxide was unstable and was rapidly decomposed with cold mineral acid into a mixture of 10- and 11- hydroxydibenz[a,c]anthracene. The oxide was converted by rat liver microsomal preparations and homogenates into a product that is probably 10,11-dihydro-10,11-dihydroxydibenz[a,c]anthracene and which was identical with the metabolite formed when dibenz[a,c]anthracene was metabolized by rat liver homogenates. The oxide did not react either chemically or enzymically with GSH. 10,11-Dihydrodibenz[a,c]anthracene and 10,11-dihydrodibenz[a,c]anthracene 12,13-oxide were both metabolized by rat liver preparations into trans-10,11,12,13-tetrahydro-10,11-dihydroxydibenz[a,c] anthracene and the oxide was converted chemically into this dihydroxy compound, and it reacted chemically but not enzymically with GSH. In the alkylation of 4-(p-nitrobenzyl)pyridine, the ;K-region' epoxide, dibenz[a,h]anthracene 5,6-oxide, was more active than either dibenz[a,c]anthracene 10,11-oxide or 10,11-dihydrobenz[a,c]anthracene 12,13-oxide.