Ethanol-dependent developmental toxicity in zebrafish

Even though ethanol has been established as a teratogen in mammals, the mechanism(s) of ethanol-mediated developmental toxicity remains unclear. It has been proposed that ethanol-metabolism dependent oxidative stress may target the nervous system leading to toxicity and dysfunction. The focus of this thesis was to test the hypothesis that ethanol metabolism, during embryogenesis, contributes to the developmental toxicity of ethanol. Experiments were conducted to determine if zebrafish embryos were responsive to ethanol. Zebrafish embryos exposed to ethanol from 3 hours post-fertilization (hpf) to either 24 or 48 hpf, resulted in similar malformations such as delayed development, pericardial and yolk sac edema. In addition, ethanol induced embryonic cell death in a dose response manner. To evaluate the direct toxicity of acetaldehyde, embryos were exposed to acetaldehyde from 3-24 hpf. Acetaldehyde was three orders of magnitude more developmentally toxic to zebrafish. The acetaldehyde- and ethanol-dependent endpoints were similar but not identical. Prior to these studies, the capacity for zebrafish to biotransform ethanol had not been evaluated. With the availability of the zebrafish genomic sequence, the ethanol metabolizing enzymes, alcohol dehydrogenase (ADH), aldehyde dehydrogenase, catalase, and cytochrome P-450 2E1 were identified. Sequence alignments and phylogenetic analyses revealed conservation throughout evolution. Since class I ADH oxidizes the majority of ethanol, two zebrafish ADHs were identified and completely characterized. To determine if ethanol metabolism is a prerequisite to produce developmental toxicity, ethanol-metabolizing enzymes were chemically inhibited in vivo . Inhibition of ethanol metabolism led to increased incidences and the severity of developmental malformations. In the presence of ethanol metabolism inhibitors, there was a two-fold increase in embryonic response to ethanol. To ascertain if ethanol-mediated oxidative stress was leading to pericardial edema and cell death, the embryos were co-treated with ethanol and several antioxidants. Glutathione and lipoic acid partially attenuated pericardial edema, but did not reduce cell death, indicating that the mechanisms underlying the two endpoints may be distinct. Thus, these studies suggest that ethanol may be directly mediating the developmental toxicity in zebrafish. The culmination of this thesis provides the opportunity to fully exploit the zebrafish model to elucidate the mechanisms by which ethanol perturbs normal vertebrate development.