Formation and Efflux of ATP-Binding Cassette Transporter Substrate 2,4-Dinitrophenyl-S-Glutathione from Cultured Human Term Placental Villous Tissue Fragments

Abstract

Upon exposure to 1-chloro-2,4-dinitrobenzene (CDNB), the human placental tissue forms its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG). The purpose of this study was to investigate the involvement of human placental ATP-binding cassette (ABC) transporters in the efflux of DNP-SG. Placental tissue samples were obtained from pregnant patients undergoing C-section deliveries following normal pregnancies; villous tissue was cultured in suspension, and DNP-SG formation and efflux upon exposure to 100 microM CDNB were measured by HPLC. DNP-SG efflux decreased by 69.1 (+/-11.3)%, 51.1 (+/-5.4)%, 56.7 (+/-8.3)% and 53.6 (+/-10.8)% (p < 0.05) in the presence of 5 mM sodium orthovanadate (ATPase inhibitor), 100 microM MK571 (MRP-inhibitor), 1 mM dipyridamole (BCRP/P-gp/MRP1-inhibitor) and 100 microM verapamil (P-gp/MRP1 inhibitor) respectively, without any change in DNP-SG formation, total tissue glutathione, GSH/GSSG ratio, tissue integrity or tissue viability. These data clearly established the role of ABC transporters in the human placental efflux of DNP-SG. To investigate the contribution of various ABC transporters toward DNP-SG transport, ATP-dependent transport of 3H-DNP-SG was determined in Sf9 membrane vesicles overexpressing P-gp, BCRP and the MRP proteins. MRP1-mediated DNP-SG transport was inhibited in the presence of sodium orthovanadate, MK571, dipyridamole and verapamil in the presence of glutathione. Furthermore, MRP1-mediated transport [K(t) = 11.3 +/- 1.3 microM and v(max) = 86.7 +/- 1.9 pmol/mg/min] was a high-affinity process compared to MRP2-mediated transport [K(t) = 168 +/- 7 microM and v(max) = 1367 +/- 18 pmol/mg/min]. The inhibition pattern and the kinetics of DNP-SG efflux in the placental villous tissue were consistent with MRP1-mediated DNP-SG efflux, suggesting a functional role and an apical localization for an MRP1-like transporter in the human placental syncytiotrophoblast.