Exploring precision-cut liver slices for comparative xenobiotic metabolism profiling in swine and cattle.

In vitro systems are useful tools for unravelling species differences in xenobiotic metabolism.The current work aimed to validate the technique of precision-cut liver slices (PCLS) for comparative studies on xenobiotic metabolism in swine and cattle.PCLS from swine (n = 3) and cattle (n = 3) were produced using a Brendel-VitronTM Tissue Slicer and cultured for 6 h. Tissue viability was preserved throughout the whole culture period.Metabolic viability was evaluated using the anthelmintics albendazole (ABZ) and fenbendazole (FBZ) as model drugs, as well as other substrates of hepatic monooxygenases: benzydamine (BZ) N-oxygenase (FMO-dependent), and the O-dealkylations of 7-ethoxyresorufin (EROD, CYP1A1-dependent) and 7-methoxyresorufin (MROD, CYP1A2-dependent).ABZ S-oxygenation resulted 6-fold (cattle) and 13.6-fold (swine) higher (p = 0.001) compared to FBZ S-oxygenation.Similar BZ N-oxygenation and EROD activities were observed in PCLS cultures from both species. MROD was 2.5-fold higher (p = 0.033) in swine than in cattle. Similarly, ABZ S-oxygenation was 1.7-fold higher (p = 0.0002) in swine than in cattle. Conversely, a 82% higher (p = 0.0003) rate of FBZ S-oxygenation was evidenced in PCLS cultures from cattle compared to those from swine.Overall, this work shows that PCLS cultures are useful to obtain relevant information on species differences in xenobiotic metabolism.

Unravelling drug-drug interactions in pigs: Induction of hepatic cytochrome P450 1A (CYP1A) metabolism after the in-feed medication with the anthelmintic fenbendazole.

The anthelmintic fenbendazole (FBZ) undergoes hepatic S­oxygenation by monooxygenases belonging to the cytochrome P450 (CYP) and flavin-monooxygenase (FMO) families. The in-feed medication with FBZ induced CYP1A-dependent metabolism in pig liver. This fact may alter the metabolism of the anthelmintic itself, and of CYP1A substrates like aflatoxin B1 (AFB1). This work evaluated the effect of the in-feed administration of FBZ on CYP1A-dependent metabolism, on its own pattern of hepatic S­oxygenation, and on the metabolism of AFB1. Landrace piglets remained untreated (n = 5) or received a pre-mix of FBZ (n = 6) in feed for 9 days. Pigs were slaughtered for preparation of liver microsomes used for: CYP content determination; monitoring the CYP1A-dependent enzyme activities, 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD); measurement of FBZ (50 µM) S­oxygenation, and AFB1 (16 nM) disappearance from the incubation medium. In microsomes of FBZ-treated animals, EROD and MROD increased 19-fold (p = 0.002) and 14-fold (p = 0.003), respectively. An enhanced (3-fold, p = 0.004) participation of the CYP pathway in FBZ S­oxygenation was observed in the liver of piglets treated with the anthelmintic (210 ± 69 pmol/min.nmol CYP) compared to untreated animals (68 ± 34 pmol/min.nmol CYP). AFB1 metabolism was 93% higher (p = 0.009) in the liver of FBZ-treated compared to untreated pigs. Positive and significant (p < 0.05) correlations were observed between CYP1A-dependent enzyme activities and FBZ or AFB1 metabolism. The sustained administration of FBZ caused an auto-induction of the CYP1A-dependent S­oxygenation of this anthelmintic. The CYP1A induction triggered by the anthelmintic could amplify the production of AFB1 metabolites in pig liver, including the hepatotoxic AFB1-derived epoxide.+.

Effects of acaricides on the activities of monooxygenases in bovine liver microsomes.

Organophosphates (OPs), pyrethrins and fipronil, are acaricides commonly used in cattle, mainly as pour on formulations. Scant information is available on their potential interactions with hepatic xenobiotic metabolizing enzymes. This work aimed to evaluate in vitro the potential inhibitory effects of widely employed acaricides on catalytic activities mediated by hepatic cytochrome P450 (CYP) and flavin-monooxygenase (FMO) enzymes in cattle. Bovine (n = 4) liver microsomes were incubated in the absence (control assays) and in presence of different OPs (fenthion, chlorpyrifos, ethion, diazinon and dichlorvos), fipronil and cypermethrin at 0.1-100 µm. Five oxidative enzyme activities were assayed by spectrofluorimetric or HPLC methods: 7-ethoxyresorufin O-deethylase (for CYP1A1), methoxyresorufin O-demethylase (for CYP1A2), benzyloxyresorufin O-debenzylase (for CYP2B), testosterone 6-beta hydroxylase (for CYP3A) and benzydamine N-oxidase (for FMO). All acaricides, particularly phosphorothionate-containing OPs, inhibited to some extent more than one enzyme activity. The most frequent inhibitor was fenthion, which inhibited (p < .05) all enzyme activities tested (from 22% at 1 µm to 72% at 100 µm). However, low inhibitory potencies (IC50s higher than 7 µm) of all acaricides studied were observed against the catalytic activities assayed. Therefore, the risk of in vivo metabolic interactions due to inhibition of monooxygenases would be low under common husbandry conditions.

Medication with fenbendazole in feed: plasma concentrations and effects on hepatic xenobiotic metabolizing enzymes in swine.

Fenbendazole (FBZ), a benzymidazole (BZD) anthelmintic drug, is used for in-feed medication in pigs. BZD-containing drugs may induce cytochrome P450 isozymes (CYPs), particularly those members of the CYP1A subfamily. The current research evaluated the plasma and liver availability and metabolism of FBZ and its metabolites, oxfendazole (OFZ) and fenbendazole sulphone (FBZSO2), after the administration of the parent drug in feed, and characterized the effect of the sustained administration of the anthelmintic on the catalytic activities of xenobiotic metabolizing enzymes in pig liver. Five female Landrace piglets remained untreated (controls), and other six were treated with a pre-mix of FBZ, combined with feed, for 9 consecutive days as usually is recommended. Blood samples were collected from each treated animal up to day 9 and analyzed by HPLC; all animals were slaughtered for preparation of liver microsomes. Plasma concentration ratios OFZ/FBZ and FBZSO2/OFZ increased significantly (p < 0.05) from the beginning to the end of drug exposure, which may indicate an enhanced conversion of FBZ into its metabolites. FBZ represented 45.8 ± 3.4% of the total anthelmintic molecules in liver tissue. Increased CYP1A-dependent 7-ethoxy (24.5-fold, p = 0.0032) and 7-methoxyresorufin (17.2-fold, p = 0.0006) O-dealkylase activities was observed in liver microsomes from FBZ-treated animals. In addition, a 64% increase (p = 0.042) in the rate of FBZ S-oxidation was observed in pigs treated with the anthelmintic drug compared to that measured in untreated animals. Thus, the continuous FBZ administration may accelerate its own in vivo hepatic metabolism through the CYP1A pathway.