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.     

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

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

Stereoselective fungal metabolism of methylated anthracenes.

The metabolism of 9-methylanthracene (9-MA), 9-hydroxymethylanthracene (9-OHMA), and 9,10-dimethylanthracene (9,10-DMA) by the fungus Cunninghamella elegans ATCC 36112 is described. The metabolites were isolated by high-performance liquid chromatography and characterized by UV-visible, mass, and 1H nuclear magnetic resonance spectral techniques. The compounds 9-MA and 9,10-DMA were metabolized by two pathways, one involving initial hydroxylation of the methyl group(s) and the other involving epoxidation of the 1,2- and 3,4- aromatic double bond positions, followed by enzymatic hydration to form hydroxymethyl trans-dihydrodiols. For 9-MA metabolism, the major metabolites identified were trans-1,2-dihydro-1,2-dihydroxy and trans-3,4-dihydro-3,4-dihydroxy derivatives of 9-MA and 9-OHMA. 9-OHMA was also metabolized to trans-1,2- and 3,4-dihydrodiol derivatives. The absolute configuration and optical purity were determined for each of the trans-dihydrodiols formed by fungal metabolism and compared with previously published circular dichroism spectral data obtained from rat liver microsomal metabolism of 9-MA, 9-OHMA, and 9,10-DMA. Circular dichroism spectral analysis revealed that the major enantiomer for each dihydrodiol was predominantly in the S,S configuration, in contrast to the predominantly R,R configuration of the trans-dihydrodiol formed by mammalian enzyme systems. These results indicate that C. elegans metabolizes methylated anthracenes in a highly stereoselective manner that is different from that reported for rat liver microsomes.     

Stereoselective fungal metabolism of methylated anthracenes

The metabolism of 9-methylanthracene (9-MA), 9-hydroxymethylanthracene (9-OHMA), and 9,10-dimethylanthracene (9,10-DMA) by the fungus Cunninghamella elegans ATCC 36112 is described. The metabolites were isolated by high-performance liquid chromatography and characterized by UV-visible, mass, and 1H nuclear magnetic resonance spectral techniques. The compounds 9-MA and 9,10-DMA were metabolized by two pathways, one involving initial hydroxylation of the methyl group(s) and the other involving epoxidation of the 1,2- and 3,4- aromatic double bond positions, followed by enzymatic hydration to form hydroxymethyl trans-dihydrodiols. For 9-MA metabolism, the major metabolites identified were trans-1,2-dihydro-1,2-dihydroxy and trans-3,4-dihydro-3,4-dihydroxy derivatives of 9-MA and 9-OHMA. 9-OHMA was also metabolized to trans-1,2- and 3,4-dihydrodiol derivatives. The absolute configuration and optical purity were determined for each of the trans-dihydrodiols formed by fungal metabolism and compared with previously published circular dichroism spectral data obtained from rat liver microsomal metabolism of 9-MA, 9-OHMA, and 9,10-DMA. Circular dichroism spectral analysis revealed that the major enantiomer for each dihydrodiol was predominantly in the S,S configuration, in contrast to the predominantly R,R configuration of the trans-dihydrodiol formed by mammalian enzyme systems. These results indicate that C. elegans metabolizes methylated anthracenes in a highly stereoselective manner that is different from that reported for rat liver microsomes.     

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Regio- and stereospecific oxidation of 9,10-dihydroanthracene and 9,10-dihydrophenanthrene by naphthalene dioxygenase: structure and absolute stereochemistry of metabolites.

The oxidation of 9,10-dihydroanthracene and 9,10-dihydrophenanthrene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas putida (NCIB 9816.4. Salicylate-induced cells of P. putida strain 9816/11 and isopropylthiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized 9,10-dihydroanthracene to (+)-cis-1R,2S)-1,2-dihydroxy-1,2,9,10-tetrahydroanthracene (> 95% relative yield; > 95% enantiomeric excess) as the major product. 9-Hydroxy-9,10-dihydroanthracene (< 5% relative yield) was a minor product formed by both organisms. The same cells oxidized 9,10-dihydrophenanthrene to (+)-cis-(3S,4R)-3,4-dihydroxy-3,4,9,10-tetrahydrophenanthrene (70% relative yield; > 95% enantiomeric excess) and (+)-(S)-9-hydroxy-9,10-dihydrophenanthrene (30% relative yield). The major reaction catalyzed by naphthalene dioxygenase with 9,10-dihydroanthracene and 9,10-dihydrophenanthrene was stereospecific dihydroxylation in which both of the previously undescribed cis-diene diols were of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The results suggest that for benzocylic substrates, the location of benzylic carbons influences the type of reaction(s) catalyzed by naphthalene dioxygenase.     

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

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

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

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

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

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

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

Metabolism of phenanthrene by the white rot fungus Pleurotus ostreatus.

The white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene, metabolized 94% of the phenanthrene added. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized to trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) (28%), 2,2'-diphenic acid (17%), and unidentified metabolites (7%). Nonextractable metabolites accounted for 35% of the total radioactivity. The metabolites were extracted with ethyl acetate, separated by reversed-phase high-performance liquid chromatography, and characterized by 1H nuclear magnetic resonance, mass spectrometry, and UV spectroscopy analyses. 18O2-labeling experiments indicated that one atom of oxygen was incorporated into the phenanthrene trans-9,10-dihydrodiol. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was 9R,10R, which is different from that of the principal enantiomer produced by Phanerochaete chrysosporium. Significantly less phenanthrene trans-9,10-dihydrodiol was observed in incubations with the cytochrome P-450 inhibitor SKF 525-A (77% decrease), 1-aminobenzotriazole (83% decrease), or fluoxetine (63% decrease). These experiments with cytochrome P-450 inhibitors and 18O2 labeling and the formation of phenanthrene trans-9R,10R-dihydrodiol as the predominant metabolite suggest that P. ostreatus initially oxidizes phenanthrene stereoselectively by a cytochrome P-450 monoxygenase and that this is followed by epoxide hydrolase-catalyzed hydration reactions.     

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.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

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

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

Initial Oxidation Products in the Metabolism of Pyrene, Anthracene, Fluorene, and Dibenzothiophene by the White Rot Fungus Pleurotus ostreatus

The initial metabolites in the degradation of pyrene, anthracene, fluorene, and dibenzothiophene by Pleurotus ostreatus were isolated by high-pressure liquid chromatography and characterized by UV-visible, gas-chromatographic, mass-spectrometric, and (sup1)H nuclear magnetic resonance spectral techniques. The metabolites from pyrene, dibenzothiophene, anthracene, and fluorene amounted to 45, 84, 64, and 96% of the total organic-solvent-extractable metabolites, respectively. Pyrene was metabolized predominantly to pyrene trans-4,5-dihydrodiol. Anthracene was metabolized predominantly to anthracene trans-1,2-dihydrodiol and 9,10-anthraquinone. In contrast, fluorene and dibenzothiophene were oxidized at the aliphatic bridges instead of the aromatic rings. Fluorene was oxidized to 9-fluorenol and 9-fluorenone; dibenzothiophene was oxidized to the sulfoxide and sulfone. Circular dichroism spectroscopy revealed that the major enantiomer of anthracene trans-1,2-dihydrodiol was predominantly in the S,S configuration and the major enantiomer of the pyrene trans-4,5-dihydrodiol was predominantly R,R. These results indicate that the white rot fungus P. ostreatus initially metabolizes polycyclic aromatic hydrocarbons by reactions similar to those previously reported for nonligninolytic fungi. However, P. ostreatus, in contrast to nonligninolytic fungi, can mineralize these polycyclic aromatic hydrocarbons. The identity of the dihydrodiol metabolites implicates a cytochrome P-450 monooxygenase mechanism.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.