Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione
Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione
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DOI：
10.1074/jbc.m510484200
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发表时间：
2006-02-24
影响因子：
4.8
通讯作者：
Cederbaum, AI
中科院分区：
文献类型：
--
作者：
Bai, JX;Cederbaum, AI
乙醇对CYP 2 E1的诱导是乙醇引起氧化应激和酒精性肝病的一种机制。尽管CYP 2 E1主要存在于内质网中，但它也位于大鼠肝脏线粒体中。在目前的研究中，慢性饮酒诱导大鼠肝线粒体CYP 2 E1。为了研究线粒体靶向CYP 2 E1在产生氧化应激和引起线粒体损伤中的作用，通过将含有缺少疏水性内质网靶向信号序列的人CYP 2 E1 cDNA的质粒转染HepG 2细胞，随后通过G418筛选，建立了线粒体中过表达CYP 2 E1的HepG 2细胞系（mE 10和mE 27细胞）。蛋白质印迹分析发现，在这些细胞的线粒体室中，有40 kDa的催化活性的NH 2-末端截短形式的CYP 2 E1（mtCYP 2 E1）。与空载体转染的细胞（pCI-neo）相比，在mE 10和mE 27细胞中，由丁硫氨酸亚砜（BSO）耗尽GSH引起的细胞死亡增加。抗氧化剂能够消除细胞活力的丧失。在BSO处理的mE 10和mE 27细胞中观察到活性氧和线粒体3-硝基酪氨酸和4-羟基壬烯醛蛋白加合物水平增加，线粒体顺乌头酸酶活性和线粒体膜电位降低。线粒体膜稳定剂环孢菌素A也能够保护这些细胞免受BSO毒性。这些结果表明，线粒体区室中的CYP 2 E1可诱导线粒体中的氧化应激，损害线粒体膜电位，并导致细胞活力丧失。饮酒诱导肝线粒体CYP 2 E1的蓄积可能在酒精性肝病中起重要作用。
Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.