ROLE OF ALDH2--TRANSGENIC MICE CARRYING ASIAN ALDH2-2 VARIANT ALLELE

ALDH2 的作用——携带亚洲 ALDH2-2 变异等位基因的转基因小鼠

• 批准号: 6431367
• 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/6431367
• 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; neurotransmitter metabolism; psychopharmacology

Organ damage caused by chronic alcohol consumption can be initiated, in part, by the accumulation of cytotoxic acetaldehyde in the target tissues, since highly reactive acetaldehyde, produced from ethanol metabolism, interacts with the free amino group of various cellular macromolecules including DNA and proteins, usually altering their physiological functions while initiating auto-immune responses. 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 the ALDH2 protein. Individuals with the genetic variation (ALDH2-2) possess reduced ALDH2 activity through dominant inactivation of the enzyme and show aversive reactions and facial flushing, due to acetaldehyde accumulation following alcohol consumption, as observed in many East Asian people. Although chemical inhibitors are frequently used in clinics, they cause many problems due to non-selective interactions with other enzymes and proteins, short duration of action due to rapid metabolism, and numerous side effects. Because of the problems associated with the chemical inhibitors of ALDH2, we have taken genetic approaches using molecular biology techniques. We hypothesized that transgenic mice carrying the dominantly negative human ALDH2-2 (hALDH2-2) transgene or knock-out mice deficient in mouse ALDH2 gene would have reduced ALDH2 activity with elevated levels of acetaldehyde upon alcohol treatment, compared to their wild-type controls. Our results showed that the recombinant human ALDH2-2 variant protein interacted with the mouse ALDH2 protein and dominantly inhibited the activity of the mouse enzyme. In addition, we produced transgenic mice carrying the hALDH2-2 using a pcDNA3 vector containing the full-length cDNA for the hALDH2-2 coding region and the mitochondrial leader sequence under the control of cytomegalovirus promoter with a polyadenylation signal and transcription termination sequences from bovine growth hormone. Expression of the hALDH2-2 transgene in the liver and brain of transgenic mice was detected by RT-PCR of mRNA, enzyme assays and immunoblot analyses using polyclonal antibodies directed against ALDH2. To our surprise, hALDH2-2 protein was expressed at low levels in the liver and brain of transgenic mice. Subsequently, mouse ALDH2 enzyme activity was slightly inhibited in these animals. Although there was no significant difference in liver or brain pathology in mice treated with 20% ethanol (4 g/kg/day) for 2 weeks, hepatic acetaldehyde levels in transgenic mice were increased 50% at 2 h after ethanol injection (p<0.03) relative to control mice while hepatic alcohol concentrations were unchanged. In addition, female transgenic mice, but not males, drank less alcohol than did control mice (p<0.03). These transgenic mice carrying the hALDH2-2 variant can be used as a model to study the role of ALDH in alcohol preference, acetaldehyde accumulation, metabolism of neurotransmitters, and acetaldehyde-mediated tissue damage after long-term alcohol treatment. Furthermore, to have clear results for the physiological roles of ALDH2, we have been trying to prepare knock-out mice deficient in mouse ALDH2 gene by gene disruption technique. During the past year, we were able to produce chimeric mice, which contain our DNA construct specifically designed to delete the mouse ALDH2 gene. We are now identifying the homozygous mice without the mouse ALDH2 gene using restriction analyses and Southern blot analyses.

由慢性酒精消耗造成的器官损害可以部分地通过靶组织中的细胞毒性乙醛积累来引发，因为由乙醇代谢产生的高反应性乙醛与乙醇代谢相互作用，与包括DNA和蛋白质在内的各种细胞大分子的自由氨基相互作用，通常会改变其物理学功能。可以通过抑制主要的醛醛，即通过化学抑制剂或遗传突变（g核苷酸取代型）和随后的glu4887 liss in n a al and Ald的变化，可以通过抑制主要的醛代谢酶，线粒体醛（ALDH2）2（ALDH2）来实现乙醛的积累。 具有遗传变异（ALDH2-2）的个体通过酶的显性失活而具有降低的ALDH2活性，并显示出由于饮酒后乙醛积累而表现出厌恶的反应和面部冲洗，如许多东亚人所观察到的那样。尽管化学抑制剂经常在诊所中使用，但由于与其他酶和蛋白质的非选择性相互作用，由于快速代谢而引起的作用持续时间短以及许多副作用，它们引起了许多问题。由于与ALDH2的化学抑制剂相关的问题，我们使用了分子生物学技术采用遗传方法。 我们假设，与其野生型对照相比，在小鼠ALDH2基因中缺乏占小鼠ALDH2基因的小鼠或敲除小鼠的转基因小鼠或缺乏小鼠ALDH2基因的敲除小鼠会降低ALDH2活性，而酒精处理后的乙醛水平升高。我们的结果表明，重组人ALDH2-2变体蛋白与小鼠ALDH2蛋白相互作用，并显着抑制了小鼠酶的活性。此外，我们使用含有HALDH2-2编码区域的全长cDNA的PCDNA3载体产生了携带HALDH2-2的转基因小鼠，在控制巨细胞病毒启动子的情况下，用多腺苷酸化信号和转录终止序列的巨细胞病毒启动子控制了巨细胞病毒启动子，来自树脂生长量。通过使用针对ALDH2的多克隆抗体进行的mRNA，酶测定和免疫印迹分析，检测到转基因小鼠肝脏和大脑中HALDH2-2转基因的表达。 令我们惊讶的是，HALDH2-2蛋白在转基因小鼠的肝脏和大脑中以低水平表达。随后，这些动物中小鼠ALDH2酶活性略有抑制。 尽管在20％乙醇（4 g/kg/天）治疗2周的小鼠中，肝脏或脑病理学无显着差异，但在乙醇注射后2小时，转基因小鼠的甲基乙醛水平增加了50％（P <0.03），而肝酒精浓度不变。此外，雌性转基因小鼠，但不是雄性的饮酒少于对照小鼠的饮酒（p <0.03）。这些携带HALDH2-2变体的转基因小鼠可以用作研究ALDH在酒精偏爱，乙醛积累，神经递质的代谢以及长期酒精治疗后乙醛介导的组织损害的模型。 此外，为了对ALDH2的生理作用取得明确的结果，我们一直在尝试通过基因破坏技术来准备缺乏小鼠ALDH2基因的小鼠。 在过去的一年中，我们能够生产嵌合小鼠，该嵌合小鼠包含专门为删除小鼠ALDH2基因而设计的DNA构建体。现在，我们正在使用限制分析和Southern印迹分析来识别没有小鼠ALDH2基因的纯合小鼠。