Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically
Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically
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DOI：
10.1093/toxsci/68.1.93
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发表时间：
2002-07-01
影响因子：
3.8
通讯作者：
Morgan, KT
中科院分区：
文献类型：
--
作者：
Merrill, CL;Ni, H;Morgan, KT
尽管已知它们是有效的抗糖尿病药物，但关于肉碱棕榈酰基转移酶-1(CPT-1)抑制剂的毒性效应，如依托莫西(ET)，鲜有报道。这些化合物通过不可逆地与CPT-1结合并阻止长链脂肪酸进入线粒体基质来抑制线粒体脂肪酸的β-氧化。基因芯片分析显示，1 mM依托托莫昔尔处理HepG2细胞6h后，细胞内几个氧化还原相关基因和细胞周期的mRNAs表达发生了显著的变化。上调的基因包括血红素加氧酶1(HO1)、8-氧鸟嘌呤DNA糖基酶1(OGG1)、谷胱甘肽还原酶(GSR)、细胞周期蛋白依赖性激酶抑制因子1(CDKN1[p21(WAF)])和锰超氧化物歧化酶前体(SOD2)，而细胞色素P450 1A1(CYP1A1)和热休克70kD蛋白1(HSPA1A)表达下调。实时定量聚合酶链式反应(RT-PCR)证实了4个mRNAs(CYP1A1、HO1、GSR、CDKN1)发生了显著的改变，并发现了另外3个mRNAs的改变：2个氧化还原相关基因：谷氨酸半胱氨酸连接酶修饰亚单位(GCLM)和硫氧还蛋白还原酶(TXNRD1)，1个DNA复制基因拓扑异构酶IIa(TOP2A)。在给药后15分钟至24小时的11个时间点上，用RT-PCR检测选定的mRNA水平的时间变化。氧化应激和线粒体能量代谢受损表现为还原型谷胱甘肽(GSH)、还原型谷胱甘肽/氧化型谷胱甘肽比值(GSH/GSSG)、线粒体膜电位(Mp)和三磷酸腺苷(ATP)水平显著降低，氧化谷胱甘肽(GSSG)和超氧化物歧化酶(O2)同时升高。这是关于依托莫西引起的氧化应激的第一个报告。
Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIa (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h post-dosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.