CONICET | Buscador de Institutos y Recursos Humanos

We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and eliminationpathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotictransporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cellswere exposed to SL (1, 5, 10, 20 or 50M) for 48 h. Expression of P-gp and itsmRNAlevels were estimatedby Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the abilityof the cells to accumulate the model substrate rhodamine 123 (Rh123, 5M), in the presence or absenceof verapamil (50M), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels weresignificantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantlyreduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally,we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistentwith P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ),a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identifythe nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leadingto a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude thatSL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. Thiseffect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of otherxenobiotics, including drugs of therapeutic use, that are P-gp substrates