RESUMEN
This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.
Asunto(s)
Cunninghamella/metabolismo , Fungicidas Industriales/farmacocinética , Oxazoles/farmacocinética , Biotransformación , IsomerismoRESUMEN
The epidemiology of the two common erythromycin-resistant methylase (erm) genes ermC and ermA was analyzed in 12 coagulase-negative Staphylococcus spp. and 34 coagulase-positive Staphylococcus spp. isolated from chicken. Southern hybridization indicated that only 2 of the 12 coagulase-negative Staphylococcus spp. strains contained the ermC gene on the plasmid; 1 strain of Staphylococcus xylosus harbored the ermC gene on a 2.5-kb plasmid, and 1 strain of Staphylococcus cohnii harbored the gene on a 4.0-kb plasmid. Twelve of the 34 strains of Staphylococcus aureus contained the ermC gene. Eleven of these strains had the ermC gene on a 2.5-kb plasmid, and 1 strain had the gene on a 4.0-kb plasmid. Ten of the 12 coagulase-negative Staphylococcus spp. and 22 of the 34 coagulase-positive Staphylococcus spp. harbored the ermA gene exclusively on the chromosome. Two different ermA EcoRI restriction fragment length polymorphisms (RFLP) were identified. A majority of the isolates was found to have two chromosomal inserts (8.0- and 6.2-kb EcoRI fragments) of ermA. One strain of S. aureus had different chromosomal inserts (6.4- and 5.8-kb EcoRI fragments) of ermA. Our results indicate that either the ermC or ermA gene, homologous to those described in human isolates, was present in all avian Staphylococcus spp. and that ermA was the predominant gene in coagulase-negative and coagulase-positive avian Staphylococcus spp. The size and copy numbers of the ermA gene were different from its human counterpart.
Asunto(s)
Antibacterianos/farmacología , Pollos/genética , ADN Bacteriano/genética , Eritromicina/farmacología , Enfermedades de las Aves de Corral/microbiología , Infecciones Estafilocócicas/genética , Secuencia de Aminoácidos , Animales , Pollos/microbiología , Farmacorresistencia Microbiana , Datos de Secuencia Molecular , Enfermedades de las Aves de Corral/genética , Staphylococcus/patogenicidadRESUMEN
A bacterial strain isolated from aquaculture pond slurry, which was extremely sensitive to erythromycin, was used to detect erythromycin at levels as low as 0.05 micrograms ml-1 in aquaculture water, sediments and soil samples. Identification of the indicator organism was attempted by 16S rRNA sequencing, biochemical profile, fatty-acid analysis and polymerase chain reaction (PCR). GenBank comparison showed that the 16S rRNA sequence of the strain was similar to those of more than 20 copies of Xanthomonas and Stenotrophomonas. The position of the strain in a phylogenetic tree based on the 16S rRNA gene sequence comparison is in a cluster of Stenotrophomonas. The fatty-acid analysis also showed that the strain is similar to Stenotrophomonas maltophilia. However, the biochemical profile of the strain is most similar to Xanthomonas campestris, except that it can utilize maltose, which is similar to S. maltophilia. Polymerase chain reaction results showed that the strain is different from X. campestris, S. maltophilia and other Xanthomonas species tested. Based on these results, the authors named this strain as Stenotrophomonas sp. strain NCTR.
Asunto(s)
Acuicultura/métodos , Bioensayo , Eritromicina/análisis , Xanthomonas/genética , Xanthomonas/aislamiento & purificación , Bioensayo/métodos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Microbiología del Agua , Contaminantes del Agua/análisisRESUMEN
The fungus Cunninghamella elegans was used to biotransform 6-nitrochrysene, a mutagen that is a widespread environmental contaminant. After 6 days, 74% of the 3H-labeled 6-nitrochrysene added had been metabolized to two isomeric sulfate conjugates. These conjugates were separated by high-performance liquid chromatography and identified by UV-visible, 1H nuclear magnetic resonance, and mass spectral techniques as 6-nitrochrysene 1-sulfate and 6-nitrochrysene 2-sulfate.
Asunto(s)
Crisenos/metabolismo , Mucorales/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Espectrofotometría UltravioletaRESUMEN
Metabolism of 2-nitrofluoranthene (2-NFA), one of the most abundant and genotoxic environmental pollutants in air, and of a mixture of 2-nitrofluoranthene and 3-nitrofluoranthene (3-NFA) was studied using (1) the fungus Cunninghamella elegans ATCC 36112 and (2) rat liver microsomes. The fungal metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and identified by 1H nuclear magnetic resonance (NMR) spectrometry, ultraviolet (UV)-visible spectroscopy, and online atmospheric-pressure chemical ionization/mass spectrometry (APCI/MS). The fungus metabolized 82% of 2-nitro-[3H]-fluoranthene to 2-nitrofluoranthene 8-sulfate and 2-nitrofluoranthene 9-sulfate. Metabolism of a mixture of 2- and 3-nitrofluoranthene by C. elegans similarly produced 2-nitrofluoranthene 8- and 9-sulfate and 3-nitrofluoranthene 8- and 9-sulfate as major metabolites. In addition, a glucoside conjugate of 3-hydroxy-2-nitrofluoranthene was tentatively identified by APCI/MS analysis. When rat liver microsomes were incubated with a mixture of 2- and 3-nitrofluoranthene for 1 h, in addition to the trans-7,8- and 9,10-dihydrodiols reported previously for 2-nitrofluoranthene, several novel metabolites were produced including 2-nitrofluoranthene trans-4,5-dihydrodiol and 2-nitrofluoranthene trans-8,9-dihydrodiol, the trans-4,5-dihydrodiol of 3-nitrofluoranthene, and phenolic products of both 2- and 3-nitrofluoranthene. The fungal metabolism of the 2- and 3-nitrofluoranthene mixture was similar to the metabolism of individual nitrofluoranthenes; however, the mammalian metabolism of the nitrofluoranthene mixture showed differences in regioselectivity at positions C4, C5, C8, and C9.
Asunto(s)
Contaminantes Atmosféricos/metabolismo , Fluorenos/metabolismo , Mucorales/metabolismo , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The metabolism of metolachlor[2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-met hyl ethyl)acetamide]by the fungus Cunninghamella elegans ATCC 36112 was determined. Thesix metabolites identified comprised 81% of the total[14C]-metolachlor metabolized by C. elegans. Thesemetabolites were separated by reversed-phase high-performance liquidchromatography and identified by 1H nuclear magnetic resonance, UV, and atmospheric pressure chemical ionization (APCI) mass spectraltechniques. Metabolites I and II were identified as stereoismers of2-chloro-N-[2-ethyl-6-hydroxymethylphenyl)]-N-(2-hydroxy-1-me thylet hyl)acetamide. Metabolites III and IV have been tentatively identified as stereoismers of2-chloro-N-[2-(1-hydroxyethyl)-6-methylphenyl]-N-(2-methoxy-1-++ +methy lethyl)acetamide. Metabolites V and VI were identified as stereoismers of2-chloro-N-(2-ethyl-6-hydroxy-methylphenyl)-N-(2-methoxy-1-me thylet hyl)acetamideand 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-hydroxy-1-methylethyl)acetam ide, respectively. The fungus Cunninghamellaelegans was able to biotransform metolachlor. Multiple site oxidation ofmetolachlor by C. elegans occurred predominantly byO-demethylation of the N-alkyl side chain and benzylichydroxylation of the arylalkyl side chain.
Asunto(s)
Acetamidas/metabolismo , Cunninghamella/metabolismo , Herbicidas/metabolismoRESUMEN
Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are direct-acting mutagens and carcinogens that are considered a risk to human health. We investigated the metabolism of 2-nitrofluorene by the fungus Cunninghamella elegans ATCC 36112. At 144 h of incubation, C. elegans had metabolized about 81% of the [9-14C]-2-nitrofluorene, resulting in 6 metabolites. The major metabolites were separated by reversed-phase high-performance liquid chromatography and identified by 1H NMR, ultraviolet (UV)-visible, and mass spectral analyses as 2-nitro-9-fluorenol, 2-nitro-9-fluorenone, 6-hydroxy-2-nitrofluorene, and sulfate conjugates of 7-hydroxy-2-nitro-9-fluorenone and 7-hydroxy-2-nitrofluorene. 2-Nitro-9-fluorenol accounted for about 62% of the total metabolism. For comparison with the microbial system, experiments with liver microsomes of rats pretreated with 3-methyl-cholanthrene were conducted. Microsomal incubations indicated formation of phenolic and ring-hydroxylated products of 2-nitrofluorene. 2-Nitrofluorene and hydroxylated metabolites have been previously implicated as direct-acting mutagens in bacterial assays and have shown sister chromatid exchanges in vivo in bone marrow cells and in vitro in ovary cells and unscheduled DNA synthesis in mammalian studies. Previous studies with other PAHs using C. elegans have shown that the phenols and glucoside and sulfate conjugates of phenols are generally less mutagenic than the parent. The results from the metabolism of 2-nitrofluorene by C. elegans suggests the detoxification potential of this fungus.
Asunto(s)
Carcinógenos/metabolismo , Fluorenos/metabolismo , Microsomas Hepáticos/metabolismo , Mucorales/metabolismo , Animales , Biodegradación Ambiental , Carcinógenos/toxicidad , Cromatografía Líquida de Alta Presión , Fluorenos/toxicidad , Hidroxilación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metilcolantreno/toxicidad , Microsomas Hepáticos/efectos de los fármacos , Ratas , Espectrofotometría UltravioletaRESUMEN
We investigated the metabolism of 3-nitrofluoranthene by filamentous fungus, Cunninghamella elegans ATCC 36112. Cunninghamella elegans metabolized about 72% of the 3-nitro[3,4-14C]fluoranthene added during 144 h of incubation to 2 major metabolites. These metabolites were separated by reversed-phase high-performance liquid chromatography and identified as 3-nitrofluoranthene-8-sulfate and 3-nitrofluoranthene-9-sulfate by 1H nuclear magnetic resonance, UV-visible, and mass spectral techniques. These results, in conjunction with previous studies on the fungal metabolism of fluoranthene, indicate that the nitro substituent at the C-3 position of fluoranthene sterically hinders epoxidation and shifts metabolism to the C-8 and C-9 positions. Since the phenolic microsomal metabolites of 3-nitrofluoranthene are mutagenic, the formation of sulfate conjugates of 8- and 9-hydroxy-3-nitrofluoranthene by C. elegans suggests that the fungal metabolic pathways may be beneficial for detoxification of this ubiquitous pollutant.
Asunto(s)
Carcinógenos/metabolismo , Fluorenos/metabolismo , Mucorales/metabolismo , Mutágenos/metabolismo , Cromatografía Líquida de Alta Presión , Contaminantes Ambientales/metabolismo , Espectroscopía de Resonancia MagnéticaRESUMEN
The metabolism of fluorene, a tricyclic aromatic hydrocarbon, by Cunninghamella elegans ATCC 36112 was investigated. Approximately 69% of the [9-14C]fluorene added to cultures was metabolized within 120 h. The major ethyl acetate-soluble metabolites were 9-fluorenone (62%), 9-fluorenol, and 2-hydroxy-9-fluorenone (together, 7.0%). Similarly to bacteria, C. elegans oxidized fluorene at the C-9 position of the five-member ring to form an alcohol and the corresponding ketone. In addition, C. elegans produced the novel metabolite 2-hydroxy-9-fluorenone.
Asunto(s)
Fluorenos/química , Fluorenos/metabolismo , Mucorales/metabolismo , Biodegradación Ambiental , Medios de Cultivo/química , Espectroscopía de Resonancia Magnética , Micología/métodosRESUMEN
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.
Asunto(s)
Acenaftenos/metabolismo , Mucorales/metabolismo , Animales , Biodegradación Ambiental , Espectroscopía de Resonancia Magnética , Microsomas Hepáticos/metabolismo , RatasRESUMEN
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.
Asunto(s)
Fluorenos/metabolismo , Mucorales/metabolismo , Mutágenos/metabolismo , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Fluorenos/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Ratas , Espectrofotometría UltravioletaRESUMEN
The fungus Cunninghamella elegans ATCC 36112 metabolized approximately 80% of the 3-14C-labeled fluoranthene (FA) added within 72 h of incubation. C. elegans metabolized FA to trans-2,3-dihydroxy-2,3-dihydrofluoranthene (trans-2,3-dihydrodiol), 8- and 9-hydroxyfluoranthene trans-2,3-dihydrodiol, 3-fluoranthene-beta-glucopyranoside, and 3-(8-hydroxyfluoranthene)-beta-glucopyranoside. These metabolites were separated by thin-layer and reversed-phase high-performance liquid chromatography and identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques. The major pathway involved hydroxylation to form a glucoside conjugate of 3-hydroxyfluoranthene and a glucoside conjugate of 3,8-dihydroxyfluoranthene which together accounted for 52% of the total ethyl acetate-soluble metabolites. C. elegans initially metabolized FA in the 2,3 position to form fluoranthene trans-2,3-dihydrodiol, which has previously been shown to be a biologically active compound in mammalian and bacterial genotoxicity tests. However, C. elegans formed predominantly glucoside conjugates of the phenolic derivatives of FA, which suggests that this fungus has the potential to detoxify FA.