Patrick J. Davis

Changes in glutathione and cellular energy as potential mechanisms of papaverine-induced hepatotoxicity in vitro.

The purpose of this study was to elucidate the mechanism of hepatotoxicity of papaverine hydrochloride (papaver) in vitro. To evaluate the role of metabolism in the toxicity of papaver, cells were pretreated with SKF-525A or benzyl imidazole (cytochrome P450 system inhibitors) for 24 hr at 1 x 10(-5) or 1 x 10(-4) M, respectively, or with phenobarbital sodium (cytochrome P450 system inducer) for 3 days at 2 x 10(-3) M. Cells then were exposed to concentrations of papaver ranging from 1 x 10(-5) to 1 x 10(-3) M for 4 to 24 hr. Cytotoxicity was evaluated by enzyme leakage (lactate dehydrogenase) and by energy status of the cells (ATP/ADP). The role of biological reactive intermediates in the toxicity of papaver was investigated by measuring changes in cellular reduced glutathione levels (GSH), by inhibiting GSH synthesis, and by determining the production of lipid peroxidation (LPX). Papaverine produced concentration- and time-dependent increases in enzyme leakage, with significant effects occurring by the 8-hr exposure period. Pretreatment with SKF-525A or benzyl imidazole increased enzyme leakage induced by papaver especially at a later time frame (24 hr), but pretreatment with phenobarbital delayed the onset of cytotoxicity from 8 to 12 hr. Decreases in GSH levels paralleled the time course of enzyme leakage. However, the administration of buthionine sulfoximine to cell cultures dramatically decreased the time by which papaver induced cellular injury (2 hr vs 8 hr). Changes in cellular energy status (ATP/ADP) were also detected earlier than enzyme leakage (4 hr vs 8 hr). In contrast, no significant production of lipid peroxidation was noted in papaver-treated cultures. We suggest that the mechanism of papaver-induced hepatotoxicity may be related to alterations in glutathione balance of the cells and to disruption of energy homeostasis.

Microbial transformations of glaucine.

Microbial transformation experiments were conducted with the aporphine alkaloid glaucine. Small-scale screening experiments provided a number of micro-organisms which produced three metabolites. In preparative scale studies, Streptomyces griseus(Ul 1158) produced norglaucine (4) and 2-O-demethylglaucine (6)(predicentrine) in 11 and 14% yield, respectively. Fusarium solani(ATCC 12823) produced didehydroglaucine (3) and a norapor-phinone (10)(an artefact) in 60 and 21% yield, respectively. With racemic glaucine, F. solani preferentially dehydrogenated (+)-(S)-glaucine, and unchanged, optically enriched (–)-(R)-glaucine was recovered from fermentations. N- and O-dealkylation did not occur in stereoselective fashion.

Microbial models of mammalian metabolism: stereospecificity of ketone reduction with pentoxifylline

The absolute configuration of pentoxifylline alcohol produced by the microbial reduction of pentoxifylline using Rhodotorula rubra (ATCC 20129) was determined by O.R.D. spectroscopy and p.m.r. studies on the R-(+)-alpha-methoxy-alpha-(trifluoromethyl) phenylacetate (MTPA) ester. The development of a reverse-phase h.p.l.c. method for resolving the diastereomeric R-(+)-MTPA esters of racemic pentoxifylline alcohol and assignment of the elution order, allowed for the determination of the stereochemical purity of pentoxifylline alcohol produced by 13 microbial cultures shown previously to reduce pentoxifylline. A marked preference for S-alcohol production was observed with five organisms exhibiting complete stereoselectivity towards S-alcohol generation, and two producing the racemic alcohol.

Toxicity assessment of paraverine hydrochloride and papaverine-derived metabolites in primary cultures of rat hepatocytes

SummaryThe present study was undertaken to assess and compare the toxic effects of papaverine hydrochloride and its metabolites. Primary cell cultures of rat hepatocytes were treated with papavarine (papaver), 3′-O-desmethyl (3′-OH), 4′-O-desmethyl (4′-OH), and 6-O-desmethyl (6-OH) papaverine at 1×10−5, 1×10−4, and 1×10−3M for 4,8, 12, and 24-h periods. Cell injury was determined by: a) cell viability using the trypan blue exclusion test; b) cytosolic enzyme leakage of lactate dehydrogenase and aspartate aminotransferase; c) morphologic alterations; and d) lactate: pyruvate (L:P) ratios. Cell cultures showed concentration-and time-dependent responses. For example, a decrease in cell viability and an increase in enzyme leakage were observed after cell treatment with 1×10−4 and 1×10−3M papaver for 8 h; 1×10−3M 6-OH papaverine for 8 h and 1×10−4M for 24 h; and 1×10−3M 4′-OH papaverine for 24 h (P<0.05). Furthermore, changes in morphology correlated to cell viability and enzyme release in those cultures treated with papaver, 4′-OH and 6-OH papaverine. Some of these changes included size deformation, cell detachment from the dishes, and cell necrosis. On the other hand, an increase in L:P ratios (P<0.05) was detected with papaver as early as 8 h with 1×10−4 and 1×10−3M and 12 h with 1×10−5M; 6-OH showed an increase, in L:P ratios at 8 h with 1×10−3M and 12 h with 1×10−4M; these changes were evident with 4′-OH at 12 h with 1×10−3M. In contrast, cells treated with 3′-OH papaverine did not show significant damage with any time period and concentration used in this study. The results of this study indicate that papaverine-derived metabolites are less cytotoxic than its parent compound, papaver. The toxicity was ranked as follows: papaver>6-OH>4′-OH>−3′-OH.

Microbial Models of Mammalian Metabolism: Stereoselective Metabolism of Warfarin in the Fungus Cunninghamella elegans

Biotransformation stereoselectivity of warfarin was studied in the fungus Cunninghamella elegans (ATCC 36112) as a model of mammalian metabolism. This organism was previously shown to produce all known phenolic mammalian metabolites of warfarin, including 6-, 7-, 8-, and 4′-hydroxywarfarin, and the previously unreported 3′-hydroxywarfarin, as well as the diastereomeric warfarin alcohols, warfarin diketone, and aliphatic hydroxywarfarins. Using S-warfarin and R-warfarin as substrates, and an HPLC assay with fluorescence detection to analyze metabolite profiles, the biotransformation of warfarin was found to be highly substrate and product stereoselective. Both aromatic hydroxylation and ketone reduction were found to be stereoselective for R-warfarin. Ketone reduction with the warfarin enantiomers exhibited a high level of product stereoselectivity in that R-warfarin was predominantly reduced to its S-alcohol, while S-warfarin was reduced primarily to the corresponding R-alcohol.

Microbial models of mammalian metabolism: Involvement of cytochrome P450 in the N-demethylation of N-methylcarbazole by Cunningham ella echin ula ta

1. As previously reported (Yang and Davis 1992), N-methylcarbazole (NMC) is converted to N-hydroxymethylcarbazole (NHMC), and 3-hydroxy-N-hydroxymethylcarbazole (3-OH-NHMC), two relatively stable carbinolamine metabolites by the fungus Cunninghamella echinulata (ATCC 9244). Decomposition of these two carbinolamines yields the corresponding dealkylated metabolites, carbazole and 3-hydroxycarbazole. In the present study, the possible involvement of cytochrome P450 in the requisite N-alkyl hydroxylation reaction was examined. 2. Carbon monoxide, a classical P450 inhibitor, markedly inhibited the formation of NHMC, as did potassium cyanide. 1-Benzylimidazole, piperonyl butoxide and SKF-525A inhibited the formation of both NHMC and 3-OH-NHMC, while beta-naphthoflavone (5,6-benzoflavone) induced their formation. 3. The source of the oxygen atom in the metabolite NHMC was examined by GC/MS analysis of NHMC formed during incubation of NMC in H218O-enriched medium which resulted in no incorporation of labelled oxygen into the metabolite. 4. An intermolecular isotope effect was not observed for the formation of NHMC suggesting that C-H bond cleavage is not a rate limiting step in the formation of this metabolite under the conditions examined. 5. It was concluded that P450 enzymes may be involved in the N-demethylation of NMC catalyzed by this fungal model of mammalian metabolism, and provides further support for biochemical and mechanistic parallels between mammalian metabolism and microbial systems catalyzing phase-1 biotransformations.

Microbial Models of Mammalian Metabolism: Production of Novel α-diketone Metabolites of Warfarin and Phenprocoumon Using Aspergillus Niger

1. The coumarin anticoagulants warfarin and phenprocoumon were metabolized by Aspergillus niger via oxidative ring cleavage to yield the corresponding alpha-diketone metabolites. 2. Structural identification was based upon physical, spectral, and chromatographic comparisons of isolated metabolites and synthetic standards generated by the oxidative cleavage of warfarin or phenprocoumon with pyridinium chlorochromate. 3. This pathway of metabolism has been previously observed for coumarin anticoagulants in mammalian systems.

Determination of pentoxifylline and its major metabolites in microbial extracts by thin-layer and high-performance liquid chromatography.

Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods have been developed for the determination of the xanthine drug, pentoxifylline, and three of its metabolites (a secondary alcohol and two carboxylic acids) in microbial extracts. The methods require initial extraction of acidified media with dichloromethane 2-propanol (4:1). Extracts are submitted to TLC development on silica gel G layers using three solvents and HPLC development on an C18 column using methanol-phosphoric acid (0.02 M, pH 5) (3:7) as mobile phase. All systems provide good separations of the drug and its metabolites. Quantitative analyses of pentoxifylline and its metabolites by HPLC were accurate and precise. The HPLC method was applied to studies of the metabolism of pentoxifylline by two microorganisms.

Inhibition of R-(−)-Apomorphine-induced Stereotypic Cage-climbing Behavior in Mice by S-(+)-Apomorphine

Apomorphine exists in both R-(−)- and S-(+)-enantiomeric forms. Only R-(−)-apomorphine (R-(−)-APO) has previously been shown to have agonistic dopaminergic activity. In this study, the ability of S-(+)-apomorphine (S-(+)-APO) to antagonize R-(−)-APO-induced stereotypic cage-climbing behavior in mice was investigated. Initial studies using a racemic mixture showed a depression of the cage-climbing behavior relative to that expected if S-(+)-APO were merely inactive. In subsequent experiments, S-(+)-APO was administered alone or one minute prior to the injection of R-(−)-APO and dose- and time-response analyses were carried out. These studies demonstrated that S-(+)-APO possessed no agonistic activity at 30 and 50 mg/kg compared to R-(−)-APO which showed an ED 50 of 4.3 mg/kg in the cage-climb model. S-(+)-APO did however, act as an antagonist of R-(−)-APO-induced stereotypic activity. Pretreatment of mice with S-(+)-APO (30 mg/kg) caused an orderly shift to the right in the dose response curve for R-(−)-APO (5 to 20 mg/kg). Furthermore, a dose-related inhibition of cage-climbing was observed when S-(+)-APO (2.5 to 60 mg/kg) pretreated mice were administered subsequently R-(−)-APO (5 mg/kg). From these experiments, an antagonist ED 50 of 7.7 mg/kg was calculated for S-(+)-APO. The S-(+)-isomer of apomorphine, thought heretofore to be pharmacologically inactive, possesses significant dopamine antagonistic activity.

In vitro metabolism and toxicity assessment of N-methylcarbazole in primary cultured rat hepatocytes

N-Methycarbazole (NMC), a carcinogen and mutagen in tobacco smoke, was converted to two major metabolites by primary cultured rat hepatocytes as measured by high performance liquid chromatography (HPLC): N-hydroxymethylcarbazole (NHMC) and carbazole. These two metabolites had comparable retention times and identical ultraviolet spectra as those of reference standards. Identical retention times and mass spectra were also observed as detected by gas chromatography-mass spectroscopy (GC-MS) for NHMC and its reference standard. The toxicities of NMC and its two metabolites were assessed by lactate dehydrogenase (LDH) leakage and neutral red (NR) uptake. The rank order of cytotoxicity of NMC and its metabolites was found to be: NHMC greater than NMC greater than carbazole. Thus, we conclude that the hydroxylation of NMC to NHMC may represent a toxification step, while the further dealkylation to carbazole is most likely a detoxication process.