MODULATION OF DRUG METABOLISING ENZYMES (DMES) AND RELATED NUCLEAR RECEPTORS (NRS) GENE EXPRESSION PROFILES IN THE LIVER AND EXTRA-HEPATIC TISSUES OF CATTLE INDUCED WITH PHENOBARBITAL (PB)

In mammals, PB induces hepatic and extra-hepatic DMEs by the activation of specific NRs [1]. Although barbiturates pharmacological effects were discovered more then 40 years ago, molecular mechanisms involved in PB induction have been only recently understood in human and laboratory species [2]; furthermore, few data about PB effects upon DMEs and NRs of veterinary species have been published [3-5]. In the present work, the transcriptional effect of PB, administered at inductive dosage regimen, was investigated upon DMEs and NRs in the liver and extra-hepatic tissues of cattle. Seven male Friesian cattle (10 months old) were used; four of them (PHEN) received PB by gavage (18 mg kg-1 body weight day-1 for 7 days), while the other three remained untreated (CTRL). The experiment was performed according to the EC Directive 86/609 and the Italian D.L. 27/01/1992, nr 116. Bovines were slaughtered the day after the suspension of PB administration. Small aliquots of liver, duodenum, kidney, lung, testis, adrenal, and muscle were collected, immediately snap frozen in liquid nitrogen, and stored at -80°C until use. Firstly, the most valid reference genes for the comparative evaluation of data from different tissues were identified, by using the NormFinder program [6] and four candidate genes were considered (β-actin, ACTB; acidic ribosomal protein large P0, RPL0; beta-2-microglobulin, β2M and peptidylprolyl isomerise A, PPIA). Thereafter, the transcriptional effect of PB upon a set of target genes was measured by using a quantitative Real Time RT-PCR approach. The target genes investigated were cytochromes P450 (CYPs) 2B22, 2C87, 2C31, 2C49 and 3A28; the constitutive androstane receptor (CAR), pregnane X receptor, retinoic X receptor-alpha (RXRα); and, finally, glutathione S-transferase A1 (GSTA1), sulfotransferase 1A1 and 2A1 (SULT1A1 and SULT2A1, respectively). Two valid housekeeping genes (ACTB and RPL0) were identified among the chosen candidates, thereby allowing a more precise comparison of transcriptional data obtained from tissues richly endowed of DMEs (liver, duodenum, kidney, lung) with those showing from low to very low level of expression (testis, adrenal, muscle). Phenobarbital and other PB-like chemicals induce liver CYP2A, 2B (mostly), 2C, 3A and as well as others DMEs, albeit to a lower extent; moreover, CAR plays a key role in such up-regulations [7]. In cattle liver, a PB-dependent significant increase (p<0.01) of CYP2B22, 2C31, 2C87 and 3A28 mRNAs was recorded, while the expression of the CYP2C49 gene was unaffected. These results agree with those previously reported in humans, rats and mice as well as with post-translational data from cattle themselves [5,8]. On the contrary, NRs gene expression profiles did not show differences among CTRL and PHEN groups. Phase II DMEs did not respond univocally to PB: a significant increase (p<0.05) was observed for GSTA1; by contrast, SULT1A1 gene expression was significantly (p<0.05) decreased, while that of SULT2A1 was unaffected. The pattern of phase II gene expression now reported resembles that described in rodents, where too PB induced GSTA1, but reduced in a dose-dependent manner both SULT1 and SULT2 [9-10]. As regards the extra-hepatic sites of drug metabolism, statistically significant changes were found only in the duodenum of PB-treated animals, where a general inhibitory trend was observed; such a down-regulation was significant for CYP2C87 (p<0.05), RXRα (p<0.05) and SULT1A1 (p<0.05). By contrast, CYP2C31 gene was not expressed at all. Altogether, these results confirm that NRs are expressed also in cattle gastrointestinal tract [11], but disagree with the transcriptional PB induction of CYPs observed in vitro and in vivo in human and rat intestine [12]. Remarkable effects attributable to PB were never found in target gene expression profiles in the kidney, lung, testis and adrenal of PHEN group, as reported in rats and humans [13]; besides, some of them were poorly expressed or undetectable. Finally, a significant inhibition of SULT1A1 gene (p<0.05) was noticed in muscle, where the increase of CYP2B22 and CAR mRNAs was not significant. Phenobarbital, at inducing dosage regimens, prompted different transcriptional effects upon cattle liver and extra-hepatic DMEs and NRs mRNAs. These results partially disagree with those reported in previous comparative studies. Confirmatory investigations are thereby needed and are planned in our laboratory.