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.     

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.     

Cometabolic oxidation of phenanthrene to phenanthrene trans-9,10-dihydrodiol by Mycobacterium strain S1 growing on anthracene in the presence of phenanthrene.

Mycobacterium strain S1, originally described as Rhodococcus strain S1 by chemotaxonomic criteria, was isolated by growth on anthracene, and is unable to use any of nine other polycyclic aromatic compounds as carbon source. Metabolism of phenanthrene during growth on anthracene as sole carbon source results in the accumulation of traces of a dihydrodiol metabolite in the growth medium, which, by comparison with authentic standards, has been tentatively identified as phenanthrene trans-9,10-dihydrodiol. Anthracene metabolites were ruled out on the basis of comparisons with authentic anthracene dihydrodiols from Pseudomonas fluorescens D1 and chemically synthesized anthrols. The original source of phenanthrene for dihydrodiol production was phenanthrene present as a < 1% contaminant in the anthracene used as carbon source. However, addition of further phenanthrene to the anthracene growth medium increased the level of phenanthrene trans-9,10-dihydrodiol formed. Mycobacterium strain S1 also produced phenanthrene trans-9,10-dihydrodiol when grown in a glucose-salts medium in the presence of phenanthrene. This dihydrodiol is a dead-end metabolite, and neither it nor its parent hydrocarbon are able to support the growth of Mycobacterium strain S1. Studies with metyrapone and ancimidol, which did not inhibit growth on anthracene but did inhibit formation of phenanthrene trans-9,10-dihydrodiol, suggest it is likely the product of a cytochrome P450 monooxygenase-like activity.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate

The metabolites of benzo[j]fluoranthene (BjF) as formed in vitro using the 9000 X g supernatant from Aroclor-pretreated rats have been identified. Two dihydrodiols, trans-4,5-dihydro-4,5-dihydroxyBjF and trans-9,10-dihydro-9,10-dihydroxyBjF have been identified as major metabolites by comparison of their spectral and chromatographic properties with those of pure synthetic standards. There was no evidence that any of the isomeric 2,3-dihydrodiol was formed as a metabolite of BjF under these incubation conditions. Neither of the metabolic dihydrodiols of BjF were formed with a high degree of stereoselectivity. The enantiomeric purity of the 4,5-dihydrodiol was 20% while that of the 9,10-dihydrodiol was 46%. At least four phenols were detected among the metabolites of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dione was also identified as a metabolite under these incubation conditions.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens

The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-¹⁴C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of ¹⁸O₂, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD circular dichroism - DMF N,N-dimethyl-formamide - GC/MS gas chromatography/mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - ODS octadecylsilane - PAH polycyclic aromatic hydrocarbon - TLC thin-layer chromatography - TMS tetramethylsilane - UV ultraviolet     

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.     

Novel evidence of cytochrome P450-catalyzed oxidation of phenanthrene in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> under ligninolytic conditions

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130 pmol mg⁻¹ in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44 ± 0.02 min⁻¹. One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

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.     

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.     

Neuromedin B: a novel bombesin-like peptide identified in porcine spinal cord

Metabolism of 1-nitrobenzo(a)pyrene (1-nitro-BaP) by rat liver microsomes yielded 1-nitro-BaP trans-7,8-dihydrodiol, 1-nitro-BaP trans-9,10-dihydrodiol and 1-nitro-BaP 7,8,9,10-tetrahydrotetrol. Formation of these metabolites suggests that a vicinal 7,8,9,10-dihydrodiol-epoxide is a metabolite of 1-nitro-BaP.     

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.     

Identification of metabolites from phenanthrene oxidation by phenoloxidases and dioxygenases of Polyporus sp. S133

Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture.     

Relative role of eukaryotic and prokaryotic microorganisms in phenanthrene transformation in coastal sediments

THE RELATIVE ROLE OF EUKARYOTIC VERSUS PROKARYOTIC MICROORGANISMS IN PHENANTHRENE TRANSFORMATION WAS MEASURED IN SLURRIES OF COASTAL SEDIMENT BY TWO DIFFERENT APPROACHES: detection of marker metabolites and use of selective inhibitors on phenanthrene biotransformation. Phenanthrene biotransformation was measured by polar metabolite formation and CO(2) evolution from [9-C]phenanthrene. Radiolabeled metabolites were tentatively identified by high-performance liquid chromatography (HPLC) separation combined with UV/visible spectral analysis of HPLC peaks and comparison to authentic standards. Both yeasts and bacteria transformed phenanthrene in slurries of coastal sediment. Two products of phenanthrene oxidation by fungi, phenanthrene trans-3,4-dihydrodiol and 3-phenanthrol, were produced in yeast-inoculated sterile sediment. However, only products of phenanthrene oxidation typical of bacterial transformation, 1-hydroxy-2-naphthoic acid and phenanthrene cis-3,4-dihydrodiol, were isolated from slurries of coastal sediment with natural microbial populations. Phenanthrene trans-dihydrodiols or other products of fungal oxidation of phenanthrene were not detected in the slurry containing a natural microbial population. A predominant role for bacterial transformation of phenanthrene was also suggested from selective inhibitor experiments. Addition of streptomycin to slurries, at a concentration which suppressed bacterial viable counts and rates of [methyl-H]thymidine uptake, completely inhibited phenanthrene transformation. Treatment with colchicine, at a concentration which suppressed yeast viable counts, depressed phenanthrene transformation by 40%, and this was likely due to nontarget inhibition of bacterial activity. The relative contribution of eukaryotic microorganisms to phenanthrene transformation in inoculated sterile sediment was estimated to be less than 3% of the total activity. We conclude that the predominant degraders of phenanthrene in muddy coastal sediments are bacteria and not eukaryotic microorganisms.     

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.     

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene. Effect of a K-region fluoro substituent on the regioselectivity of cytochromes P-450 in liver microsomes from control and induced rats

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene (6-FB[c]Ph) was compared with that of benzo[c]phenanthrene (B[c]Ph) to elucidate the enhancement of carcinogenicity of B[c]Ph by the 6-fluoro substituent. Liver microsomes from untreated (control), phenobarbital-treated, and 3-methylcholanthrene-treated rats metabolized 6-FB[c]Ph at rates of 3.5, 1.5, and 7.7 nmol of products/nmol of cytochrome P-450/min, respectively. The rates of metabolism of B[c]Ph by the same microsomes were 2.9, 1.6, and 5.5 nmol of products/nmol of cytochrome P-450/min, respectively. Whereas the K-region 5,6-dihydrodiol was the major metabolite of B[c]Ph, the major metabolite of 6-FB[c]Ph was the K-region 7,8-oxide, which underwent slow rearrangement to an oxepin. Thus, the 6-fluoro substituent blocks oxidation at the 5,6-double bond and inhibits hydration of the K-region 7,8-oxide by epoxide hydrolase. Substitution with fluorine at C-6 caused an almost 2.5-fold increase in the percentages of the putative proximate carcinogens, i.e. benzo-ring dihydrodiols with bay-region double bonds, when liver microsomes from 3-methylcholanthrene-treated rats were used. Little or no increase was observed in their formation by liver microsomes from control or phenobarbital-treated rats. Interestingly, liver microsomes from control rats formed almost 3-fold as much 3,4-dihydrodiol as isosteric 9,10-dihydrodiol. The R,R-enantiomers of the 3,4- and 9,10-dihydrodiols and the S,S-enantiomer of the 7,8-dihydrodiol were predominantly formed by all three microsomal preparations.