Previous studies by this laboratory have indicated that expression of the multidrug resistance (mdr) gene can be increased in vivo by exposure to a variety of xenobiotics. Because of the nature of these compounds, it was proposed that mdr gene expression might, at least in part, be regulated by the arylhydrocarbon (Ah) receptor. In the present study, we used a primary hepatocyte culture model to examine the relationship between induction of cytochrome P450IA and mdr expression in vitro. Both 3-methylcholanthrene (MC) and 2-acetylaminofluorene (AAF) were efficient inducers of mdr expression in this model. Induction of mdr gene expression by both MC and AAF obeyed a log10 concentration/response relationship. In contrast, 2,3,7,8-tetrachlorodibenzo-P-dioxin did not induce mdr expression at concentrations that yielded maximum induction of cytochrome P450IA expression. These data suggest that mdr induction was not mediated via the Ah receptor. Nuclear run-off analysis indicated that both AAF and MC induced mdr expression by increasing transcription. Primer extension analysis indicated that mdr gene transcription was initiated at one major site 151 bp upstream of the ATG site in both the uninduced and induced state in vivo and in vitro. The sequence of the primer and the site of initiation of gene transcription indicate that the main gene induced was the mdr 1b gene.
P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant (mdr) gene family. The physiological function of this protein is unknown. Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis. Both obstructive and alpha-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver. Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain. Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription. alpha-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes. To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats. Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil. These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.
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