Tissue distribution and phenobarbital induction of target SLC- and ABC-transporters in cattle

Introduction. Phenobarbital (PB) is a prototypical cytochrome P450 (CYP) inducer, eliciting a number of pleiotropic effects (Handshin & Meyer, 2003). Besides CYPs, PB has been shown to affect some drug transporters (DTs) gene expression (Johnson et al., 2002; Patel et al., 2003). In cattle, little is still known about DTs mRNA tissue distribution and transcriptional effects of a general inducer such as PB. In this study, the constitutive expression and modulation of the solute carrier organic anion transporter family 1, member 3 (SLCO1B3), SLCO2B1 and SLC10A1 as well as ATP-binding cassette transporter B1 (ABCB1), ABCB11, ABCC2 and ABCG2 were investigated, for the first time, in liver and foremost extrahepatic tissues of control and PB-treated cattle. Target genes were chosen on a literature basis, and were represented by hepatic DTs known to be explicitly modulated by PB in human and laboratory species. Materials and Methods. Seven male Friesian cattle (10 months old) were used; four of them (PHEN) received PB by oral gavage (18 mg kg-1 body weight day-1 for 7 days), while the other ones were used as controls (CTRL). Bovines were slaughtered the day after the suspension of oral PB administration. 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. Target genes tissue distribution and transcriptional effects of PB were measured by using a quantitative Real Time PCR (qPCR) approach. Results. Two valid internal control genes (β-actin, acidic ribosomal protein large P0 and β-2-microglobulin) were indetified by using geNormPLUS algorithm (Vandesompele et al., 2002). Only two out of the seven target DTs (SLCO1B3 and SLC10A1) were not constitutively expressed in cattle tissues. On a comparative basis, most of DTs (SLCO2B1, ABCB1, ABCC2, ABCG2) were expressed in the liver, followed by testis (SLCO2B1, ABCB1, ABCG2), adrenal gland (SLCO2B1, ABCB1, ABCG2), lung (ABCB1, ABCG2), kidney and skeletal muscle (ABCG2). Phenobarbital never altered DTs mRNA levels, except for an increase of hepatic ABCC2 mRNA and a down-regulation of renal ABCG2. Conclusions. Present data only partially confirm those obtained in humans and laboratory species, and should be considered a preliminary step for further in depth molecular investigations about species-differences in DTs gene expression, regulation and function. Acknowledgements. This work was supported by grants from Università degli Studi di Padova (60A08‐9881) to M.D., and a PhD fellowship from Università degli Studi di Padova to V.Z. References. Handschin, C. & Meyer, UA. (2003). Induction of drug metabolism: the role of nuclear receptors. Pharmacological Reviews, 55, 649-673. Johnson, D.R., Habeebu, S.S. & Klaassen, C.D. (2002) Increase in bile flow and biliary excretion of glutathione-derived sulfhydryls in rats by drug-metabolizing enzyme inducers is mediated by multidrug resistance protein 2. Toxicological Sciences, 66, 16-26. Patel, N.J., Zamek-Gliszczynski, M.J., Zhang, P., Han, Y.H., Jansen, P.L., Meier, P.J., Stieger, B. & Brouwer, K.L. (2003) PB alters hepatic Mrp2 function by direct and indirect interactions. Molecular Pharmacology, 64, 154-159. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, 34.