Ethanol teratogenesis in Japanese medaka: effects at the cellular level

The adverse effects of alcohol on the developing humans represent a spectrum of structural and neurobehavioral abnormalities, most appropriately termed as fetal alcohol spectrum disorder (FASD). The mechanism by which ethanol induces FASD is unknown. Human studies of FASD are very limited due to ethical constraints; however, several animal models from nematodes to mammals are utilized to understand the molecular mechanism of this disorder. We have used Japanese medaka (Oryzias latipes) embryo-larval development as a unique non-mammalian model to study the molecular mechanism of FASD. Fertilized medaka eggs were exposed to ethanol (0-400 mM) for 48 h post fertilization (hpf) and then maintained in regular embryo rearing medium without ethanol. Viable embryos were harvested on 0, 2, 4 and 6 day post fertilization (dpf) and analyzed for DNA, RNA and protein contents of the embryos. By applying semi-quantitative RT-PCR (rRT-PCR) and quantitative real-time RT-PCR (qRT-PCR), RNA samples were further analyzed for seven transcription factors, emx2, en2, iro3, otx2, shh, wnt1 and zic5 which are expressed in the neural tube of medaka embryo during early phase of development. RNA and protein contents of the embryos were significantly reduced by ethanol at 400 mM dose on 4 and 6 dpf compared to the control (no ethanol), and 100 mM ethanol treated embryos. However, significant reduction of DNA was observed only in 4 dpf embryos. Total protein contents of yolk remained unaltered after ethanol treatment. Expression pattern of emx2, en2, iro3, otx2, shh, wnt1, and zic5 mRNAs were found to be developmentally regulated, however, remained unaltered after ethanol treatment. It is therefore concluded that alteration of nucleic acid and protein contents of medaka embryo by ethanol could be used as an indicator of embryonic growth retardation which might be the result of disruption of specific gene function during development.