Function Of The Mitochondrial Aldehyde Dehydrogenase 2

长期饮酒通常会对人体的各种器官造成损害。酒精介导的组织损伤被认为主要是由于在饮酒过程中和饮酒后目标组织中氧化还原状态的变化、乙醛、氧化应激和过氧化脂质水平的升高、自由基代谢产物、内毒素诱导的库普弗细胞激活导致各种细胞因子的释放、星状细胞的激活以及目标组织中抗氧化剂水平的降低。尽管有这些理论，但很少有动物(啮齿动物)酒精诱导的器官损伤模型可以证实各种概念。在过去的几年里，我们假设乙醛和脂醛(如细胞毒性的4-羟基壬烯醛和丙二醛)以及乙醇诱导的细胞色素P450 2E1(CYP2E1)和其他酶引起的氧化应激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有毒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，启动自身免疫反应和DNA突变。乙醛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)来实现，可以通过化学抑制剂或遗传突变(G到A核苷酸替换)来实现，从而改变ALDH2蛋白中的Glu487Lys。遗传变异的个体通过酶的显性失活而导致ALDH2活性降低，并在接触酒精时表现出厌恶反应，就像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，由于快速代谢导致作用持续时间短，我们采用了分子生物学技术的遗传方法。我们假设小鼠ALDH2基因缺陷的敲除小鼠不应该具有ALDH2活性，导致与酒精暴露时背景水平相比，乙醛水平极高。此外，在适当的实验条件下，ALDH2基因敲除的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。过去，我们制备了三种不同的DNA载体，通过电穿孔的方法将其导入小鼠ES细胞，然后通过DNA Southern分析筛选出具有G-418(一种抗生素)抗性的胚胎干细胞。前两个DNA构建没有产生任何阳性的ES细胞。DNA Southern分析证实，构建的第三个DNA载体获得了3个阳性的ES细胞。我们鉴定了3个带有我们的ALDH2基因敲除结构的阳性ES细胞，将它们注射到胚泡细胞中，然后对雌性小鼠进行体外受精。通过对子代小鼠DNA的DNA Southern和巢式PCR分析，我们证实了嵌合体小鼠的产生(混合基因类型)，其中包含我们专门设计用于删除小鼠ALDH2基因的敲除DNA构建体。在阳性嵌合小鼠与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。我们现在正在与这些F1小鼠交配，以产生纯合的基因敲除小鼠，然后用DNA Southern分析进行确认。

Long-term alcohol consumption generally causes damage to various organs in humans. The alcohol-mediated tissue damage is believed to result largely from changes in redox states, elevated levels of acetaldehyde, oxidative stress and lipid peroxides, free radical metabolites, endotoxin-induced activation of Kupffer cells leading to a release of various cytokines, activation of stellate cells, and reduction in anti-oxidant levels in target tissues during and after alcohol consumption. Despite these theories, few animal (rodent) models for alcohol-induced organ damage are available to confirm the various concepts. During the last couple of years, we hypothesized that elevated acetaldehyde and lipid aldehydes (such as cytotoxic 4-hydroxynonenal and malondialdehyde) along with increased oxidative stress caused by ethanol-inducible cytochrome P450 2E1 (CYP2E1) and other enzymes 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 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 the genetic variation possess reduced ALDH2 activity through dominant inactivation of the enzyme and show aversive reactions with flushing responses upon exposure to alcohol, 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, short duration of action due to rapid metabolism, we have taken genetic approaches using molecular biology techniques. We hypothesized that knock-out mice deficient in the mouse ALDH2 gene should not possess ALDH2 activity, leading to extremely high levels of acetaldehyde compared with background levels upon alcohol exposure. In addition, 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. In the past, we have prepared three different DNA constructs that were transfected by electroporation into mouse ES cells followed by screening of the G-418 (an antibiotic) resistant embryonic stem (ES) cells by DNA Southern analyses. The first two DNA constructs did not produce any positive ES cells. However, the third DNA construct led to three positive ES cells verified by DNA Southern analysis. Three positive ES cells with our ALDH2 knock-out construct were identified, injected into blastocyst cells, and then subjected to in vitro fertilization into female mice. By DNA Southern and nested PCR analyses of DNA isolated from offspring mice, we confirmed the production of chimera mice (mixed genotypes), which contain our knock-out DNA constructs specifically designed to delete the mouse ALDH2 gene. After mating between the positive chimeric mice and C57/BL mice, we verified that seven male and eleven female heterozygous mice contained the ALDH2- knockout construct. We are now mating these F1 mice to produce the homozygous knockout mice followed by confirmation with DNA Southern analysis.