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.
