Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.     

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.     

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The formation of dihydrodiols from benzo[alpha]pyrene by oxidation with an ascorbic acid/ferrous sulphate/EDTA system.

In the oxidation of benzo[alpha]pyrene in an abscorbic acid-ferrous sulphate-EDTA system, four dihydrodiols were detected. Three, trans-4,5-dihydro-4,5-dihydroxybenzo[alpha]pyrene, trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene were identified by their UV spectra and by direct comparisons of their chromatographic properties, using HPLC, with those of the authentic compounds. The fourth compound appeared to be trans-11,12-dihydro-11,12-dihydroxybenzo[alpha]pyrene since its ultraviolet spectrum was identical to that of the cis-dihydrodiol. Time-course experiments showed that the maximum amounts of products were obtained after 8 h of oxidation. A re-examination of the dihydrodiols formed from benzo[alpha]pyrene by rat-liver microsomal fractions failed to show the formation of the trans-11,12-dihydrodiol.     

Polycyclic aromatic hydrocarbon (PAH) ortho-quinone conjugate chemistry: kinetics of thiol addition to PAH ortho-quinones and structures of thioether adducts of naphthalene-1,2-dione.

Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of an NADP+ dependent oxidation of non-K-region trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase (EC 1.3.1.20). Since these PAH o-quinones could be detoxified by non-enzymatic or enzymatic conjugation with cellular thiols, their reactivity with 2-mercaptoethanol, cysteine and glutathione (GSH) was examined by ion-pair reverse phase high pressure liquid chromatography (RP-HPLC). Second-order rate constants for the addition of these thiols to naphthalene-1,2-dione (NPQ) in water ranging from 4.9 x 10(3) - 1.1 x 10(4) min-1 M-1 and the reactions were complete within 10 min. When these reactions were conducted at near physiological pH (50 mM potassium phosphate buffer pH 7.0), the rate constants increased by 2-orders of magnitude. When benzo[a]pyrene-7,8-dione (BPQ) was substituted in these reactions the second-order rate constants decreased by 2-3 orders of magnitude and the reactions took several hours to reach completion. The decrease in reactivity can be explained by the presence of the bay region in BPQ. Methylation influenced the reactivity of PAH o-quinones with GSH and the following order of reactivity was observed: 7,12-dimethyl-benz[a]anthracene-3,4-dione (7,12-DMBAQ) > 12-methyl-BAQ, 7-methyl-BAQ and BAQ > BPQ. Of these quinones 7,12-dimethyl-BAQ was almost equi-reactive with NPQ. This suggests that methyl substitution in the bay and peri regions enhances reactivity with GSH. Using NPQ as a model for other PAH o-quinones, N-acetyl-L-cysteine, L-cysteine and GSH conjugates of NPQ were synthesized and characterized by [1H]- and [13C]NMR. Evidence for Michael type 1,4-addition products was obtained in which the resultant adduct could exist as either a catechol or o-quinone. By contrast, L-cysteine was able to form adducts via S- or N-attack and N-attack gave a purple p-iminoquinone. There was no evidence for the formation of bis-N-acetyl-L-cysteinyl-, bis-glutathionyl adducts or phenolic coupled products. The toxicity of thiol conjugates of NPQ remains to be explored.     

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 preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent M_r of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with K_m values in the range of 1.4 to 7.1 μM, and exhibited a much higher Michaelis constant for NAD⁺ (K_m of 160 μM). At pH 7.0, the specificity constant ranged from (1.3 ± 0.1) × 10⁶ M⁻¹ s⁻¹ with benz[a]anthracene 1,2-dihydrodiol to (20.0 ± 0.8) × 10⁶ M⁻¹ s⁻¹ with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.     

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

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.     

Effects of 1-ethynylpyrene and related inhibitors of P450 isozymes upon benzo[a]pyrene metabolism by liver microsomes

The effects of three aryl acetylenes, 1-ethynylpyrene (EP), 2-ethynylnaphthalene (EN) and 3-ethynylperylene (EPE), upon the metabolism of benzo[a]pyrene (BaP) by microsomes isolated from rat liver were investigated. These aryl acetylenes all inhibited the total metabolism of BaP. Formation of BaP 7,8-dihydrodiol and BaP tetrol products by microsomal preparations from rats that had been pretreated with 3-methylcholanthrene (3MC) were preferentially inhibited. The effects of EP upon the metabolism of BaP 7,8-dihydrodiol by microsomes from rat liver were also studied. This aryl acetylene strongly inhibited the formation of BaP tetrols from BaP 7,8-dihydrodiol by liver microsomes both from untreated rats and from rats pretreated with 3MC, but enhanced the conversion of the BaP dihydrodiol into other metabolites.     

Two K-regions of 5-methylchrysene are sites of oxidative metabolism

Two K-region trans-dihydrodiols were identified as products formed in the metabolism of 5-methylchrysene by liver microsomes from phenobarbital-treated male Sprague-Dawley rats. These two dihydrodiols were isolated from a mixture of metabolites by reversed-phase and normal-phase high-performance liquid chromatographies. Both K-region dihydrodiols were characterized by ultra-violet, mass, and circular dichroism spectral analyses. Chiral stationary phase high-performance liquid chromatographic analyses indicated that 5-methylchrysene 5,6-dihydrodiol and 11,12-dihydrodiol contain (S,S): (R,R) enantiomer ratios of 2:98 and 12:88, respectively. Although it is a bay-region dihydrodiol, the hydroxyl groups of 5-methylchrysene trans-5,6-dihydrodiol adopt a quasidiequatorial conformation.     

Characterization of mercapturic acid and glutathionyl conjugates of benzo[a]pyrene-7,8-dione by two-dimensional NMR.

Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine, and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dione and either N-acetyl-L-cysteine or glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[a]pyrene (a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[a]pyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[a]pyrenyl-peptide conjugate.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Microsomal metabolism of picene

Picene, a polycyclic aromatic hydrocarbon (PAH) of environmental relevance has recently been predicted to be carcinogenic, based on quantum mechanical calculation, although in several animal studies no carcinogenicity could be detected. In order to find out if the metabolism of this PAH can provide an explanation for its lack of carcinogenicity, picene was incubated with the hepatic microsomal fraction of Sprague-Dawley rats, which had been pretreated with Aroclor 1254. Sixteen ethyl acetate-extractable metabolites could be separated by reversed-phase high-performance liquid chromatography. Comparison of the chromatographic behavior and the UV and mass spectral properties of the metabolites with those of synthetic derivatives of picene allowed the identification of trans-1,2-, -3,4-, -5,6-dihydrodiol as well as 2- and 4-phenol as microsomal metabolites of picene. At a substrate concentration of 2.7 microM and an amount of 68 micrograms microsomal protein per ml incubation volume, 4-picenol was the main microsomal metabolite with 32.2% of total metabolic conversion, followed by the 1,2-(bay-region)dihydrodiol with 16.7%, the 3,4-(M-region)dihydrodiol with 15.9%, 2-picenol with 9.1% and the 5,6-(K-region)dihydrodiol with 1.6%. In this respect the metabolism of picene is not significantly different from that of the carcinogenic PAH benzo[a]pyrene and dibenz[a,h]anthracene. The M-region dihydrodiols, potential precursors of electrophilically reactive dihydrodiol bay-region epoxides, are formed from all three PAHs at 11-16% of total metabolic conversion. From the 2.8- to 4.4-fold lower amounts of polar and water-soluble metabolites of picene as compared to dibenz[a,h]anthracene and benzo[a]pyrene it is deduced that dihydrodiol epoxides are generated from picene to a much smaller extent than from the two carcinogenic PAHs. The lacking carcinogenicity of picene could therefore result from the inability of microsomal enzymes to transform its M-region dihydrodiol to dihydrodiol bay-region epoxides in amounts necessary to initiate carcinogenesis.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.