Background. Fenbendazole (FBZ) and triclabendazole (TCBZ) are benzimidazole drugs (BZDs) widely used in veterinary practice as anthelmintics. Members of cytochrome P450 (CYP) and flavin monooxygenase (FMO) superfamilies of drug metabolizing enzymes are primarily responsible of their biotransformation1. The xenobiotic-dependent up-regulation of CYPs is well documented, while FMOs are generally considered not inducible2. In the present study, the effect of FBZ and TCBZ (alone or in combination) on CYP1A1/2 and FMO1/3 mRNA levels were measured on precision-cut bovine liver slices (bLS). Methods. Precision-cut bLS from 6 male cattle were obtained according to Maté et al3. Following the fine-tuning of an absolute quantification protocol for target genes4, bLS were incubated for 0, 6 and 12 h with FBZ and TCBZ (50 µM), alone or in combination. β-naphthoflavone (βNF, 25 µM) was used as positive control to confirm gene induction. Target gene mRNA levels were measured by qPCR. The TATA Box binding protein and ribosomal protein lateral stalk subunit P0/β-actin were used as internal control genes to normalize FBZ/TCBZ and βNF data, respectively. Results. At T0, CYP1A2 mRNA levels were 25-fold higher than CYP1A1; however, FMO1 and 3 were equally represented. βNF up-regulated CYP1A1 (P<0.05; 4-fold vs control) after 6 h of incubation, while increasing amounts (P<0.05) of CYP1A2, FMO1/3 mRNAs were noticed after 12 h (4-, 2.5- and 3.5-fold vs control, respectively). Concerning BZDs, FBZ increased CYP1A1/2 mRNAs (P<0.05) after 12 h of incubation, whereas TCBZ up-regulated only FMO3 (P<0.05) and after 6 h. No transcriptional effect was ever noticed following bLS exposure to BZDs combination. Conclusion. Likewise to humans, bLS appear to be a reliable model to study CYP1A induction by NF and other potential aryl hydrocarbon receptor agonists. Meanwhile, for the first time we proved cattle FMO1-3 up-regulation by NF. About BZDs, FBZ and TCBZ were shown to affect CYP1A1/2 and FMO3 gene expression. Confirmatory studies on CYP1A and FMOs catalytic activities are actually underway.
Fenbendazole and triclabendazole effects on CYP1A1/1A2 and FMO1/3 mRNAs in cattle liver slices: preliminary results
GIANTIN, MERY;TOLOSI, ROBERTA;DACASTO, MAURO;
2017
Abstract
Background. Fenbendazole (FBZ) and triclabendazole (TCBZ) are benzimidazole drugs (BZDs) widely used in veterinary practice as anthelmintics. Members of cytochrome P450 (CYP) and flavin monooxygenase (FMO) superfamilies of drug metabolizing enzymes are primarily responsible of their biotransformation1. The xenobiotic-dependent up-regulation of CYPs is well documented, while FMOs are generally considered not inducible2. In the present study, the effect of FBZ and TCBZ (alone or in combination) on CYP1A1/2 and FMO1/3 mRNA levels were measured on precision-cut bovine liver slices (bLS). Methods. Precision-cut bLS from 6 male cattle were obtained according to Maté et al3. Following the fine-tuning of an absolute quantification protocol for target genes4, bLS were incubated for 0, 6 and 12 h with FBZ and TCBZ (50 µM), alone or in combination. β-naphthoflavone (βNF, 25 µM) was used as positive control to confirm gene induction. Target gene mRNA levels were measured by qPCR. The TATA Box binding protein and ribosomal protein lateral stalk subunit P0/β-actin were used as internal control genes to normalize FBZ/TCBZ and βNF data, respectively. Results. At T0, CYP1A2 mRNA levels were 25-fold higher than CYP1A1; however, FMO1 and 3 were equally represented. βNF up-regulated CYP1A1 (P<0.05; 4-fold vs control) after 6 h of incubation, while increasing amounts (P<0.05) of CYP1A2, FMO1/3 mRNAs were noticed after 12 h (4-, 2.5- and 3.5-fold vs control, respectively). Concerning BZDs, FBZ increased CYP1A1/2 mRNAs (P<0.05) after 12 h of incubation, whereas TCBZ up-regulated only FMO3 (P<0.05) and after 6 h. No transcriptional effect was ever noticed following bLS exposure to BZDs combination. Conclusion. Likewise to humans, bLS appear to be a reliable model to study CYP1A induction by NF and other potential aryl hydrocarbon receptor agonists. Meanwhile, for the first time we proved cattle FMO1-3 up-regulation by NF. About BZDs, FBZ and TCBZ were shown to affect CYP1A1/2 and FMO3 gene expression. Confirmatory studies on CYP1A and FMOs catalytic activities are actually underway.
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