Diah S K, Smitherman P K, Townsend A J, Morrow C S
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA.
Toxicol Appl Pharmacol. 1999 Jun 1;157(2):85-93. doi: 10.1006/taap.1999.8672.
We examined the roles of glutathione S-transferase (GST) P1-1 and the glutathione S-conjugate (GS-X) transporter, multidrug resistance protein 1 (MRP1), singly or in combination, in the detoxification of 1-chloro-2,4-dinitrobenzene (CDNB). Derivatives of MCF7 breast carcinoma cells expressing GST P1-1 and MRP1 alone or in combination were developed. Detoxification was measured in cells as formation of the glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathione (DNP-SG), efflux of DNP-SG, and ultimately protection from CDNB cytotoxicity. MRP1 expression in the absence of GST P1-1 confers a three- to fourfold resistance to CDNB, which is associated with a >10-fold increase in the maximum rate of DNP-SG efflux. DNP-SG efflux in MRP1-expressing MCF7 cells was ATP-dependent and exhibited an apparent Km for DNP-SG of 95 microM. MRP1 expression alone, however, had no effect on DNP-SG formation. Combined expression of GST P1-1 and MRP1 increased the rates of DNP-SG formation when cells were exposed to 10 microM CDNB. Moreover, combined expression of GSTP1-1 with MRP1 moderately augmented MRP1-mediated resistance to CDNB but only during short term (10 min) exposures to CDNB where IC50 values were in the 8-10 microM range. In contrast, expression of GST P1-1 in the absence of MRP1 slightly sensitized cells to the toxicity of CDNB (10 min exposures), despite increasing rates of DNP-SG formation. The sensitization to CDNB in cells expressing GST P1-1 alone was associated with increased intracellular accumulation of DNP-SG, indicating that DNP-SG may contribute to CDNB toxicity. The potential toxicity of DNP-SG is also suggested by the finding that inhibition of DNP-SG formation by prior glutathione depletion confers resistance to CDNB cytotoxicity in MRP1-poor MCF7 cells. Altogether, our results demonstrate that glutathione conjugation and MRP1-mediated conjugate efflux can operate together to confer resistance to CDNB. The data indicate that MRP1-mediated conjugate efflux is required for cytoprotection from CDNB because its conjugate (DNP-SG), when present at high intracellular levels, may also be toxic to cells.
我们研究了谷胱甘肽S-转移酶（GST）P1-1和谷胱甘肽S-共轭物（GS-X）转运蛋白多药耐药蛋白1（MRP1）单独或联合作用对1-氯-2,4-二硝基苯（CDNB）解毒的作用。构建了单独或联合表达GST P1-1和MRP1的MCF7乳腺癌细胞衍生物。通过测定细胞中CDNB的谷胱甘肽共轭物S-(2,4-二硝基苯基)-谷胱甘肽（DNP-SG）的形成、DNP-SG的外排以及最终对CDNB细胞毒性的保护作用来衡量解毒情况。在缺乏GST P1-1时，MRP1的表达使细胞对CDNB产生三到四倍的抗性，这与DNP-SG最大外排速率增加10倍以上相关。在表达MRP1的MCF7细胞中，DNP-SG的外排依赖于ATP，其对DNP-SG的表观Km为95 microM。然而，单独表达MRP1对DNP-SG形成没有影响。当细胞暴露于10 microM CDNB时，GST P1-1和MRP1的联合表达增加了DNP-SG的形成速率。此外，GSTP1-1与MRP1的联合表达适度增强了MRP1介导的对CDNB的抗性，但仅在短期（10分钟）暴露于CDNB且IC50值在8-10 microM范围内时。相反，在缺乏MRP1时，GST P1-1的表达使细胞对CDNB的毒性稍有敏感（10分钟暴露），尽管DNP-SG的形成速率增加。单独表达GST P1-1的细胞对CDNB的敏感性增加与细胞内DNP-SG积累增加有关，表明DNP-SG可能导致CDNB毒性。预先耗尽谷胱甘肽抑制DNP-SG形成可使MRP1含量低的MCF7细胞对CDNB细胞毒性产生抗性，这一发现也提示了DNP-SG的潜在毒性。总之，我们的结果表明谷胱甘肽共轭作用和MRP1介导的共轭物外排可以共同作用赋予对CDNB的抗性。数据表明，MRP1介导的共轭物外排对于防止CDNB的细胞毒性是必需的，因为其共轭物（DNP-SG）在细胞内高浓度存在时可能对细胞也有毒性。
