Reactive oxygen species participate in mdr1b mRNA and P-glycoprotein overexpression in primary rat hepatocyte cultures

P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several facto rs shown to induce mdr1 overexpression (UV irradiation, epidermal growth fa ctor, tumour necrosis factor a, doxorubicin) have been associated with the generation of reactive oxygen species (ROS), In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the m dr1b gene were used as a model system to investigate whether ROS might part icipate in the regulation of intrinsic mdr1b overexpression. Addition of H2 O2 to the culture medium resulted in a significant increase in mdr1b mRNA a nd P-glycoprotein after 3 days of culture, with maximal (similar to 2-fold) induction being observed with 0.5-1 mM H2O2, Furthermore, H2O2 led to acti vation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA st rand breaks, indicating that ROS reached the nuclear compartment, Thus, ext racellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdr1b mRNA and P-glycoprotein expression. Conversely , antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycopr otein overexpression, Intracellular steady-state levels of the mdr1 substra te rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreate d with H2O2 Or aminotriazole and decreased in antioxidant-treated cells. Th e induction of mdr1b mRNA and of functionally active mdr1-type P-glycoprote ins by elevation in intracellular ROS levels and the repression of intrinsi c mdr1b mRNA and P-glycoprotein overexpression by antioxidant compounds sup port the conclusion that the expression of the mdr1b P-glycoprotein is regu lated in a redox-sensitive manner.