Wang Z, Dey S, Rosen B P, Rossman T G
Nelson Institute of Environmental Medicine and Kaplan Cancer Center, New York University Medical Center, New York 10016, USA.
Toxicol Appl Pharmacol. 1996 Mar;137(1):112-9. doi: 10.1006/taap.1996.0062.
Several Chinese hamster V79 cell line variants resistant to arsenite and one arsenite-hypersensitive variant have been isolated. The basis for the variation in arsenite sensitivity was studied by transport experiments using radiolabeled arsenite. Two arsenite-resistant variants (As/R7 and As/R27) exhibited decreased accumulation of arsenite, and the hypersensitive variant (As/S5) exhibited increased arsenite accumulation compared with the parental line. Cells depleted of endogenous energy reserves were loaded with radiolabeled arsenite, and the rate of arsenic efflux was measured. Arsenite-resistant variants exhibited an increased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with the protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase also decreased arsenite efflux, suggesting the involvement of an arsenite-glutathione complex. However, separation of the products of extrusion and the intracellular arsenic species by paper chromatography followed by autoradiography failed to show the appearance of an arsenite-glutathione complex in either case. Rather, all label in the product of the transport reaction appeared to be arsenite whether cells were loaded with arsenate or arsenite, indicating first that intracellular reduction of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was associated with high-molecular-weight material, possibly protein. Our results demonstrate the existence of an energy-dependent arsenical efflux pump in mammalian cells and show that arsenic is extruded as arsenite.
已分离出几种对亚砷酸盐具有抗性的中国仓鼠V79细胞系变体以及一种对亚砷酸盐敏感的变体。通过使用放射性标记亚砷酸盐的转运实验研究了亚砷酸盐敏感性变化的基础。与亲本细胞系相比，两种抗亚砷酸盐变体（As/R7和As/R27）表现出亚砷酸盐积累减少，而敏感变体（As/S5）表现出亚砷酸盐积累增加。耗尽内源性能量储备的细胞用放射性标记的亚砷酸盐加载，并测量砷流出率。抗亚砷酸盐变体表现出流出率增加，而敏感变体表现出流出率降低。在用质子载体羰基氰化物间氯苯腙孵育的细胞中，流出减少，表明其能量依赖性。两种谷胱甘肽S-转移酶抑制剂也降低了亚砷酸盐流出，表明涉及亚砷酸盐-谷胱甘肽复合物。然而，通过纸色谱法随后进行放射自显影分离挤出产物和细胞内砷物种，在两种情况下均未显示出亚砷酸盐-谷胱甘肽复合物的出现。相反，无论细胞加载的是砷酸盐还是亚砷酸盐，转运反应产物中的所有标记似乎都是亚砷酸盐，这首先表明细胞内发生了As(V)还原为As(III)，其次表明亚砷酸盐是以未结合的形式转运的。所有细胞内标记都与高分子量物质（可能是蛋白质）相关。我们的结果证明了哺乳动物细胞中存在能量依赖性砷流出泵，并表明砷以亚砷酸盐形式被挤出。
