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.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

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.     

Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[a]anthracene

We have examined the selectivity of rat liver microsomal epoxide hydrolase (EC 3.3.2.3) toward all of the possible positional isomers of benzo-ring diol epoxides and tetrahydroepoxides of benz[a]anthracene, as well as the 1,2-diol 3,4-epoxides of triphenylene. This set includes compounds with no bay region in the vicinity of the benzo-ring, a bay-region diol group, a bay-region epoxide group, and (for the triphenylene derivatives) both a bay-region diol and a bay-region epoxide. In all cases where both the tetrahydroepoxides and the corresponding diol epoxides were examined, there is a large retarding effect of hydroxyl substitution on the rate of the enzyme-catalyzed hydration. When the tetrahydroepoxides are fair or poor substrates (epoxide group in the 1,2-, 8,9-, or 10,11-position), the additional retardation introduced by adjacent hydroxyl groups causes the enzyme-catalyzed hydrolysis of the corresponding diol epoxides to be insignificantly slow or nonexistent. In contrast, a benz[a]anthracene derivative with an epoxide group in the 3,4-position, (-)-tetrahydrobenz[a]anthracene (3R,4S)-epoxide, has been identified as the best substrate known for epoxide hydrolase, with a Vmax at 37 degrees C and pH 8.4 of 6800 nmol/min/mg of protein, and the two diastereomeric (+/-)-benz[a]anthracene 1,2-diol 3,4-epoxides, unlike all the other diol epoxides examined to date, are moderately good substrates for epoxide hydrolase. This novel observation is accounted for by the fact that the very high reactivity of the tetrahydrobenz[a]anthracene 3,4-epoxide system towards epoxide hydrolase is large enough to overcome a kinetically unfavorable effect of hydroxyl substitution. The enantioselectivity and positional selectivity of the enzyme have been determined for the tetrahydro-1,2- and -3,4-epoxides of benz[a]anthracene as well as the 1,2-diol 3,4-epoxides. When the epoxide is located in the 3,4-position, the benzylic carbon is the preferred site of attack, whereas for the enantiomers of the bay-region tetrahydro-1,2-epoxides, the chemically less reactive non-benzylic carbon is preferred. The regio- and enantioselectivity of epoxide hydrolase are discussed in terms of a possible model for the hydrophobic binding site of this enzyme.     

Comparative studies of the metabolic activation of chrysene in rodent and human skin

Metabolism and activation of chrysene was examined in mouse, rat and human skin using a short-term organ culture technique. Mouse skin released larger quantities of free dihydrodiols into the culture medium than either rat or human skin and greater quantities of chrysene metabolites became covalently bound to the DNA of mouse skin. The stereochemistry of the chrysene-1,2-diol that was formed by each skin type was examined using high-performance liquid chromatography (HPLC) with a chiral stationary phase to resolve the enantiomers. It was found that in each case the (-)-enantiomer predominated. When hydrolysates of DNA extracted from rodent or human skin that had been treated with 3H-labelled chrysene were chromatographed on Sephadex LH-20 columns, the elution profiles of the hydrocarbon-DNA adducts were found to vary between the species studied. Further examination using HPLC showed that some of the adducts formed in skin had the chromatographic characteristics of adducts formed when the anti-isomer of the 'bay-region' diol-epoxide of chrysene (r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene) reacted with DNA and that others had the characteristics of triol-epoxide adducts.     

Gender-specific metabolism of benz[a]anthracene in hepatic microsomes from Long-Evans and Hooded Lister rats

The cytochrome P450 isoforms responsible for the regio-selective metabolism of benz[a]anthracene (BA) are poorly defined but as with other polycyclic aromatic hydrocarbons (PAHs) may include members of the CYP2C sub-family. Since the expression of some of these is regulated in a gender-specific manner and may be altered by age, rat strain or by phenobarbital treatment, the effects of these variables on metabolism of BA to diols was investigated. These studies used hepatic, microsomal membranes from immature and adult Long-Evans rats and adult Hooded Lister rats. BA-diols were resolved by normal phase HPLC into three discrete peaks identified as benz[a]anthracene-5,6-diol (BA-5,6-diol), benz[a]anthracene-10, 11-diol (BA-10,11-diol) and a mixture of benz[a]anthracene-3,4- and -8,9-diols (BA-3,4-diol and BA-8,9-diol and termed Peak(3/8)). Significant gender-related differences were found in the rates of diol formation in adults of both the Long-Evans and Hooded Lister rat strains. Formation of BA-10,11-diol and to a lesser extent the components of Peak(3/8) were greater in the male compared to female animals by factors of at least 14 and two, respectively. An age-dependent effect is also observed in the Long-Evans rat since these differences are still apparent in prepubertal animals but to a lesser extent (gender ratio male:female BA-10,11-diol 9X; Peak(3/8) 1.4X). In contrast BA-5,6-diol was formed at similar rates by membranes from female and male rats whether mature (Long-Evans and Hooded Lister) or immature (Long-Evans). Phenobarbital treatment of the adult Long-Evans rats resulted in a moderate increase in the formation of each diol other than at the 10,11-position and the induction was not gender specific. The rate of formation of BA-10, 11-diol was decreased in phenobarbital-treated male rats suggesting modulation of a male specific isoform. Measurement of microsomal epoxide hydrolase revealed no gender or age differences and suggests that this enzyme is not rate limiting in BA-diol formation and thus is not responsible for the differences in BA-diol formation observed. The results suggest that CYP2C11 along with a male-specific isoenzyme not regulated by age are important in the formation of BA-10,11-diol and a component(s) of Peak(3/8) in males. CYPs 2B2 and/or 2C6 appear to be involved in formation of BA-5,6-diol in male and female. Identification of the CYPs involved in the regio-selective metabolism of BA may lead to an explanation of the lower carcinogenic potency of this PAH compared to dimethylbenz[a]anthracene and this study provides novel clues concerning the identities of the CYPs, which are important.     

Reactions of polycyclic hydrocarbon epoxides with RNA and polyribonucleotides

RNA, poly(G) and poly(A) were reacted with benz[a]anthracene 5,6-oxide or with 7,12-dimethylbenz[a]anthracene 5,6-oxide and hydrolysates of the alkylated polymers examined using a combination of Sephadex LH20 column chromatography and thin-layer chromatography on silica gel. The results show that two RNA products are formed in reactions with benz[a]anthracene 5,6-oxide, one resulting from reaction with guanine and the other from reaction with adenine. With 7,12-dimethylbenz[a]anthracene 5,6-oxide, six RNA products appeared to be formed, two resulting from reactions with guanine and three from alkylation of adenine; the other product has not been identified.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation of benz[a]anthracene 8,9-oxide and 10,11-dihydrobenz[a]anthracene 8,9-oxide and their metabolism by rat liver preparations

The syntheses of 10,11-dihydrobenz[a]anthracene 8,9-oxide, benz[a]anthracene 8,9-oxide and 9-hydroxybenz[a]anthracene are described, together with those of a number of related compounds. The epoxides react both chemically and enzymically with water to yield the corresponding dihydrodiols and with reduced glutathione to form glutathione conjugates, and they react chemically with N-acetylcysteine to yield the corresponding mercapturic acids. 8,9-Dihydro-8,9-dihydroxybenz[a]anthracene, formed enzymically from benz[a]anthracene 8,9-oxide, was identical with a dihydrodiol formed when benz[a]anthracene was metabolized by rat liver homogenates. Similarly 10,11-dihydrobenz[a]anthracene 8,9-oxide yielded a dihydrodiol identical with the product formed when 10,11-dihydrobenz[a]anthracene was metabolized.     

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.     

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.     

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 oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.     

Additional evidence for the involvement of the 3,4-diol 1,2-oxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin

The role of vicinal diol-epoxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene to intermediates that react with nucleic acids was investigated using Sephadex LH-20 column chromatography and high pressure liquid chromatography. The results show that some of the hydrocarbon-DNA products formed in mouse skin treated in vivo with 7,12-dimethylbenz[a]anthracene arise from the reaction of DNA with 3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a]anthracene 1,2-oxides which, on the basis of this and other evidence, appears to be a biologically-active metabolite of 7,12-dimethylbenz[a]anthracene. However, since other nucleic acid-hydrocarbon adducts were also present that have not been identified as resulting from the reaction of the 3,4-diol 1,2-oxides with DNA, other mechanisms may also be involved in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin.     

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.     

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 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.     

Benz[j]aceanthrylene: a novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (congruent to 10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.     

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.     

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.