ATP-dependent transport of aflatoxin B-1 and its glutathione conjugates by the product of the multidrug resistance protein (MRP) gene

Glutathione-S-transferase-catalyzed conjugation of glutathione (GSH) to aflatoxin B-1-8,9-epoxide plays an important role in preventing binding of this ultimate carcinogen to target macromolecules. Once formed, the aflatoxin B-1-epoxide-GSH conjugates are actively extruded from the cell by an unidentified ATP-dependent export pump or pumps. Two possible candidates for this GSH conjugate pump are the 190-kDa multidrug resistance protein (MRP) and the 170-kDa P-glycoprotein. Both proteins belong to the ATP-binding cassette superfamily of transmembrane transport proteins and confer resistance to a similar spectrum of natural-product drugs. Using membrane vesicles from MRP-transfected cells, we found that MRP transports GSH conjugates of both the endo-isomers and exo-isomers of aflatoxin B-1-8,9-epoxide in an ATP-dependent, osmotically sensitive manner (V-max = 180 pmol/mg/min, K-m = 189 nM). Membrane vesicles from P-glycoprotein-overexpressing cells showed very low levels of transport, MRP-mediated transport was inhibited by an MRP-specific monoclonal antibody and by a variety of GSH derivatives and cholestatic steroid glucuronides. ATP-dependent transport of unmodified aflatoxin B-1 by MRP-enriched membrane vesicles was low but markedly enhanced in the presence of 5 mM GSH, even though GSH conjugates of aflatoxin B-1 were not formed by the vesicles. These data demonstrate that MRP is capable of energy-dependent transport of aflatoxin B-1 and its GSH conjugates and suggest a potential protective role for MRP in mammalian chemical carcinogenesis.