Zhang Wei, St Clair Daret, Butterfield Allan, Vore Mary
*Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536;
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506.
Toxicol Sci. 2016 May;151(1):44-56. doi: 10.1093/toxsci/kfw021. Epub 2016 Jan 27.
Doxorubicin (DOX) induces dose-dependent cardiotoxicity in part due to its ability to induce oxidative stress. We showed that loss of multidrug resistance-associated protein 1 (Abcc1/Mrp1) potentiates DOX-induced cardiac dysfunction in mice in vivo Here, we characterized DOX toxicity in cultured cardiomyocytes (CM) and cardiac fibroblasts (CF) derived from C57BL wild type (WT) and Mrp1 null (Mrp1-/-) neonatal mice. CM accumulated more intracellular DOX relative to CF but this accumulation did not differ between genotypes. Following DOX (0.3-4 μM), Mrp1-/- CM, and CF, especially CM, showed a greater decrease in viability and increased apoptosis and DNA damage, demonstrated by higher caspase 3 cleavage, poly (ADP-ribose) polymerase 1 (PARP) cleavage and phosphorylated histone H2AX (γH2AX) levels versus WT cells. Saline- and DOX-treated Mrp1-/- cells had significantly higher intracellular GSH and GSSG compared with WT cells (P < .05), but the redox potential (Eh) of the GSH/GSSG pool did not differ between genotypes in CM and CF, indicating that Mrp1-/- cells maintain this major redox couple. DOX increased expression of the rate-limiting GSH synthesis enzyme glutamate-cysteine ligase catalytic (GCLc) and regulatory subunits (GCLm) to a significantly greater extent in Mrp1-/- versus WT cells, suggesting adaptive responses to oxidative stress in Mrp1-/- cells that were inadequate to afford protection. Expression of extracellular superoxide dismutase (ECSOD/SOD3) was lower (P < .05) in Mrp1-/- versus WT CM treated with saline (62% ± 8% of WT) or DOX (43% ± 12% of WT). Thus, Mrp1 protects CM in particular and CF against DOX-induced toxicity, potentially by regulating extracellular redox states.
阿霉素（DOX）会诱导剂量依赖性心脏毒性，部分原因是其诱导氧化应激的能力。我们发现多药耐药相关蛋白1（Abcc1/Mrp1）缺失会增强DOX在体内诱导的小鼠心脏功能障碍。在此，我们对源自C57野生型（WT）和Mrp1基因敲除（Mrp1-/-）新生小鼠的培养心肌细胞（CM）和心脏成纤维细胞（CF）中的DOX毒性进行了表征。相对于CF，CM积累了更多的细胞内DOX，但这种积累在不同基因型之间没有差异。在用DOX（0.3 - 4μM）处理后，Mrp1-/- CM和CF，尤其是CM显示出更大的活力下降、凋亡增加和DNA损伤，通过与WT细胞相比更高的半胱天冬酶3裂解、聚（ADP - 核糖）聚合酶1（PARP）裂解和磷酸化组蛋白H2AX（γH2AX）水平得以证明。与WT细胞相比，盐水和DOX处理的Mrp1-/-细胞具有显著更高的细胞内谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）（P < 0.05），但在CM和CF中，GSH/GSSG池的氧化还原电位（Eh）在不同基因型之间没有差异，表明Mrp1-/-细胞维持了这一主要的氧化还原对。与WT细胞相比，DOX在Mrp1-/-细胞中比在WT细胞中更大程度地增加了限速GSH合成酶谷氨酸 - 半胱氨酸连接酶催化亚基（GCLc）和调节亚基（GCLm）的表达，这表明Mrp1-/-细胞中对氧化应激的适应性反应不足以提供保护。在用盐水（为WT的62% ± 8%）或DOX（为WT的43% ± 12%）处理的Mrp1-/- CM与WT CM中，细胞外超氧化物歧化酶（ECSOD/SOD3）的表达较低（P < 0.05）。因此，Mrp1尤其能保护CM和CF免受DOX诱导的毒性，可能是通过调节细胞外氧化还原状态来实现的。
