Mouflon (Ovis musimon) dicrocoeliosis: effects of parasitosis on the activities of biotransformation enzymes and albendazole metabolism in liver.

Parasitic infections can modify the host's ability to metabolize drugs and other xenobiotics by altering the biotransformation enzymes; these changes may have various pharmacological, toxicological or physiological consequences. In our study, several activities of liver biotransformation enzymes and in vitro metabolism of albendazole (ABZ) were tested and compared in non-infected mouflons (Ovis musimon) and in mouflons infected by lancet fluke (Dicrocoelium dendriticum). Subcellular fractions of liver homogenates were isolated from 5+5 mouflon rams (1-year-old) parasitologically negative or naturally infected by fluke. From the eight enzyme activities that were assayed, only two activities significantly differ in the case of Dicrocoelium-infected versus non-infected animals. In infected mouflons, a significant increase (53%) of thiobenzamide-S-oxidase (TBSO) activity, corresponding mainly to the activity of flavine monooxygenase (FMO), and significant decrease (60%) of glutathione-S-transferase (GST) activity was observed. In addition, dicrocoeliosis caused the enhancement of ABZ hepatic biotransformation. The velocity of the formation of (+)-ABZ sulfoxide and ABZ sulfone was significantly increased. However, the shifts in ABZ biotransformation were very mild that undesirable alterations in ABZ pharmacokinetic are not expected. From this point of view, the use of ABZ in the therapy of mouflon dicrocoeliosis in young animals can be recommended. The treatment of the same mouflons by other drugs that are mainly conjugated with glutathione, seems to be more problematic; hence, all consequences of documented reduced GST activity should be accounted.

Activities of biotransformation enzymes in pheasant (Phasianus colchicus) and their modulation by in vivo administration of mebendazole and flubendazole.

Basal activities of certain pheasant hepatic and intestinal biotransformation enzymes and modulation of their activities by anthelmintics flubendazole (FLBZ) and mebendazole (MBZ) were investigated in subcellular fractions that were prepared from liver and small intestine of control and FLBZ or MBZ treated birds. Several oxidation, reduction and conjugation enzyme activities were assessed. In the liver, treatment of pheasants by FLBZ or MBZ caused very slight or no changes in monooxygenase activities and conjugation enzymes. More significative changes were detected in small intestine. Metyrapone and daunorubicin reductase activities were increased by both substances in the liver. This is the first evidence that certain benzimidazoles modulate reductases of carbonyl group. With respect to the relatively slight extent of the changes caused by FLBZ or MBZ we can assume that repeated administration of therapeutic doses of both FLBZ and MBZ has probably no serious influence on pheasant biotransformation enzyme system.

Liver microsomal biotransformation of albendazole in deer, cattle, sheep and pig and some related wild breeds.

Albendazole (ABZ) biotransformation was studied in vitro in liver microsomes of adult noncastrated male farm animals (ram, buck, bull and boar), castrated adult males (wether, billy and hog), and free living males (fallow buck, red deer stag, mouflon ram, roe buck and wild boar). Liver microsomal fractions were incubated with either ABZ or racemic albendazole sulphoxide (ABZSO). ABZ was extensively metabolized to the (+) and (-) enantiomers of ABZSO, whereas ABZSO underwent a slow oxidation to albendazole sulphone (ABZSO2) in all species. In all species both ABZSO enantiomers were detected. The chiral ratio, (+)-ABZSO/(-)-ABZSO, was greater than one in farm animals, mouflon and wild boar, and less than one in three species of deer. For total ABZ sulphoxidation, deer like species had lower values compared to the other species. Mouflon ram and ram had lower total sulphoxidation rates compared to wethers, as well as ABZ suphoxidation towards (+)-ABZSO. No significant difference occurred comparing ABZSO formation in mouflon ram and ram, but ABZSO2 formation rate in mouflon ram was higher than in rams and wethers. Roe deer stag, fallow buck and red deer stag did not differ in both total-ABZSO and (-)-ABZSO synthesis rates and roe deer stag and fallow buck did not differ in synthesis rates of (+)-ABZSO and ABZSO2. The bull differed from other species in all metabolites studied, except for red deer stag and boar in (-)-ABZSO synthesis rate. The extent of ABZSO sulphonation to ABZSO2 in bull microsomes was more than twice that of other species.

Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon).

Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites - pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance.

The effect of new lipophilic chelators on the activities of cytosolic reductases and P450 cytochromes involved in the metabolism of anthracycline antibiotics: studies in vitro.

A major obstacle to the therapeutic use of anthracyclines, highly effective anticancer agents, is the fact that their administration results in dose-dependent cardiomyopathy. According to the currently accepted hypothesis, anthracyclines injure the heart by generating oxygen free radicals. The ability of pyridoxal isonicotinoyl hydrazone (PIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) -- new iron chelators -- to protect against peroxidation as well as their suitable biological, physical and chemical properties make the compounds promising candidates for pre-clinical and clinical studies. Activities of carbonyl reductase CR (1.1.1.184), dihydrodiol dehydrogenase DD2 (1.3.1.20), aldehyde reductase ALR1 (1.1.1.2) and P450 isoenzymes (CYP1A1, CYP1A2, CYP2B, CYP3A) involved in the metabolism of daunorubicin, doxorubicin and other drugs or xenobiotics were studied. Various concentrations of the chelators were used either alone or together with daunorubicin or doxorubicin for in vitro studies in isolated hepatocytes. A significant decrease of activity was observed for all enzymes only at PIH and SIH concentrations higher than those presumed to be used for therapy. The results show that PIH and SIH have no effect on the activities of the enzymes studied in vitro and allow us to believe that they will not interfere with the metabolism of co-administered drugs and other xenobiotics. Daunorubicin (Da) and doxorubicin (Dx) significantly reduce cytochrome P450 activity, but the addition of SIH and PIH chelators (50 microM) reverses the reduction and restores the activity to 70-90 % of the activity of relevant controls.

The effects of fenbendazole, flubendazole and mebendazole on activities of hepatic cytochromes P450 in pig.

Fenbendazole (FBZ), flubendazole (FLBZ) and mebendazole (MBZ) are benzimidazole anthelmintics widely used in veterinary medicine. The effects of these drugs on cytochromes P450 (CYP) were investigated in primary cultures of swine (Sus scrofa f. domestica) hepatocytes. After 48-h incubation of hepatocytes with benzimidazoles (0.1-2.5 microm), ethoxyresorufin O-deethylation (EROD), benzoxyresorufin O-dearylation (BROD), testosterone hydroxylase (6beta-TOH) and testosterone oxidase (17-TO) activities were measured and the CYP1A and 3A protein levels were determined by Western blotting. FBZ produced a significant, concentration-dependent increase of CYP1A activity (EROD) and protein level. No enhancement of CYP1A was observed after exposure to FLBZ and MBZ. All benzimidazoles tested did not cause any induction of CYP3A (BROD, 6beta-TOH, 17-TO activities and protein content). On the other hand, MBZ produced a significant, concentration-dependent decrease of CYP3A (BROD, 6beta-TOH and 17-TO) activities. Pharmacological and toxicological consequences of CYP1A induction and CYP3A inhibition should be taken into account in treatment of pigs with FBZ and MBZ.

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.

Benzimidazole drugs and modulation of biotransformation enzymes.

Benzimidazole drugs (e.g., anthelmintics albendazole, fenbendazole, oxfenbendazole, thiabendazole, mebendazole; inhibitors of proton pump omeprazole, lansoprasole, pantoprasole) represent substances used in both human and veterinary medicine; however, from the point of view of induction and inhibition of biotransformation enzymes, research has been carried out mainly due to the initiative of human pharmacologists. The purpose of the present review is to inform about inductive and inhibitive effects of benzimidazole drugs in man, animals and cell cultures. Pharmacological and toxicological consequences of modulation of biotransformation enzymes are discussed and the significance of studies in the field of modulation of biotransformation enzymes in food-producing animals is explained. Since the modulating effect of benzimidazoles strongly varies depending on structure of the individual substances, the particular attention is paid to structure-modulation relationships.

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.

Stereospecific biotransformation of albendazole in mouflon and rat-isolated hepatocytes.

The anthelmintic albendazole (ABZ) undergoes a two-step oxidation resulting first in the formation of chiral albendazole sulfoxide (ABZSO) followed by its transformation to albendazole sulfone (ABZSO2) in many farm and laboratory animal species. Although cloven-hoofed game are also treated with ABZ, limited information concerning ABZ biotransformation in these species is available. The present study focused on in vitro ABZ sulfoxidation in hepatocytes from wild sheep-mouflon (Ovis musimon) and comparison of ABZ sulfoxidation in mouflon and rat (Rattus norvergicus) hepatocytes. ABZ was used as a substrate for primary cultures of mouflon and rat hepatocytes. Time-dependent stereospecific consumption of ABZSO and ABZSO2 formation has been investigated. The metabolites were determined by high-performance liquid chromatography with both achiral and chiral stationary phases. Although total-ABZSO formation did not significantly differ between mouflon and rat, after separation of the (+)-ABZSO and (-)-ABZSO enantiomers a significant difference between species was found. The enantiomeric ratio of (+)/(-)-ABZSO in mouflon hepatocytes was 2.8-3.8, while rat hepatocytes biotransformed ABZ to almost racemic ABZSO, with an enantiomeric ratio of 1.0-1.1. The ratio were similar for two concentrations of substrate used and stable over several time intervals. The formation of ABZSO2 was more extensive in rat (approximately five times) than in mouflon hepatocytes.