Biotransformation of the antimelanoma agent betulinic acid by Bacillus megaterium ATCC 13368.

Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11alpha-hydroxy-lup-20(29)-en-28-oic acid, 1beta-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3beta,7beta, 15alpha-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).

Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea.

Resveratrol (trans-3,4',5-trihydroxystilbene) is produced by grapes (Vitis spp.) in response to microbial attack by the fungal grapevine pathogen Botrytis cinerea. Several reports indicate that pathogenic B.cinerea strains are capable of biotransforming resveratrol into an assortment of unidentified oxidized metabolites as a means of reducing the antifungal effects of resveratrol and facilitating Botrytis invasion into host-plant tissues. Studies utilizing growing incubations of Botrytis cinerea ATCC 11542 with resveratrol resulted in the production of three new (restrytisols A-C) (1-3) and three known (resveratrol trans-dehydrodimer, leachinol F, and pallidol) oxidized resveratrol dimers. All of the metabolites were evaluated for their anti-HIV-1, cytotoxic, and cyclooxygenase (COX) I and COX II inhibitory activities.

Microbial transformations of the antimelanoma agent betulinic acid.

Microbial transformation studies of the antimelanoma agent betulinic acid (1) were conducted. Screening experiments showed a number of microorganisms capable of biotransforming 1. Three of these cultures, Bacillus megaterium ATCC 14581, Cunninghamella elegans ATCC 9244, and Mucor mucedo UI-4605, were selected for preparative scale transformation. Bioconversion of 1 with resting-cell suspensions of phenobarbital-induced B. megaterium ATCC 14581 resulted in the production of the known betulonic acid (2) and two new metabolites: 3beta,7beta-dihydroxy-lup-20(29)-en-28-oic acid (3) and 3beta,6alpha, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (4). Biotransformation of 1 with growing cultures of C. elegans ATCC 9244 produced one new metabolite characterized as 1beta,3beta, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (5). Incubation of 1 with growing cultures of M. mucedo UI-4605 afforded metabolite 3. Structure elucidation of all metabolites was based on NMR and HRMS analyses. In addition, the antimelanoma activity of metabolites 2-5 was evaluated against two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).

Glucosidation of betulinic acid by Cunninghamella species.

Microbial transformation of the antimelanoma agent betulinic acid (1) was studied. Preparative scale biotransformation with resting-cell suspensions of Cunninghamella species NRRL 5695 resulted in the production of a fungal metabolite of 1, which has been characterized as 28-O-beta-D-glucopyranosyl 3beta-hydroxy-lup-20(29)-en-28-oate (2) based on spectral and enzymic data. The in vitro cytotoxicity assay of metabolite 2 revealed no activity against several human melanoma cell lines.

Biotransformation of resveratrol to piceid by Bacillus cereus.

Microbial transformation of resveratrol (1), trans-3,4', 5-trihydroxystilbene, was studied. Preparative scale biotransformation of 1 with whole-cell suspensions of Bacillus cereus UI 1477 resulted in the production of metabolite 2 which was identical in all respects to an authentic sample of piceid, resveratrol 3-O-beta-D-glucoside.

Comparative toxicity of (+)-(R)- and (-)-(S)-1,2-dibromo-3-chloropropane.

The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC).

Hepatotoxicity of germander (Teucrium chamaedrys L.) and one of its constituent neoclerodane diterpenes teucrin A in the mouse.

The hepatotoxicity of the herbal plant germander and that of one of its major furanoneoclerodane diterpenes, teucrin A, were investigated in mice. Teucrin A was found to cause the same midzonal hepatic necrosis as observed with extracts of the powedered plant material. Evidence that bioactivation of teucrin A by cytochromes P450 (P450) to a reactive metabolite(s) is required for initiation of the hepatocellular damage is provided by results of experiments on the induction and inhibition of P450 and from studies on the effects of glutathione depletion. Pretreatment of mice with the P450 inducer phenobarbital enhanced the hepatotoxic response, as indicated by an increase in plasma alanine aminotransferase (ALT) levels and hepatic necrosis, while pretreatment with the P450 inhibitor piperonyl butoxide markedly attenuated the toxic response. Hepatotoxicity of teucrin A also was increased following pretreatment with the inhibitor of glutathione synthesis buthionine sulfoximine. Most importantly, the tetrahydrofuran analog of teucrin A, obtained by selective chemical reduction of the furan ring, was not hepatotoxic, a result that provides strong evidence that oxidation of the furan ring moiety of the neoclerodane diterpenes is involved in the initiation of hepatocellular injury caused by germander.

Identification of four biliary metabolites of the diterpene sclareol in the laboratory rat.

1. Ag.l.c. method was developed to determine sclareol (1) and its microbial metabolites: 3-keto-sclareol (2), 2 alpha-hydroxysclareol (3), 3 alpha-hydroxysclareol (4), 3 beta-hydroxysclareol (5), 18-hydroxysclareol (6), and 2 alpha, 18-dihydroxysclareol (7) in both microbial cultures and biological fluids of the laboratory rat. 2. Metabolism of the diterpene (1) was studied in the laboratory rat. This in vivo study was facilitated by the availability of microbial metabolites of sclareol as reference standards, and the g.l.c. assay for sclareol and its metabolites in biological fluids. 3. Following i.v. treatment (100 mg/kg), the disappearance of (1) from rat plasma was rapid and biphasic. No microbial metabolites of sclareol were detectable in plasma. 4. Sclareol (1) and its microbial metabolites were not detected in rat urine following either i.v. or oral treatments; unchanged (1) was excreted in rat faeces to the extent of 9% of an oral dose in 48 h. 5. Following i.v. treatment, 0.02% dose was recovered in bile as unchanged (1). Four biliary metabolites of (1) (0.4% dose) were identified as (2), and (4)-(6) based on g.l.c.-mass spectrometry and comparisons with reference standards. All four biliary metabolites of (1) in rat have been identified as microbial metabolites of (1).

Hydroxylation and glucoside conjugation in the microbial metabolism of the diterpene sclareol.

1. The microbial metabolism of sclareol (1), a labdane diterpene ditertiary alcohol, was studied. Preliminary screening identified a number of microorganisms capable of metabolizing sclareol. 2. Preparative scale fermentation of growing cultures of Bacillus cereus UI-1477 resulted in the production of seven metabolites which have been characterized as 3 beta-hydroxysclareol (2), 2 alpha-hydroxysclareol (3), 18-hydroxysclareol (4), 2 alpha,18-dihydroxysclareol (6), 8 alpha,13 beta-dihydroxy-labd-14-en-3 beta-O-beta-D-glucoside (5), 8 alpha,13 beta-dihydroxy-labd-14-en-18-O-beta-D-glucoside (7), and 8 alpha,13 beta-dihydroxy-labd-14-en-2 alpha-O-beta-D-glucoside (10) by chemical, enzymic, and spectral data, especially 2D-n.m.r. techniques and thermospray liquid chromatography-mass spectrometry analysis.

Microbial models of mammalian metabolism: fungal metabolism of the diterpene sclareol by Cunninghamella species.

Microbial metabolism of the diterpene sclareol was studied. Screening studies have shown a number of microorganisms capable of metabolizing sclareol. Preparative scale fermentation with Cunninghamella species NRRL 5695 has resulted in the production of two fungal metabolites that have been characterized as 3 beta-hydroxysclareol and 18-hydroxy-sclareol with the use of 2D nmr techniques. The yield of the two metabolites was improved by utilizing resting-cell suspensions of Cunninghamella species NRRL 5695.