Molecular mechanism of P-glycoprotein induction by Genistein in rat hepatocyte primary culture

SEMENIUK M; BUCCI MUÑOZ M; CERÉ L; TABORDA D; CATANIA VA; RIGALLI JP; RUIZ ML

Congreso; Reunión Anual SAFIS 2019; 2019

Background and Aim: Previously, we found that hepatic P-glycoprotein (P-gp), an ATP binding cassette (ABC)-transporter involved in xenobiotic excretion, was induced by genistein (GNT) treatment. GNT is a phytoestrogen that behaves as a ligand of nuclear receptors, such as estrogen receptor (ER) and pregnane X receptor (PXR), and interacts with transcription factors (e.g. Nrf2) involved in the regulation of ABC transporters. The aim of this study was to elucidate the molecular mechanism of P-gp induction by GNT.Methods: Isolated rat hepatocytes were cultured and incubated with GNT (10 µM) for 6 h, control hepatocytes (C) were incubated with GNT vehicle (dimethyl sulfoxide, 0.1 % v/v). To evaluate ER or PXR participation in P-gp regulation by GNT, ER antagonist (ICI182/780, 1 µM) or PXR inhibitor (sulforaphane, SFN, 10 µM), both added 30 min before GNT, or vehicle, were used. Involvement of Nrf2 was evaluated by siRNA knock down (KD). P-gp encoding genes: Mdr1a and Mdr1b, and Nrf2 mRNA levels were measured by Real Time PCR. The efficiency of Nrf2 KD was evaluated by measuring mRNA levels of Nrf2.Results: GNT (10 µM) increased Mdr1a levels (GNT: 136±15 % vs C: 100±4 %). Mdr1b levels remained unchanged. ER antagonist did not prevent Mdr1a induction by GNT (ICI182/780 + GNT: 161±8 % vs ICI182/780: 100±14 %). SFN prevented Mdr1a induction by GNT (SFN + GNT: 129±27 % vs SFN: 100±20 %). Nrf2 KD did not prevent Mdr1a induction by GNT (Nrf2 KD + GNT: 173±33 % vs Nrf2 KD: 100±29 %). All results are presented as mean ± SD, n = 3-4, p < 0.05.Discussion: GNT induced Mdr1a levels in hepatocyte primary culture. Since the ER antagonist, ICI 182/780, and the KD of Nrf2 did not prevent GNT from increasing Mdr1a mRNA levels, it can be suggested that GNT is acting through an ER and Nrf2 independent mechanism. Our results suggest PXR as a potential candidate to mediate P-gp up-regulation by GNT, since its antagonist, sulforaphane, prevented Mdr1a mRNA induction.