EFFLUX-MEDIATED RESISTANCE TO ARSENICALS IN ARSENIC-RESISTANT AND ARSENIC-HYPERSENSITIVE CHINESE-HAMSTER CELLS

Several Chinese hamster V79 cell line variants resistant to arsenite a nd one arsenite-hypersensitive variant have been isolated. The basis f or the variation in arsenite sensitivity was studied by transport expe riments using radiolabeled arsenite. Two arsenite-resistant variants ( As/R7 and As/R27) exhibited decreased accumulation of arsenite, and th e hypersensitive variant (As/S5) exhibited increased arsenite accumula tion compared with the parental line. Cells depleted of endogenous ene rgy reserves were loaded with radiolabeled arsenite, and the rate of a rsenic efflux was measured. Arsenite-resistant variants exhibited an i ncreased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with th e protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase al so decreased arsenite efflux, suggesting the involvement of an arsenit e-glutathione complex. However, separation of the products of extrusio n and the intracellular arsenic species by paper chromatography follow ed by autoradiography failed to show the appearance of an arsenite-glu tathione complex in either case. Rather, all label in the product of t he transport reaction appeared to be arsenite whether cells were loade d with arsenate or arsenite, indicating first that intracellular reduc tion of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was a ssociated with high-molecular-weight material, possibly protein. Our r esults demonstrate the existence of an energy-dependent arsenical effl ux pump in mammalian cells and show that arsenic is extruded as arseni te. (C) 1996 Academic Press, Inc.