Metabolism of 7,12-dimethylbenz[a]anthracene by Cunninghamella elegans

The metabolites of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, in cultures of Cunninghamella elegans were isolated by high-pressure liquid chromatography and characterized by UV spectroscopy and mass spectrometry. The major metabolites were DMBA-trans-8,9-dihydrodiol and DMBA-trans-3,4-dihydrodiol. The 7-hydroxymethyl and the 12-hydroxymethyl derivatives of these dihydrodiol metabolites were also formed. The metabolic profile described in this report contrasts with those obtained in our earlier experiments in which the incubation of DMBA with Pseudomonas aeruginosa and Penicillium notatum produced no dihydrodiol metabolites but only methyl-hydroxylated metabolites.     

Metabolism of 7,12-dimethylbenz[a]anthracene by Cunninghamella elegans.

The metabolites of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, in cultures of Cunninghamella elegans were isolated by high-pressure liquid chromatography and characterized by UV spectroscopy and mass spectrometry. The major metabolites were DMBA-trans-8,9-dihydrodiol and DMBA-trans-3,4-dihydrodiol. The 7-hydroxymethyl and the 12-hydroxymethyl derivatives of these dihydrodiol metabolites were also formed. The metabolic profile described in this report contrasts with those obtained in our earlier experiments in which the incubation of DMBA with Pseudomonas aeruginosa and Penicillium notatum produced no dihydrodiol metabolites but only methyl-hydroxylated metabolites.     

Sulfation of naringenin by Cunninghamella elegans

A new flavonoid sulfate, naringenin-7-sulfate, was obtained by fermentation of naringenin using the fungus Cunninghamella elegans NRRL 1392 in 23% yield. Structural elucidation of the metabolite was achieved using EIMS, UV, IR, 1D and 2D NMR spectroscopy beside acid and enzyme hydrolyses.     

Fungal transformation of fluoranthene.

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.     

Fungal transformation of fluoranthene

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.     

PAH biotransformation in terrestrial invertebrates--a new phase II metabolite in isopods and springtails

Soil-living invertebrates are exposed to high concentrations of contaminants accumulating in dead organic matter, such as polycyclic aromatic hydrocarbons (PAHs). The capacity for PAH biotransformation is not equally developed in all invertebrates. In this paper, we compare three species of invertebrates, Porcellio scaber (Isopoda), Eisenia andrei (Lumbricidae) and Folsomia candida (Collembola), for the metabolites formed upon exposure to pyrene. Metabolic products of pyrene biotransformation in extracts from whole animals or isopod hepatopancreas were compared to those found in fish bile (flounder and plaice). An optimized HPLC method was used with fluorescence detection; excitation/emission spectra were compared to reference samples of 1-hydroxypyrene and enzymatically synthesized conjugates. Enzymatic hydrolysis after fractionation was used to demonstrate that the conjugates originated from 1-hydroxypyrene. All three invertebrates were able to oxidize pyrene to 1-hydroxypyrene, however, isopods and collembolans stood out as more efficient metabolizers compared to earthworms. In contrast to fish, none of the invertebrates produced pyrene-1-glucuronide as a phase II conjugate. Both Collembola and Isopoda produced significant amounts of pyrene-1-glucoside, whereas isopods also produced pyrene-1-sulfate. A third, previously unknown, conjugate was found in both isopods and springtails, and was analysed further using electrospray and atmospheric pressure chemical ionisation mass spectrometry. Based on the obtained mass spectra, a new conjugate is proposed: pyrene-1-O-(6"-O-malonyl)glucoside. The use of glucose-malonate as a conjugant in animal phase II biotransformation has not been described before, but is understandable in the microenvironment of soil-living invertebrates. In the earthworm, three other pyrene metabolites were observed, none of which was shared with the arthropods, although two were conjugates of 1-hydroxypyrene. Our study illustrates the great variety of the still unexplored metabolic diversity of invertebrate xenobiotic metabolism.     

Transformation of amoxapine by Cunninghamella elegans

We examined Cunninghamella elegans to determine its ability to transform amoxapine, a tricyclic antidepressant belonging to the dibenzoxazepine class of drugs. Approximately 57% of the exogenous amoxapine was metabolized to three metabolites that were isolated by high-performance liquid chromatography and were identified by nuclear magnetic resonance and mass spectrometry as 7-hydroxyamoxapine (48%), N-formyl-7-hydroxyamoxapine (31%), and N-formylamoxapine (21%). 7-Hydroxyamoxapine, a mammalian metabolite with biological activity, now can be produced in milligram quantities for toxicological evaluation.     

Synthesis of sulfate esters of phosphatidylglycerol (diphytanyl ether analog)

Synthesis of 1-sn-phosphatidyl-3'-sn-glycero-1'-sulfate (phosphatidylglycero-1-sulfate) was achieved by monosulfation of 1-sn-phosphatidyl-3'-sn-glycerol (diphytanyl ether analog) with an equimolar amount of SO(3)-pyridine complex at room temperature; with excess sulfation reagent at 60 degrees C, the 1',2'-disulfate ester was obtained. The phosphatidyl-glycero-2-sulfate isomer was synthesized by an unambiguous route starting from the bacterial 2,3-di-O-phytanyl-sn-glycerol. The synthetic phosphatidylglycerosulfates were characterized by analytical, chromatographic, optical rotatory, and spectral (infrared and NMR) data and compared with the phosphatidylglycerosulfate isolated from Halobacterium cutirubrum.     

An expeditious preparation of various sulfoforms of the disaccharide beta-D-Galp-(1-->3)-D-Galp, a partial structure of the linkage region of proteoglycans, as their 4-methoxyphenyl beta-D-glycosides

An expeditious preparation of various sulfoforms of the disaccharide 4-methoxyphenyl O-(beta-D-galactopyranosyl)-(1-->3)-beta-D-galactopyranoside, namely the 4(I)- and 6(I)-sulfate, the 4(II)- and 6(II)-sulfate, and the 6(I),6(II)-disulfate derivatives, is reported for the first time. These molecules will be useful for the study of the early steps of the biosynthesis and sorting of proteoglycans. All target compounds were readily obtained from the common key intermediate 4-methoxyphenyl O-(2,3-di-O-benzoyl-4,6-di-O-levulinoyl-beta-D-galactopyranosyl)-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-galactopyranoside, easily prepared from the common starting material 4-methoxyphenyl 4,6-O-benzylidene-beta-D-galactopyranoside. Noticeable is the possible preparation of the different 6-O-sulfonated species through a one-pot procedure starting from a tetrol precursor.     

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.     

Microbial metabolism. Part 13. Metabolites of hesperetin

The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin (1) to four metabolites: 4'-methoxy-5,7,8,3'-tetrahydroxyflavanone (8-hydroxyhesperetin) (2), 5,7,3',4'-tetrahydroxyflavanone (eriodictyol) (3), 4'-methoxy-5,3'-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,3'-trihydroxyflavanone 4'-O-α-quinovopyranoside (eriodictyol 4'-O-α-quinovopyranoside) (5). The structures were established by spectroscopic methods.     

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.     

Microbial transformations of alpha-santonin

Fungal biotransformations of alpha-santonin (1) were conducted with Mucor plumbeus (ATCC 4740), Cunninghamella bainieri (ATCC 9244), Cunninghamella echinulata (ATCC 9245), Curvularia lunata (ATCC 12017) and Rhizopus stolonifer (ATCC 10404). Rhizopus stolonifer (ATCC 10404) metabolized compound 1 to afford 3,4-epoxy-alpha-santonin (2) and 4,5-dihydro-alpha-santonin (3) while Cunninghamella bainieri (ATCC 9244), Cunninghamella echinulata (ATCC 9245) and Mucor plumbeus (ATCC 4740) were capable of metabolizing compound 1 to give a reported metabolite, 1,2-dihydro-alpha-santonin (4). The structures of these transformed metabolites were established with the aid of extensive spectroscopic studies. These fungi regiospecifically reduced the carbon-carbon double bond in ring A of alpha-santonin.     

Fungal Biotransformation of 6-Nitrochrysene

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.     

内生真菌Paraconiothyrium brasiliense代谢产物β-咔啉生物碱及其黄嘌呤氧化酶抑制活性研究

本研究通过前体介导调控一株内生真菌的次级代谢产物,采用正相硅胶柱色谱和制备型HPLC等方法分离纯化,利用NMR、MS等波谱学方法鉴定化合物结构,从中分离鉴定了10个生物碱类化合物,鉴定结果为:川芎哚(1)、1-(1',2'-二脱氧-α-D-核吡喃糖基)-β-咔啉(2)、flazin(3)、tangutorid E(4)...     

On the structures of hydroxylated metabolites of estradiol 17-sulfate by rat liver microsomes

The metabolism of estradiol 17-sulfate (ES) by hepatic microsomes of female rats produced four new metabolites in addition to 2- and 4-hydroxyestradiol 17-sulfates (2- and 4-OH-ES), which were detected on an HPLC chromatogram. By comparison with synthetic specimens, three of these compounds were identified as 6alpha-, 6beta-, and 7beta-hydroxyestradiol 17-sulfates. To elucidate the structure of the remaining metabolite, a large-scale incubation of ES was carried out, followed by isolation using preparative HPLC to give the single material, which was assigned as 15beta-hydroxyestradiol 17-sulfate by instrumental analyses. On the other hand, when ES was incubated with the microsomes of male rats, 2-OH-ES was produced accompanied by two minor products: 4-OH-ES and a metabolite of unknown structure. The results show clearly that the metabolism of ES by rat hepatic microsomes is remarkably different between the sexes.     

Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans

Microbial transformation of adrenosterone (1) by suspended-cell cultures of the filamentous fungus Cunninghamella elegans resulted in the production of five metabolites 2-6, which were identified as 9alpha-hydroxyadrenosterone (2), 11-ketotestosterone (3), 6beta-hydroxyadrenosterone (4), 9alpha-hydroxy-11-ketotestosterone (5), and 6beta-hydroxy-11-ketotestosterone (6). Structures of new metabolites 2, 5, and 6 were established by single-crystal X-ray diffraction analysis.     

Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans.

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.     

Microbial transformation of acetyl-11-keto-β-boswellic acid and their inhibitory activity on LPS-induced NO production

The capabilities of 20 strains of fungi to transform acetyl-11-keto-β-boswellic (AKBA) were screened. And biotransformation of AKBA by Cunninghamella blakesleana AS 3.970 afforded five metabolites (1–5), while two metabolites (6, 7) were isolated from biotransformation of Cunninghamella elegans AS 3.1207. The chemical structures of these metabolites were identified by spectral methods including 2D NMR and their structures were elucidated as 7β-hydroxy-3-acety-11-keto-β-boswellic acid (1), 21β-dihydroxy-3-acety-11-keto-β-boswellic acid (2), 7β,22α-dihydroxy-3-acety-11-keto-β-boswellic acid (3), 7β,16α-dihydroxy-3-acety-11-keto-β-boswellic acid (4), 7β,15α-dihydroxy-3-acety-11-keto-β-boswellic acid (5); 7β,15α,21β-trihydroxy-3-acety-11-keto-β-boswellic acid (6) and 15α,21β-dihydroxy-3-acety-11-keto-β-boswellic acid (7). All these products are previously unknown. Their primary structure–activity relationships (SAR) of inhibition activity on LPS-induced NO production in RAW 264.7 macrophage cells were evaluated.