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氧化还原状态的变化、乙醛水平升高、氧化应激和脂质过氧化物、自由基代谢物、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在饮酒期间和之后靶组织中抗氧化剂水平的降低。尽管有这些理论，酒精诱导的器官损伤的动物（啮齿动物）模型很少，以确认各种概念。在过去的几年里，我们假设乙醛和脂质醛的升高（如细胞毒性的4-羟基壬烯醛和丙二醛）沿着乙醇诱导的细胞色素P450 2 E1（CYP 2 E1）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，因为在乙醇代谢过程中产生的高反应性和毒性的醛，与细胞蛋白质和DNA的游离氨基相互作用，通常改变其靶点的生理功能，引发自身免疫反应和DNA突变。乙醛的积累可以通过抑制主要的醛代谢酶，线粒体醛脱氢酶2（ALDH 2），通过化学抑制剂或基因突变（G至A核苷酸取代），随后改变ALDH 2蛋白中的Glu 487 Lys来实现。具有遗传变异的个体通过酶的显性失活而具有降低的ALDH 2活性，并且在暴露于酒精时表现出厌恶的潮红反应，正如在许多东亚人中观察到的那样。由于与ALDH 2的化学抑制剂相关的问题，例如与其他酶和蛋白质的非选择性相互作用，由于快速代谢而导致的作用时间短，我们已经采用了使用分子生物学技术的遗传方法。我们假设敲除小鼠ALDH 2基因缺陷的小鼠不应具有ALDH 2活性，导致与酒精暴露后的背景水平相比，乙醛水平极高。此外，在适当的实验条件下，ALDH 2基因敲除小鼠可能更容易受到高剂量酒精和其他肝毒性药物引起的组织损伤。为了测试这些假设并开发模拟人类条件的动物模型，我们使用基因破坏技术来特异性地删除小鼠ALDH 2基因。在过去，我们已经制备了三种不同的DNA构建体，通过电穿孔转染到小鼠ES细胞中，然后通过DNA Southern分析筛选G-418（抗生素）抗性胚胎干（ES）细胞。前两个DNA构建体不产生任何阳性ES细胞。然而，第三个DNA构建体导致三个阳性ES细胞，通过DNA Southern分析验证。我们的ALDH 2敲除构建体的三个阳性ES细胞被鉴定，注射到囊胚细胞中，然后进行体外受精成雌性小鼠。通过对从后代小鼠中分离的DNA进行DNA Southern和巢式PCR分析，我们证实了嵌合体小鼠（混合基因型）的产生，其含有我们专门设计用于删除小鼠ALDH 2基因的敲除DNA构建体。在阳性嵌合小鼠和C57/BL小鼠之间交配后，我们验证了7只雄性和11只雌性杂合小鼠含有ALDH 2敲除构建体。我们现在将这些F1小鼠交配以产生纯合敲除小鼠，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.