Induction by Phenobarbital of Phase I and II Xenobiotic-Metabolizing Enzymes in Bovine Liver: An Overall Catalytic and Immunochemical Characterization

Abstract

In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs.

Keywords: cattle; drug-metabolizing enzymes; enzyme activity; hepatic drug metabolism; induction; phenobarbital; species differences.

Figure 1

Hepatic CYP2B22 (A), 3A (B), 2C88 (C), 2C31 (D), and 2C42 (E) mRNA levels in untreated control (UT, n = 3) and phenobarbital-treated (PB, n = 4) cattle. Data (arithmetic means ± SD) are expressed as n-fold change (arbitrary units, A.U.) normalized to ΔΔCt mean value of β-actin (ACTB, the chosen internal control gene, ICG), to which an arbitrary value of 1 was assigned. * p < 0.05; ** p < 0.01; *** p < 0.001 (unpaired t-test).

Figure 2

Hepatic CYP2B22 protein expression (A) and in vitro metabolism of CYP2B22 marker substrates benzphetamine (N-demethylation; (B)), 7-EFMC (O-demethylation; (C)), 7-benzyloxyresorufin (O-debenzylation; (D)) in untreated control (UT, n = 3) and phenobarbital-treated (PB, n = 4) cattle. In the radar plot (E), data are expressed in arbitrary units (A.U.), and a value of 1 was attributed to UT cattle. In the bar charts, data are expressed as arithmetic means ± SD. 7-EFMC: 7-ethoxy-4-trifluoromethylcoumarin. ** p < 0.01; *** p < 0.001 (unpaired t-test).

Figure 3

Hepatic CYP2C protein expression (A) and in vitro metabolism of CYP2C marker substrates aminopyrine (N-demethylation; (B)), chlorpheniramine (N-demethylation; (C)), and 7-MFMC (O-demethylation; (D)) in untreated control (UT, n = 3) and phenobarbital-treated (PB, n = 4) cattle. In the radar plot (E), data are expressed in arbitrary units (A.U.), and a value of 1 was attributed to UT cattle. In the bar charts, data are expressed as arithmetic means ± SD. 7-MFMC: 7-methoxy-4-trifluoromethylcoumarin. ** p < 0.01; *** p < 0.001 (unpaired t-test).

Figure 4

Hepatic CYP3A protein expression (A) and in vitro metabolism of CYP3A marker substrates TST (6β- and 16β-hydoxylation; (B)), erythromycin (N-demethylation; (C)), ethylmorphine (N-demethylation; (D)), TAO (N-demethylation; (E)), and monensin (O-demethylation; (F)) in untreated control (UT, n = 3) and phenobarbital-treated (PB, n = 4) cattle. In the radar plot (G), data are expressed in arbitrary units (A.U.), and a value of 1 was attributed to UT cattle. In the bar charts, data are expressed as arithmetic means ± SD. 6b-TSTOH: 6β-hydroxylated testosterone; 16b-TSTOH: 16β-hydroxylated testosterone; TAO: triacetyloleandomycin. * p < 0.05; ** p < 0.01; *** p < 0.001 (unpaired t-test).

Figure 5

Proteasome chymotrypsin-like activity in untreated control (UT, n = 3) and phenobarbital-treated (PB, n = 4) cattle. Data are expressed as arithmetic means ± SD. A.U.: arbitrary unit. *** p < 0.001 (unpaired t-test).