Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism.
Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism.
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DOI：
10.1016/j.taap.2006.05.003
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发表时间：
2006-10
影响因子：
3.8
通讯作者：
Hai Wu;P. Cai;D. Clemens;T. Jerrells;G. Ansari;B. Kaphalia
中科院分区：
文献类型：
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作者：
Hai Wu;P. Cai;D. Clemens;T. Jerrells;G. Ansari;B. Kaphalia
慢性酒精滥用是一个主要的健康问题，会导致肝脏和胰腺疾病，并会损害肝脏乙醇脱氢酶（ADH）。肝脏ADH催化乙醇氧化是乙醇在体内处置的主要途径。肝微粒体细胞色素P450（CYP 2 E1），诱导慢性酒精滥用，也报告氧化乙醇。然而，已知大鼠模型中受损的肝脏ADH活性促进非氧化代谢，导致乙醇的非氧化代谢物例如脂肪酸乙酯（FAEE）通过FAEE合酶催化的非氧化途径形成。因此，在HepG 2细胞和转染ADH（VA-13）、CYP 2 E1（E47）或ADH + CYP 2 E1（VL-17 A）的重组HepG 2细胞中确定了乙醇诱导细胞毒性的代谢基础。Western印迹分析显示，与ADH过表达的VA-13和VL-17 A细胞相比，HepG 2和E47细胞中ADH缺乏。将附着的HepG 2细胞和重组细胞与乙醇一起孵育，并通过测量FAEEs的形成来确定乙醇的非氧化代谢。在细胞培养物中，在所有浓度的乙醇（100-800 mg%）孵育6 h（FAEE合成的最佳时间）时，HepG 2和E47细胞中的FAEE合成水平显著高于VA-13和VL-17 A细胞。这些结果表明，ADH催化的乙醇氧化代谢是其处置的主要机制，无论CYP 2 E1过表达。另一方面，ADH活性降低有利于乙醇非氧化代谢为FAEE，如在E47细胞中发现的，无论CYP 2 E1过表达。因此，CYP 2 E1介导的乙醇氧化可能是乙醇处置的次要机制。仅在HepG 2和VA-13细胞中进行的进一步研究表明，HepG 2细胞中的乙醇处置和ATP浓度低于VA-13细胞，中性脂质积累和细胞毒性（凋亡）高于VA-13细胞。在与乙醇孵育的HepG 2与VA-13细胞中观察到的细胞凋亡似乎是通过激活半胱天冬酶-9和半胱天冬酶-3释放线粒体细胞色素c介导的。这些结果有力地支持了我们的假设，即肝脏ADH活性降低有利于乙醇的非氧化代谢，乙醇非氧化代谢产物通过内源性途径引起HepG 2细胞凋亡。
Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100–800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.