J. Velík

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.

The effects of albendazole and its metabolites on hepatic cytochromes P450 activities in mouflon and rat

Albendazole (ABZ) is a benzimidazole anthelmintic widely used in veterinary medicine. The effects of ABZ on cytochromes P450 were investigated in primary cultures of mouflon (Ovis musimon) and rat (Rattus norvegicus) hepatocytes. Besides ABZ, its two main metabolites (albendazole-sulphoxide, ABZSO and albendazole-sulphone, ABZSOO) were tested to clarify which compound is responsible for the induction potency of this benzimidazole drug. After 48 h incubation of hepatocytes with benzimidazoles (0.2-25 microM), ethoxyresorufin O-deethylation (EROD) and benzoxyresorufin O-dearylation (BROD) were measured and the P4501A and 3A protein levels were determined by Western blotting. All benzimidazoles provoked a significant increase of EROD and BROD activities in rat hepatocytes. ABZSO and ABZSOO seemed to be responsible for the induction effect of ABZ on P450s in rat. In mouflon, no pharmacologically significant induction of EROD and BROD activities by benzimidazoles tested was observed. From this point of view, anthelmintic therapy of mouflons with ABZ seems to be safe.

The stereospecificity of flobufen metabolism in isolated guinea pig hepatocytes

BackgroundFlobufen (F) is an original nonsteroidal anti-inflammatory drug with one center of chirality. 4-Dihydroflobufen (DHF), compound with two chiral centers, is the main metabolite of F in microsomes and cytosol in all standard laboratory animals. This work describes the biotransformation of F enantiomers and DHF stereoisomers in isolated male guinea pig hepatocytes. Guinea pigs were chosen with respect to similarities in F metabolism as in Man found earlier. R-F, S-F, (2R;4S)-DHF, (2S;4R)-DHF, (2S;4S)-DHF and (2R;4R)-DHF, structurally very similar compounds, served as substrates in order to observe their interaction with enzymes. Stereospecificity of the respective enzymes was studied in vitro, using hepatocytes monolayer. Chiral HPLC using R,R-ULMO column as chiral stationary phase was used for detection and quantitation of metabolites.Results(2R;4S)-DHF and (2S;4S)-DHF were the principle stereoisomers detected after incubation with rac-F, R-F and S-F. The ratio of (2R;4S)-DHF/(2S;4S)-DHF ranged from 1.1 to 2.4 depending on the substrate used. (2R;4S)-DHF was the major stereoisomer found after incubation with (2S;4S)-DHF and (2R;4R)-DHF. (2S;4S)-DHF was the principle stereoisomer found after incubation with (2R;4S)-DHF and (2S;4R)-DHF. Besides DHF stereoisomers, other metabolites (M-17203, UM-1 and UM-2) were also detected after incubation of hepatocytes monolayer with F. Interestingly, these metabolites were not found in incubation of all F forms and DHF with fresh liver homogenate.ConclusionsDifferent activities and stereospecificities of the respective enzymes were observed for each substrate in primary culture of hepatocytes. Cell integrity is crucial for formation of secondary metabolites M-17203, UM-1 and UM-2.