to H2O2 for 24 h led to a significant decrease in active caspase 3 levels when compared with corresponding control cells treated with H2O2. Reduced cell death 
in MRP1 silenced cells treated with H2O2 under conditions of higher cellular GSSG may in part ” be MRP1-Mediated GSH Efflux in RPE Cells due to increased glutathione reductase resulting in increased conversion of GSSG to GSH. Overall, these data support the conclusion that inhibition of MRP1 protects RPE cells from H2O2-induced cell death which is mediated by changes in thiol status and GR. Increased GSH efflux and susceptibility to cell death in MRP1 overexpressing cells We next overexpressed human MRP1 in ARPE-19 cells to study whether MRP1 overexpression would affect GSH and GSSG release. Real-time PCR and immunoblot analyses established the level of overexpression in MRP1 transfected cells. GSH release was significantly higher in MRP1 overexpressing than vector controls treated with H2O2 for 5 h. There was no significant change in LDH release in MRP1 overexpressing cells when compared with control cells indicating that GSH release was not due to toxicity. Intracellular GSH levels in MRP1 overexpressing cells were significantly lower than vector control cells. We further examined the effect of H2O2 exposure for 5 h, 24 h, and 36 h in control and MRP1 overexpressing cells. The extent of cell death did not differ between control and MRP1 overexpressing cells at a shorter duration of H2O2 treatment. However, at 24 h and 36 h of H2O2 treatment, a progressive increase in cell death was seen in control cells. Oxidant-induced cell toxicity in MRP1 overexpressing cells was significantly higher than that seen in vector alone control cells. This finding was corroborated by levels of caspase 3 activation which progressively increased as the duration of H2O2 exposure increased. To explore the mechanism of cell death, we determined the GSH and GSSG levels in MRP1 overexpressed cells treated with H2O2 for 36 h. Cellular GSH levels were reduced by 32% in MRP1 overexpressed cells compared to vector control cells. H2O2 treatment further significantly decreased cellular GSH levels by 25% and 62%, respectively in vector control cells and MRP1 overexpressed cells. However, GSSG levels were markedly lower in MRP1 stressed as well as unstressed cells when compared to vector alone cells. With regard to efflux, MRP1 overexpressed cells effluxed significantly higher amounts of GSH vs vector controls 9301676 with or without exposure to H2O2. On the other hand, GSSG release was very low in MRP1 overexpressed cells under stressed as well as unstressed conditions. These data show that MRP1 overexpression enhances RPE susceptibility to oxidant MRP1-Mediated GSH Efflux in RPE Cells induced cell death due to low cellular GSH by increased GSH efflux. Discussion RPE cells and retina from a-crystallin KO mice are highly susceptible to oxidant injury. Though multiple molecular mechanisms have been proposed to account for the function of crystallins in apoptosis, the role of GSH or thiols in this process has not Piceatannol chemical information received much attention. Depending on the severity of oxidant injury, cells undergo either GSH-dependent apoptosis or GSH-independent necrosis. We have demonstrated that H2O2-induced cell death in a-crystallin KO RPE cells was due to apoptosis. The dose of H2O2 used in the current study was previously shown by us to induce ROS production in RPE cells. Here we show that apoptosis induced by H2O2 decreased significan
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