Function Of The Mitochondrial Aldehyde Dehydrogenase 2

线粒体醛脱氢酶 2 的功能

• 批准号: 6530276
• 负责人: BYOUNG-JOON SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6530276
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; aldehyde dehydrogenases; alleles; animal breeding; behavioral genetics; enzyme activity; ethanol; gender difference; gene targeting; genetically modified animals; isozymes; laboratory mouse; model design /development; neurotransmitter metabolism; psychopharmacology; southern blotting

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.

长期饮酒通常会损害人类的各种器官。人们认为，酒精介导的组织损伤主要是由于氧化还原态的变化，乙醛升高，氧化应激和脂质过氧化物的水平升高，自由基代谢产物，内毒素诱导的库普弗细胞激活的激活，导致各种细胞因子的释放，抗氧化细胞的激活，在抗氧化剂中释放抗氧化剂和抗氧化剂，并在抗氧化剂中，并在抗氧化剂中及其氧化剂，并在抗氧化剂中及其氧化氧化物，并在抗氧化剂中及其氧化剂，并在抗氧化剂中及其氧化细胞的活化。尽管有这些理论，但很少有用于酒精诱导器官损伤的动物（啮齿动物）模型来确认各种概念。在过去的几年中，我们假设乙醛和脂质醛升高（例如细胞毒性4-羟基苯甲酸和丙二醛），以及乙醇可诱导的细胞核P450 2E1（cyp2e1）的氧化剂造成的氧化剂，以及乙醇可诱导的氧气（cyp2e1）的造成效果，并造成了氧气的造成效果，氧化剂的氧化剂量强烈，耐受的氧气疗法，这是对乙醇的氧化作用，并且在乙醇代谢期间产生的反应性和有毒醛与游离氨基蛋白和DNA相互作用，通常会改变其靶标的生理功能，并启动自身免疫反应和DNA突变。可以通过抑制主要的醛醛代谢酶，即通过化学抑制剂或遗传突变（g核苷酸取代）与glu4887 lisdh2的变化，可以通过抑制主要的醛代谢酶，线粒体醛脱氢酶2（ALDH2）来实现乙醛。具有遗传变异的个体通过酶的显性失活而具有降低的ALDH2活性，并在暴露于酒精时表现出厌恶的反应，与许多东亚人中观察到的那样。由于与ALDH2的化学抑制剂有关的问题，例如与其他酶和蛋白质的非选择性相互作用，因此由于快速代谢而引起的作用持续时间短，因此我们使用分子生物学技术采取了遗传方法。我们假设缺乏小鼠ALDH2基因的敲除小鼠不应具有ALDH2活性，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，ALDH2敲除小鼠可能更容易受到高剂量的酒精和另一种肝毒性剂引起的组织损伤。为了检验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专门删除小鼠ALDH2基因。过去，我们制备了三种不同的DNA构建体，这些构建体通过电穿孔到小鼠ES细胞中转染，然后通过DNA Southern分析筛选G-418（一种抗生素）耐药胚胎（ES）细胞。前两个DNA构建体没有产生任何阳性ES细胞。但是，第三个DNA构建体导致通过DNA Southern分析验证的三个阳性ES细胞。鉴定了三个带有ALDH2敲除构建体的阳性ES细胞，并注入胚泡细胞，然后在雌性小鼠中受到体外受精。通过从后代小鼠分离的DNA的DNA南部和嵌套的PCR分析，我们确认了嵌合小鼠的产生（混合基因型），其中包含我们专门设计用于删除小鼠ALDH2基因的基因敲除DNA构建体。在阳性嵌合小鼠和C57/BL小鼠之间交配后，我们验证了七个男性和11位雌性杂合小鼠包含Aldh2-敲除构建体。现在，我们正在交配这些F1小鼠，以产生纯合敲除小鼠，然后通过DNA Southern分析进行确认。