The metabolic activation of benz[a]anthracene in hamster embryo cells: evidence that diol-epoxides react with guanosine, deoxyguanosine and adenosine in nucleic acids

The principal nucleoside-hydrocarbon adducts present in hydrolysates of RNA and DNA isolated from hamster embryo cells treated with benz[a]anthracene (BA) were examined by chromatography on Sephadex LH 20 and by high pressure liquid chromatography (HPLC) on Spherisorb 5 ODS. The results extend the previous finding that a non-'bay-region' diol-epoxide, anti-BA-8,9-diol 10,11-oxide (r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a] anthracene) is involved in the binding of BA to cellular nucleic acids and show that this diol-epoxide most probably reacts with guanosine and adenosine in RNA and with deoxyguanosine in DNA. The results also show that a 'bay-region' diol-epoxide anti-BA-3,4-diol 1,2-oxide (t-3,-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene to DNA in some situations, reacts mainly with deoxyguanosine.     

The contribution of specific cytochromes P-450 in the metabolism of 7,12-dimethylbenz[a]anthracene in rat and human liver microsomal membranes

The role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) has been studied in microsomal membranes from rat and human liver. An antibody inhibition study using membranes from phenobarbital-treated rats demonstrates that a member(s) of the CYP2C family accounts for up to 90% of the formation of the proximate carcinogen, DMBA-3,4-diol, and makes significant contributions to the formation of DMBA-5,6-diol and DMBA-8,9-diol. In these membranes the formation of DMBA-5,6-diol can be entirely accounted by the combined activity of members of the CYP2C and CYP2B families. The metabolism of DMBA has been investigated in human using microsomes from 10 individuals and the metabolites formed by these membranes were found to be mainly hydroxymethyl- and -diol products. The rates of formation of each metabolite show considerable interindividual variation and there was no correlation between these rates for any pairing of metabolites. The CYP content in these membranes of specific members of families 1, 2, 3 and 4 did correlate with the rates of formation of individual metabolites. Surprisingly there was no correlation between the content of CYP2C and formation of DMBA-3,4-diol but an antibody to rat CYP2C6 partially inhibited the formation of this metabolite. The results indicate that in human both inducible sub-families of CYPs, particularly of the PB-type, and constitutively expressed CYPs may be important in DMBA metabolism and that each metabolite may be produced by the combined activity of several CYP isoforms.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

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.     

Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

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.     

Degradation of benz[<Emphasis Type="Italic">a</Emphasis>]anthracene by <Emphasis Type="Italic">Mycobacterium vanbaalenii</Emphasis> strain PYR-1

Cultures of Mycobacterium vanbaalenii strain PYR-1 grown in mineral salts medium and nutrients in the presence of benz[a]anthracene metabolized 15% of the added benz[a]anthracene after 12days of incubation. Neutral and acidic ethyl acetate extractable metabolites were isolated and characterized by high performance liquid chromatography (HPLC) and uv–visible absorption, gas chromatography/mass (GC/MS) and nuclear magnetic resonance (NMR) spectral analysis. Trimethylsilylation of the metabolitesfollowed by GC/MS analysis facilitated identification of metabolites. The characterization of metabolites indicated that M. vanbaalenii initiated attack of benz[a]anthracene at the C-1,2-, C-5,6-, C-7,12- and C-10,11-positions to form dihydroxylated and methoxylated intermediates. The major site of enzymatic attack was in the C-10, C-11 positions. Subsequent ortho- and meta-cleavage of each of the aromatic rings led to the accumulation of novel ring-fission metabolites in the medium. The major metabolites identified were 3-hydrobenzo[f]isobenzofuran-1-one (3.2%), 6-hydrofuran[3,4-g]chromene-2,8-dione (1.3%), benzo[g]chromene-2-one (1.7%), naphtho[2,1-g]chromen-10-one (48.1%), 10-hydroxy-11-methoxybenz[a]anthracene (9.3%), and 10,11-dimethoxybenz[a]anthracene (36.4%). Enzymatic attack at the C-7 and C-12 positions resulted in the formation of benz[a]anthracene-7,12-dione, 1-(2-hydroxybenzoyl)-2-naphthoic acid, and 1-benzoyl-2-naphthoic acid. A phenyl-naphthyl metabolite, 3-(2-carboxylphenyl)-2-naphthoic acid, was formed when M. vanbaalenii was incubated with benz[a]anthracene cis-5,6-dihydrodiol, indicating ortho-cleavage of 5,6-dihydroxybenz[a]anthracene. A minor amount of 5,6-dimethoxybenz[a]anthracene was also formed. The data extend and propose novel pathways for the bacterial metabolism of benz[a]anthracene.     

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.     

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 oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.     

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.     

Cytochrome P-450-catalyzed stereoselective epoxidation at the K region of benz[a]anthracene and benzo[a]pyrene

The enantiomers of K-region benz[a]anthracene (BA) 5,6-epoxide and benzo[a]pyrene (BP) 4,5-epoxide were resolved by chiral stationary-phase high-performance liquid chromatography (CSP-HPLC). The K-region epoxides formed in the metabolism of BA by liver microsomes from untreated (control), phenobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats were determined by CSP-HPLC to have a 5R,6S/5S,6R enantiomer ratio of 25:75, 21:79, and 4:96, respectively. The K-region 4,5-epoxide formed in the metabolism of BP by the same rat liver microsomal preparations contained a 4R,5S/4S,5R enantiomer ratio of 48:52 (control), 40:60 (PB), and 5:95 (MC), respectively. The results indicate that various cytochrome P-450 isozymes of rat liver exhibit different stereoselective properties in catalyzing the epoxidation reactions at the K region of BA and of BP.     

The induction of sister-chromatid exchanges in Chinese hamster ovary cells by K-region epoxides and some dihydrodiols derived from benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene

Experiments were performed to investigate the effects of 3 polycyclic aromatic hydrocarbons, benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene and K-regio epoxides and some of their related dihydrodiols on the chromosomes of Chinese hamster ovary cells in vitro. Of the 3 hydrocarbons only benz[a]anthracene showed any activity in inducing sister-chromatid exchanges. The K-region epoxide and the 3,4-dihydrodiol have been found to be more active than the corresponding K-region or the other non K-region dihydrodiols derived from benz[a]anthracene. Athough dibenz[a,c]anthracene was almost inactive, the K-region 5,6-epoxide and all 3 possible dihydrodiols, the 1,2-, 3,4- and 10,11-diols were active in inducing increased numbers of sister-chromatid exchanges in the chromosomes of these cells. The 3,4-dihydrodiol of dibenz[a,h]anthrecene was also active in inducing sister-chromatid exchanges whereas the 1,2- and 5,6-dihydrodiols were only weakly active. This study provides some support for the suggestiion that the activation of these 3 hydrocarbons proceeds by the metabolic conversion of non K-region dihydrodiols into vicinal diol-epoxides.     

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.     

Bacterial mutagenicity of new cyclopenta-fused cata-annelated polycyclic aromatic hydrocarbons, and identification of the major metabolites of benz[j]acephenanthrylene formed by Aroclor-treated rat liver microsomes

Three novel cyclopenta-fused polycyclic aromatic hydrocarbons were synthesized, benz[d]aceanthrylene, benz[k]aceanthrylene, and benz[j]acephenanthrylene, and evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. The two benzaceanthrylene derivatives were active at low S9 concentrations in strain TA98 (4 and 27 rev/nmole respectively), as had been predicted from the calculated delta Edeloc/beta values of the carbocations derived from opening of the cyclopenta-fused epoxide rings, but the majority of this mutagenicity appeared to be due to free-radical decomposition products of spontaneous endo-peroxide formation. These compounds were therefore not further investigated. Benz[j]acephenanthrylene was also an indirect-acting frameshift mutagen (8-12 rev/nmole in strain TA98), but unlike most of the previously assayed cyclopenta-fused polycyclic aromatic hydrocarbons exhibited no peak of activity at low S9 protein concentration. The principal metabolites formed from this compound by microsomes from Aroclor-treated rat liver were benz[j]acephenanthrylene-4,5-dihydro-4,5-diol (necessarily derived from hydration of benz[j]acephenanthrylene 4,5-oxide) and benz[j]acephenanthrylene-9,10-dihydro-9,10-diol (precursor to benz[j]acephenanthrylene-9,10-dihydrodiol 7,8-oxide, the bay-region diol-epoxide). Consideration of the reduced activity of this compound compared to the related structure chrysene, the S9 dependence curves, and the predicted delta Edeloc/beta values of the postulate active species, suggests that in contrast to most other cyclopenta-fused polycyclic aromatic hydrocarbons, bay-region diol-epoxide formation plays a greater role than epoxidation of the cyclopenta-fused ring in the metabolic activation of benz[j]acephenanthrylene.     

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.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Hydroperoxide-dependent epoxidation of 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene by ram seminal vesicle microsomes and by hematin

Addition of arachidonic acid to ram seminal vesicle microsomes oxidizes 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene (BA-3,4-diol) to five more polar products. Four of the products are identified by chromatographic and spectroscopic analysis as tetrahydrotetraols, which are solvolysis products of dihydrodiolepoxides. The fifth product is a 10-methyl ether formed by methanolysis of the anti-diolepoxide. Quantitation of the individual products indicates that anti-diolepoxides predominate over syn-diolepoxides by approximately 2:1. Identical product profiles are detected from the reaction of BA-3,4-diol with hematin and 13-hydroperoxy-octadecadienoic acid in the presence of Tween 20. No other products are detected in either system, which indicates that peroxyl radicals oxidize BA-3,4-diol exclusively by epoxidation of the 1,2-double bond. The stereochemical and regiochemical differences between oxidation of BA-3,4-diol by peroxyl radicals and cytochrome P-450 are dramatic and suggest that BA-3,4-diol is uniquely suited as a probe to quantitate peroxyl radical-dependent epoxidation in vitro and in vivo.     

The involvement of a non-'bay-region' diol-epoxide in the formation of benz(a)anthracene-DNA adducts in a rat-liver microsomal system

The metabolic activation of benz(a)anthracene was investigated by incubating [³H]-benz(a)anthracene with DNA, a NADPH-generating system and rat-liver microsomes. When hydrolysates of the DNA were chromatographed on Sephadex LH20 columns, three hydrocarbon-nucleoside adduct peaks were resolved and these were further examined using HPLC. One adduct probably results from the reaction of the non-bay-region diol-epoxide $\text{r}$-8,$\text{t}$-9-dihydroxy-$\text{t}$-10,11-oxy-8,9,10,11-tetrahydrobenz(a)anthracene $\text{(}\text{anti}$-BA-8,9-diol 10,11-oxide) with DNA. The other two adducts did not co-chromatograph with adducts formed from any of the four possible isomeric diolepoxides that can be formed in the 8,9,10,11-ring of benz(a)anthracene.     

Regioselectivity in rat microsomal metabolism of benzo[a]pyrene: evidence for involvement of two distinct binding sites

The metabolic profile of benzo[a]pyrene (BP) in cumene hydroperoxide-(CHP)-dependent reaction by male rat liver microsomes was dependent on CHP concentration. At 0.05 mM CHP, 3-hydroxy-BP was the major metabolite. Increase in CHP reduced 3-hydroxy-BP formation but increased BP quinone formation simultaneously. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene (PY) selectively inhibited quinone formation but enhanced 3-hydroxy-BP formation. Naphthalene (NP) had no effect on BP quinone formation but inhibited BP 3-hydroxylation. Phenanthrene (PA) and benz[a]anthracene (BA) inhibited effectively 3-hydroxy-BP formation but only slightly quinone formation. BP binding to microsomal protein correlated to quinone formation and not BP 3-hydroxylation. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less efficient than the male. Quinones were consistently predominant metabolites and their formation was also inhibited by pyrene. Our data provide evidence that regioselectivity in BP metabolism involves at least two distinct binding sites. One site recognizes the benzo region of BP in BP 3-hydroxylation and the other recognizes the pyrene region in quinone formation. The different ratios of 3-hydroxy-BP to quinone formation by male and female rat liver microsomes suggest that the two binding sites are probably located at separate cytochrome P-450 isozymes.