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.
