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

由于乙醇代谢产生的高活性乙醛与包括DNA和蛋白质在内的各种细胞大分子的游离氨基相互作用，在启动自身免疫反应的同时，通常改变它们的生理功能，因此长期饮酒引起的器官损害在一定程度上可以由靶组织中细胞毒性乙醛的积累引起。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)，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。具有遗传变异(ALDH2-2)的个体通过酶的显性失活而具有ALDH2活性降低，并表现出厌恶反应和脸红，这是由于酒精消费后乙醛积累，在许多东亚人中观察到的。虽然化学抑制剂在临床上经常使用，但由于与其他酶和蛋白质的非选择性相互作用，代谢速度快，作用时间短，以及大量的副作用，导致了许多问题。由于与ALDH2的化学抑制剂相关的问题，我们采用了使用分子生物学技术的遗传方法。我们假设，与野生型对照相比，携带主要阴性的人类ALDH2-2(hALDH2-2)转基因小鼠或缺乏小鼠ALDH2基因的转基因小鼠在酒精处理时，随着乙醛水平的升高，ALDH2活性会降低。我们的结果表明，重组人ALDH2-2变异体蛋白与小鼠ALDH2蛋白相互作用，并明显抑制小鼠的酶活性。此外，我们还利用含有hALDH2-2编码区全长cDNA3和线粒体前导序列的pcDNA3载体，在巨细胞病毒启动子的控制下获得了携带hALDH2-2的转基因小鼠，并带有牛生长激素的转录终止序列。利用针对ALDH2的多克隆抗体，通过RT-PCR、酶分析和免疫印迹分析检测转基因小鼠肝和脑中hALDH2-2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脑中低水平表达。随后，小鼠的ALDH2酶活性在这些动物中受到轻微的抑制。20%乙醇(4g/kg/d)灌胃2周后，小鼠肝脏和脑组织病理改变无显著差异，但转基因小鼠肝脏乙醛水平在注射乙醇后2 h较对照组升高50%(p&lt；0.03)，而肝脏酒精浓度无明显变化。此外，雌性转基因小鼠，而不是雄性小鼠，比对照小鼠喝的酒精更少(p&lt；0.03)。这些携带hALDH2-2突变体的转基因小鼠可以作为研究长期酒精处理后ALDH在酒精偏好、乙醛蓄积、神经递质代谢和乙醛介导的组织损伤中的作用的模型。此外，为了对ALDH2的生理作用有明确的结果，我们一直试图通过基因破坏技术来制备小鼠ALDH2基因缺陷的敲除小鼠。在过去的一年里，我们能够培育出嵌合小鼠，其中包含我们专门设计的DNA结构，以删除小鼠的ALDH2基因。我们现在正在使用限制分析和Southern印迹分析来鉴定没有小鼠ALDH2基因的纯合子小鼠。

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.