Catalytic, immunochemical and molecular characterization of the xenobiotic-metabolising enzyme modulation by phenobarbital in the bovine liver

Introduction In mammalian species, phenobarbital (PB) has long been known to induce liver phase I metabolism, via activation of specific nuclear receptors (NRs), and phase II biotransformations as well. Nevertheless, the pattern of the in vivo modulation of oxidative, hydrolytic and conjugative drug metabolizing enzymes (DMEs) by this barbiturate has never been characterized in cattle. In the present study DME catalytic activity and expression were determined in liver subfractions from PB-induced or untreated cattle. Samples from the same animals were used for RNA isolation, cDNA cloning and sequencing of some bovine liver CYPs and their transcription factor (i.e. NR) as reported in Cantiello et al. (1). The ubiquitin (Ub)-dependent 26S proteasome system was also investigated, in view of the key role played in the proteolytic disposal of structurally aberrant, abnormal, and/or otherwise malformed proteins, including cytochrome P450s (CYPs) which may be damaged as the result of their catalytic activity. Materials and methods Seven male 10 months old-Friesian cattle were obtained from local farms. Four individuals received PB (18 mg/Kg b.w./day for 7 days) by oral gavage, while the remainders remained untreated (UT). Liver subcellular fractions were isolated by differential ultracentrifugation (3) and used to measure both the haemoprotein content and the rate of the in vitro metabolism of several phase I and phase II substrates (3, 4), that, based on studies performed in human, rodent and food producing species (5, 6), are reported to be markers for a number of oxidative or conjugative isozymes. Flavin-containing monooxygenase (FMO) activities were measured with methimazole or ethylene thiourea as substrates, according to published procedures. Apoprotein levels were measured by western blotting analysis as described (3). Proteasome chymotrypsin-like activity in liver extracts was assayed by monitoring in continuo the production of 7-amino-4-methylcoumarin (amc) from the fluorogenic peptide Suc-LLVY-amc (7). Data were analysed by Student’s t-test. Results and discussion As partly depicted in Table 1, PB enhanced most of the tested microsomal enzyme activities (1- to 19-fold increase). It is relevant to note that, as previously observed in primary cultures of hepatocytes from ruminant species (8), PB failed to significantly increase the rate of pentoxyresorufin O-depentylation, which is reported to be an extremely specific marker for CYP 2B activity in rodents (9). As regards testosterone hydroxylation, there was an increase in the generation of 2β- (about 4-fold), 6β- (3-fold) and 16β- (3-fold) OH-derivatives in treated vs control samples. As expressed as per nmole of CYP (turnover number) statistically significant changes were obtained for benzyloxyresorufin O-deethylase (7.5-fold; P<0.01), aminopyrine N-demethylase (2-fold; P<0.05) and 7-methoxy-4-trifluoromethylcoumarin O-demethylase (9-fold; P<0.001). Western immunoblots revealed a consistent increase in the expression of CYP2B-, CYP2C- and CYP3A-related proteins (2.6, 2.2 and 3.4 fold, respectively). As expected, PB did not augment FMO activities measured with either substrate, both in the presence or in the absence of CYP inhibitors (n-octylamine). Proteasome activity in liver extracts from PB-treated animals was about twice as that found in untreated controls (P<0.001), suggesting a PB-mediated increase in the amount of damaged hemoproteins. In line with results of previous experiments performed in calves (10), microsomal carboxylesterase activities toward different aromatic esters were slightly induced by the treatment, showing a significant increase only in the case of -naphthylacetate (about 1.5-fold; P<0.05). Glutathione S-transferase was not induced by the treatment, either using 1-chloro-2,4-nitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) as the substrates, whereas a modest rise (about 1.4-fold) in uridinediphosphoglucuronosyltransferase (UGT) activity was observed using p-nitrophenol (pNP) as substrate. It is well known that some isoenzymes of UGT1 family are inducible by PB, although this phenomenon may be underestimated because of the considerable overlapping substrate specificity displayed by the multiple transferase isoforms. As an example, either pNP or 1-naphthol are routinely used to assess the conjugative capacity of certain UGT1 isoenzymes (UGT1A6 and UGT1A1, respectively); however, neither of them can be considered as a specific substrate of any UGT (11). Further research is in progress to evaluate CYP mRNA levels and to confirm the involvement of CAR and other NRs in the induction mechanisms.