Metabolism of 6-nitrochrysene by intestinal microflora.

Since bacterial nitroreduction may play a critical role in the activation of nitropolycyclic aromatic hydrocarbons, we have used batch and semicontinuous culture systems to determine the ability of intestinal microflora to metabolize the carcinogen 6-nitrochrysene (6-NC). 6-NC was metabolized by the intestinal microflora present in the semicontinuous culture system to 6-aminochrysene (6-AC), N-formyl-6-aminochrysene (6-FAC), and 6-nitrosochrysene (6-NOC). These metabolites were isolated and identified by high-performance liquid chromatography, mass spectrometry, and UV-visible spectrophotometry and compared with authentic compounds. Almost all of the 6-NC was metabolized after 10 days. Nitroreduction of 6-NC to 6-AC was rapid; the 6-AC concentration reached a maximum at 48 h. The ratio of the formation of 6-AC to 6-FAC to 6-NOC at 48 h was 93.4:6.3:0.3. Interestingly, compared with results in the semicontinuous culture system, the only metabolite detected in the batch studies was 6-AC. The rate of nitroreduction differed among human, rat, and mouse intestinal microflora, with human intestinal microflora metabolizing 6-NC to the greatest extent. Since 6-AC has been shown to be carcinogenic in mice and since nitroso derivatives of other nitropolycyclic aromatic hydrocarbons are biologically active, our results suggest that the intestinal microflora has the enzymatic capacity to generate genotoxic compounds and may play an important role in the carcinogenicity of 6-NC.     

Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium.

Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G + C content of the DNA, and high-molecular-weight mycolic acids (C58 to C64). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30 degrees C and minimally at 35 degrees C. No growth was observed at 5 or 42 degrees C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl- and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.     

Fungal metabolism and detoxification of the nitropolycyclic aromatic hydrocarbon 1-nitropyrene.

Nitropolycyclic aromatic hydrocarbons are ubiquitous environmental pollutants, many of which are potent mutagens in bacterial and mammalian cells and carcinogenic to rodents. In this study, we investigated the fungal metabolism of 1-nitropyrene and determined the mutagenic activity of the metabolites toward Salmonella typhimurium TA98, TA98NR, and TA100. Cunninghamella elegans metabolized 1-nitropyrene to form glucoside conjugates of 6-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene. The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by application of UV absorption, 1H-nuclear magnetic resonance, and mass spectroscopy. Mutagenicity assays performed on samples extracted from incubations of C. elegans with 1-nitropyrene indicated that mutagenic activity decreased with time. Consistent with the loss in mutagenic activity, the glucoside conjugates of 6- and 8-hydroxy-1-nitropyrene were nonmutagenic in the Salmonella reversion assay. The results indicate that the fungus C. elegans metabolizes 1-nitropyrene to detoxified products.     

Bacterial degradation of low concentrations of phenanthrene and inhibition by naphthalene

Phenanthrene-degrading bacteria were isolated from enrichment cultures of soils contaminated with creosote and jet fuel. The isolates from the creosote enrichments were classified by fatty acid methyl ester profiles as Acidovorax delafieldii and Sphingomonas paucimobilis; the bacterium from the jet fuel-contaminated soil was not identified and was designated strain JFD 11. All three isolates used phenanthrene as a sole carbon and energy source, and two of the isolates used fluoranthene as a sole carbon and energy source. Anthracene and fluorene were cometabolized by all three strains, but pyrene was not transformed. Naphthalene inhibited all of the strains, and 28-h cultures of A. delafieldii were inhibited by naphthalene concentrations as low as 5 ppm. Short-term degradation experiments were undertaken with center-well flasks and concentrations of phenanthrene ranging from 1.2 to 12.0 m. Since initial degradation rates were not a function of phenanthrene concentration, it was inferred that the half-saturation constants were less than the lowest phenanthrene concentration tested. Correspondence to: C.E. Cemiglia.     

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.     

Algal oxidation of aromatic hydrocarbons: formation of 1-naphthol from naphthalene by Agmenellum quadruplicatum, strain PR-6.

Hydroxyurea induces repair replication in human lymphoblastoid NC37 BaEV cells during incubation with liver microsomes and NADPH. Catalase reduces hydroxyurea-induced repair by more than 95%, suggesting that hydrogen peroxide derived from hydroxyurea in the presence of the metabolic activation system is involved in the DNA damage.     

Mutagenicity of azo dyes: structure-activity relationships.

Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.     

Localization of the azoreductase ofClostridium perfringens by immuno-electron microscopy

An antibody againstClostridium perfringens azoreductase was used with protein A (gold-labeled) to locate the site of synthesis of extracellular azoreductase in this bacterium. Electron microscopy of immunogold-stained thin sections ofC. perfringens cells showed an average of 134 gold particles per cell, distributed throughout the cytoplasm and not associated with any organized structures.     

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 a novel metabolite in phenanthrene metabolism by the fungus Cunninghamella elegans

The metabolism of phenanthrene by the fungus Cunninghamella elegans was investigated. Kinetic experiments using [9-14C]phenanthrene showed that after 72 h, 53% of the total radioactivity was associated with a glucoside conjugate of 1-hydroxyphenanthrene (phenanthrene 1-O-beta-glucose). This metabolite was isolated by reversed-phase high-performance liquid chromatography and characterized by the application of UV absorption, 1H nuclear magnetic resonance, and mass spectral techniques. The results show that aromatic ring oxidation followed by glucosylation is a predominant pathway in the metabolism of the polycyclic aromatic hydrocarbon phenanthrene by C. elegans.