Adenosine triphosphate-dependent transport of estradiol-17β(β-D-glucuronide) in membrane vesicles by MDR1 expressed in insect cells

MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1-expressing multidrug-resistant cells display increased resistance to estradiol-17 beta(beta-D-glucuronide) (E(2)17G). MDR1 substrates/ modulators inhibit adenosine triphosphate (ATP)-dependent transport of E(2)17G in the rat canalicular membrane and protect against E(2)17G-mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E(2)17G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP-dependent transport of E(2)17G (10 mu mol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1-baculovirus. This transport was saturable (K-m = 62 mu mol/L) and occurred into an osmotically sensitive space. ATP-dependent transport of E(2)17G (10 mu mol/L) was inhibited 63% by 10 mu mol/L daunomycin, but not by 100 mu mol/L S-(2,4-dinitrophenyl)glutathione (GS-DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]), Glucuronide conjugates of the estrogen D-ring (100 mu mol/L), estriol-17 beta(beta-D-glucuronide) (E(3)17G) and estriol-16 alpha(beta-D-glucuronide) (E(3)16G), inhibited MDR1-mediated E(2)17G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol-3-(beta-D-glucuronide) (E(2)3G) or estradiol-3-sulfate-17 beta(beta-D-glucuronide) (E(2)3SO(4)17G), had no effect. E(2)17G neither stimulated MDR1 ATPase activity nor inhibited verapamil-stimulated ATPase activity Infusion of 1.5 mu mol/L doxorubicin or 1 mu mol/L taxol protected against cholestasis induced by E(3)16G and E(3)17G in isolated perfused rat liver. These studies identify E(2)17G, and probably E(3)16G and E(3)17G, as endogenous substrates for MDR1.