Functional Role Of The Mitochondrial Aldehyde Dehydrogen

尽管酒精导致器官损伤的机制多种多样，但很少有动物(啮齿动物)模型来评估这些概念。在过去的三年里，我们假设乙醛和脂醛(如细胞毒性4-羟基壬烯醛(HNE)和丙烯醛)的增加以及氧化应激的增加可能有助于酒精诱导的组织损伤的发展，因为乙醇代谢过程中产生的高活性和毒性的醛与细胞蛋白质和DNA的游离氨基相互作用，通常改变其靶标的生理功能，并启动自身免疫反应和DNA突变。乙醛和其他羰基化合物的积累可以通过抑制主要的乙醛代谢酶线粒体乙醛脱氢酶2(ALDH2)，通过化学抑制剂或遗传突变(G到A核苷酸替换)来实现，随后ALDH2蛋白中的Glu487Lys发生变化。带有这种基因变异的个体通过酶的显性失活而具有ALDH2活性降低，并表现出对酒精消费的厌恶反应和脸红反应，正如在许多东亚人中观察到的那样。由于与ALDH2的化学抑制剂相关的问题，如与其他酶和蛋白质的非选择性相互作用以及作用时间短，我们采取了遗传方法。我们假设，小鼠ALDH2基因缺陷的敲除小鼠不应该具有ALDH2活性，导致酒精暴露时乙醛水平极高。在适当的实验条件下，ALDH2基因敲除小鼠可能更容易受到高剂量酒精和另一种肝毒剂造成的组织损伤。为了验证这些假设，并建立一个模拟人类条件的动物模型，我们使用了基因中断技术来特异性地删除小鼠的ALDH2基因。我们已经培育出嵌合体小鼠(混合基因类型)，其中包含我们专门设计的DNA结构，以删除小鼠的ALDH2基因。在与C57/BL小鼠交配后，我们证实了7只雄性和11只雌性杂合子小鼠含有ALDH2-基因敲除结构。我们正在对这些F1小鼠进行交配，以产生纯合的基因敲除小鼠，然后用DNA Southern分析进行确认。与这种遗传方法类似，我们还研究了ALDH同工酶对HNE介导的细胞损伤的作用，因为我们观察到培养细胞对细胞毒性HNE的不同敏感性。我们的结果表明，一些细胞对HNE相当敏感，而另一些细胞对HNE相对敏感。我们的免疫印迹结果显示，对HNE的区分敏感性取决于ALDH同工酶的存在或不存在。例如，ALDH2和ALDH1同工酶的存在使培养的细胞对HNE介导的细胞死亡具有抵抗力，而某些对HNE敏感的细胞不包含ALDH同工酶。此外，用ALDH的化学抑制剂如二硫纶抑制ALDH同工酶显著增加了HNE介导的耐药细胞的损伤率。这些结果可能表明ALDH同工酶在细胞防御脂醛机制中发挥重要作用，脂醛是在接触有毒化学品和大剂量酒精后产生的。我们正在测定参与HNE代谢的其他酶的水平，以评估ALDH同工酶在HNE介导的损伤中的作用。

Despite various mechanisms for alcohol-mediated organ damage, few animal (rodent) models exist to evaluate those concepts. During the last three years, we hypothesized that elevated acetaldehyde and lipid aldehydes such as cytotoxic 4-hydroxynonenal (HNE) and acrolein along with increased oxidative stress may contribute to the development of alcohol-induced tissue damage, since highly reactive and toxic aldehydes, produced during ethanol metabolism, interact with free amino group of cellular proteins and DNA, usually altering their physiological functions of the targets and initiating auto-immune responses and DNA mutations. Accumulation of acetaldehyde and other carbonyl compounds can be achieved through inhibition of the major aldehyde metabolizing enzyme, the mitochondrial aldehyde dehydrogenase 2 (ALDH2), by either chemical inhibitors or genetic mutation (G to A nucleotide substitution) with a subsequent change in Glu487Lys in ALDH2 protein. Individuals with this genetic variation possess reduced ALDH2 activity through dominant inactivation of the enzyme and show aversive reactions and flushing response to alcohol consumption, as observed in many East Asian people. Because of the problems associated with the chemical inhibitors of ALDH2 such as non-selective interactions with other enzymes and proteins and short duration of action, we have taken genetic approaches. We hypothesized that knock-out mice deficient in mouse ALDH2 gene should not possess ALDH2 activity, leading to extremely high levels of acetaldehyde than the background mice upon alcohol exposure. Under proper experimental conditions, the ALDH2 knock-out mice may be more susceptible to tissue damage caused by a high dose of alcohol and another hepatotoxic agent. In order to test these hypotheses and to develop an animal model simulating human conditions, we used gene disruption techniques to specifically delete the mouse ALDH2 gene. We have produced chimera mice (mixed genotypes), which contain our DNA construct specifically designed to delete the mouse ALDH2 gene. After mating between the positive chimer mice and C57/BL mice, we verified that seven male and eleven female heterozygous mice contained the ALDH2- knockout construct. We are in the middle of mating these F1 mice to produce the homozygous knockout mice followed by confirmation with DNA Southern analysis. Parallel to this genetic approach, we also studied the role of ALDH isozymes against HNE-mediated cell damage, because we observed a differential sensitivity of cultured cells toward cytotoxic HNE. Our results showed that some cells are quite resistant while others are relatively sensitive to HNE. Our immunoblot results revealed that the differential sensitivity toward HNE depends on the presence or absence of ALDH isozymes. For instance, the presence of ALDH2 and ALDH1 isozymes makes the cultured cells resistant to HNE-mediated cell death, while certain cells sensitive to HNE did not contain ALDH isozymes. In addition, inhibition of ALDH isozymes with chemical inhibitors of ALDH such as disulfiram significantly increased the rate of HNE-mediated damage in the resistant cells. These results may suggest an important role of ALDH isozymes in cellular defense mechanism against lipid aldehydes, which are being produced after exposure to toxic chemicals and large doses of alcohol. We are determining the levels of other enzymes involved in the metabolism of HNE to evaluate the role of the ALDH isozymes in the HNE-mediated damage.