Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

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.     

Reactions of stereoisomeric and structurally related bay region diol epoxide derivatives of benz[a]anthracene with DNA. Conformations of noncovalent complexes and covalent carcinogen-DNA adducts

The modes of reaction of the tumorigenic bay region diol epoxide anti-BADE [+/-)-trans-3,4-diol-anti-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthr acene) and the less potent tumor initiating diastereomer syn-BADE [+/-)-trans-3,4-diol-syn-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthra cene) with native, double-stranded DNA were compared. The bay-region diol epoxide derived from 3-methylcholanthrene (3-MCDE, racemic trans-9,10-diol-anti-7,8-epoxy-7,8,9,10-tetrahydromethylcholanthrene+ ++) was included in this study in order to assess the effects of the methyl and methylene substituents on the reactivity with DNA. Utilizing linear dichroism and other spectroscopic methods, it is shown that all three diol epoxides forn non-covalent complexes with DNA. The diastereomers anti-BADE and syn-BADE form intercalative physical complexes, but the association constant K of the syn-diastereomer is about 6-7 times smaller than for anti-BADE; this effect is ascribed to the bulky quasi-diaxial conformation of the diol epoxide ring in the syn diastereomer. The value of K (4000 M-1) is similar for anti-BADE and 3-MCDE, although the latter is not intercalated in the classical sense since the short axis of the molecule is tilted closer to the axis of the DNA double helix. The conformations of the covalent DNA adducts are interpreted in terms of a quasi-intercalative conformation (site I), and a conformation in which the long axes of the polycyclic molecules are tilted closer to the axis of the helix (site II). Both tumorigens, anti-BADE and 3-MCDE, undergo a marked re-orientation from a non-covalent site I to a covalent site II conformation upon binding chemically with the DNA bases, although a small fraction of the covalent anti-BADE adducts remains quasi-intercalated; in contrast, the alkyl substituents in 3-MCDE not only prevent the formation of intercalative physical complexes, but also the formation of site I covalent adducts. In the case of the less tumorigenic syn-BADE, both the non-covalent complexes and the covalent adducts are of the site I-type. The bay-region diol epoxide of benz[a]anthracene and of 3-methylcholanthrene display a similar pattern of reactivities and covalent adduct conformations as the bay region diol epoxide derivatives of benz[a]pyrene, suggesting that adduct conformation might be an important factor in determining the levels of mutagenic and tumorigenic activities of this class of compounds.     

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.     

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.     

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

Effect of methylation on the conformer stability and reactivity of the bay-region diol-epoxides of chrysene

The relative stabilities of conformers of the bay-region tetrahydroepoxide of methylated chrysene have been calculated. From these calculations on tetrahydroepoxides, one infers that substitution of a methyl group in the same bay-region as the epoxide should destabilize both syn-diaxial and anti-diequatorial bay-region diol-epoxide diastereomers with respect to the syn-diequatorial and anti-diaxial diastereomers. The results of these calculations, together with recent experimental observations, suggest that the enhanced in vivo binding to DNA of the isomer having the methyl group and the epoxide in the same bay-region (1,2-diol-3,4-epoxide of 5-MeC) might be partially due to this destabilization of the syn-diaxial diastereomer. The carbocation delocalization energies associated with epoxide ring opening of the methylated bay-region tetrahydroepoxide isomers of chrysene are also given.     

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.     

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.     

Location of the epoxide function determines specificity of the allelic variants of human glutathione transferase Pi toward benzo[c]chrysene diol epoxide isomers

Carcinogenic activity of many polycyclic aromatic hydrocarbons (PAHs) is mainly attributed to their respective diol epoxides, which can be classified as either bay or fjord region depending upon the location of the epoxide function. The Pi class human glutathione (GSH) transferase (hGSTP1-1), which is polymorphic in humans with respect to amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), plays an important role in the detoxification of PAH-diol epoxides. Here, we report that the location of the epoxide function determines specificity of allelic variants of hGSTP1-1 toward racemic anti-diol epoxide isomers of benzo[c]chrysene (B[c]C). The catalytic efficiency (k(cat)/K(m)) of V104,A113 (VA) and V104,V113 (VV) variants of hGSTP1-1 was approximately 2.3- and 1.7-fold higher, respectively, than that of the I104,A113 (IA) isoform toward bay region isomer (+/-)-anti-B[c]C-1,2-diol-3,4-epoxide. On the other hand, the IA variant was approximately 1.6- and 3.5-fold more efficient than VA and VV isoforms, respectively, in catalyzing the GSH conjugation of fjord region isomer (+/-)-anti-B[c]C-9,10-diol-11,12-epoxide. The results of the present study clearly indicate that the location of the epoxide function determines specificity of the allelic variants of hGSTP1-1 in the GSH conjugation of activated diol epoxide isomers of B[c]C.     

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.     

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.     

Epoxide hydrolase activity of Chryseomonas luteola for the asymmetric hydrolysis of aliphatic mono-substituted epoxides

Asymmetric hydrolysis of a homologous range of straight chain 1,2-epoxyalkanes was achieved using whole cells of Chryseomonas luteola. Depending on the chain length, hydrolyses of the racemic epoxides afforded optically active epoxides and diols with varying degrees of optical purity. In the case of 1,2-epoxyoctane, the enantiomeric excess of the remaining (S)-epoxide and formed (R)-diol was excellent (ees > 98% and eep = 86%). This is the first report of a bacterial epoxide hydrolase with such unusual enantioselectivity for terminal mono-substituted epoxides bearing no directing group on the chiral C-2 carbon. Benzyl glycidyl ether and the 2,2-disubstituted epoxide, 2-methyl-1,2-epoxyheptane, were hydrolysed, but no enantioselectivity was observed. © Rapid Science Ltd. 1998     

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.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

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.     

Stereoselective metabolism of the optical isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene to bay-region diol epoxides by rat liver microsomes

Enantiomerically pure isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene have been obtained by chromatographic separation of their diastereomeric bis esters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. Liver microsomes from control rats, as well as rats treated with phenobarbital or 3-methylcholanthrene, metabolize these dihydrodiols to a pair of diastereomerically related bay-region 1,2-diol-3,4-epoxides in which the benzylic hydroxyl group and the epoxide oxygen are either cis (isomer-1) or trans (isomer-2) to each other. In general, diol epoxide-1 was the major metabolite of the (+)-(1S,2S)-dihydrodiol, whereas diol epoxide-2 was the major metabolite of the (?)-(1R-2R)-dihydrodiol. The extent of this stereoselectivity is dependent on the source of the microsomes and is greatest for liver microsomes from 3-methylcholanthrene-treated rats; the ratio of diol epoxide-1 relative to diol epoxide-2 was 5.6 : 1 with the (+)-enantiomer as substrate and 1 : 5.5 with the (?)-enantiomer as substrate. For a given microsomal preparation, rates of metabolism were independent of the enantiomer composition of the substrate. Relative to microsomes from control animals, treatment of rats with 3-methylcholanthrene enhanced rates of metabolism by about 40%, whereas treatment with phenobarbital decreased rates to a similar extent when the amounts of metabolites formed per nanomole of cytochrome P?450 were compared. The failure of treatment by 3-methylcholanthrene to enhance markedly the rate of metabolism of a polycyclic aromatic hydrocarbon substrate is unusual.     

HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[a,h]anthracene in skin.

Mouse skin and human skin have been treated in vivo or in short-term organ culture with dibenz[a,h]anthracene (DB[a,h]A), the related 3,4- or 5,6-diols or the anti- or syn-3,4-diol 1,2-oxides. DNA hydrolysates have been 32P-postlabelled and the adducts present examined by HPLC using a phenyl-modified reverse phase column and, for comparison, by PEI-cellulose TLC and autoradiography. The adducts formed when the diol-epoxides were reacted with salmon sperm DNA were also examined. The results show that in mouse skin treated in vivo, the major adducts formed from DB[a,h]A and the 3,4-diol were the same and that two of them were more polar than those formed in skin or in DNA that had been treated with the related anti- or syn-diol epoxides. Human skin treated with DB[a,h]A in culture yielded an adduct profile that was qualitatively similar to the profiles obtained with mouse skin.     

CP-arene oxides: the ultimate, active mutagenic forms of cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs)

The bacterial mutagenic response (Ames-assay, Salmonella typhimurium strain TA98+/-S9-mix) of a series of monocyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) identified in combustion exhausts, viz. cyclopenta[cd]pyrene (1), acephenanthrylene (2), aceanthrylene (3) and cyclopenta[hi]chrysene (4), is re-evaluated. The mutagenic effects are compared with those exerted by the corresponding partially hydrogenated derivatives, 3,4-dihydrocyclopenta[cd]pyrene (5), 4,5-dihydroacephenanthrylene (6), 1,2-dihydroaceanthrylene (7) and 4,5-dihydrocyclopenta[hi]chrysene (8). It is shown that the olefinic bond of the externally fused five-membered ring of 1, 3 and 4 is of importance for a positive mutagenic response. In contrast, whilst CP-PAH 2 is found inactive, its dihydro analogue (6) shows a weak metabolism-dependent response. The importance of epoxide formation at the external olefinic bond in the five-membered ring is substantiated by the bacterial mutagenic response of independently synthesized cyclopenta[cd]pyrene-3,4-epoxide (9), acephenanthrylene-4,5-epoxide (10), aceanthrylene-1,2-epoxide (11) and cyclopenta[hi]chrysene-4,5-epoxide (12). Their role as ultimate, active mutagenic forms, when CP-PAHs 1, 3 and 4 exhibit a positive mutagenic response, is confirmed. Semi-empirical Austin Model 1 (AM1) calculations on the formation of the CP-arene oxides (9-12) and their conversion into the monohydroxy-carbocations (9a-12a and 9b-12b) via epoxide-ring opening support our results. For 2 and 4, which also possess a bay-region besides an annelated cyclopenta moiety, the calculations rationalize that epoxidation at the olefinic bond of the cyclopenta moiety is favoured.