Li Shi-Yan, Gomelsky Mark, Duan Jinhong, Zhang Zhaojie, Gomelsky Larissa, Zhang Xiaochun, Epstein Paul N, Ren Jun
Division of Pharmaceutical Sciences, Department of Molecular Biology and Microscopic Facilities, University of Wyoming, Laramie, Wyoming 82071-3375.
J Biol Chem. 2004 Mar 19;279(12):11244-52. doi: 10.1074/jbc.M308011200. Epub 2004 Jan 13.
Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken beta-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0-200 microm) elicited ROS generation and apoptosis in HUVECs in a time- and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehyde-induced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant alpha-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.
乙醛是主要的乙醇代谢产物，其毒性和反应性远高于乙醇，据推测它是酒精诱导组织和细胞损伤的原因。本研究旨在探讨促进乙醛代谢是否会影响乙醛诱导的氧化应激和细胞凋亡。在鸡β-肌动蛋白启动子控制下构建了编码将乙醛转化为乙酸的人乙醛脱氢酶2（ALDH2）的转基因，并将其转染到人脐静脉内皮细胞（HUVECs）中。使用绿色荧光蛋白和ALDH2酶活性测定法验证了ALDH2转染的效果。使用氯甲基-2',7'-二氯二氢荧光素二乙酸酯测量活性氧（ROS）的产生。通过4',6'-二脒基-2'-苯基吲哚二盐酸盐荧光显微镜、定量DNA片段化和半胱天冬酶-3测定评估细胞凋亡。乙醛（0 - 200微摩尔）以时间和浓度依赖性方式诱导HUVECs产生ROS和细胞凋亡，这与应激信号分子ERK1/2和p38丝裂原活化蛋白（MAP）激酶的激活有关。在乙醛诱导的ROS产生和细胞凋亡之间观察到密切的线性相关性。有趣的是，ALDH2转基因或抗氧化剂α-生育酚可预防乙醛诱导的ROS产生、细胞凋亡、ERK1/2激活和p38 MAP激酶激活。选择性激酶抑制剂U0126、SB203580和SB202190证实了ERK1/2和p38 MAP激酶参与乙醛诱导的细胞凋亡。总体而言，我们的数据表明，通过ALDH2转基因过表达促进乙醛代谢可能预防乙醛诱导的细胞损伤和应激信号激活。这些结果表明ALDH2酶在预防和解毒乙醛或酒精诱导的细胞损伤方面具有治疗潜力。
