Ethanol teratogenesis in Japanese medaka: effects at the cellular level

Abstract

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.

Figure 1

Effect of ethanol on DNA, RNA and protein contents of the medaka embryo during development. Each bar represents the mean± SEM of four to six separate experiments. Bar head with pound symbol (#) indicates that the results are significantly different (p<0.05) from zero dpf; different lower case letters (a, b and c) represent the data are significantly different from the corresponding control, 100 or 400 mM groups at 2dpf. A= DNA, B= RNA, C= embryo protein D= Yolk protein

Figure 2

Representative gel pictures of semi-quantitative RT-PCR analysis of emx2, en2, otx2, shh, wnt1, zic5, and iro3 mRNAs of medaka embryos developmentally exposed to ethanol. A= emx2, B=en2, C=otx2, D= shh, E= wnt1, F=zic5 and G= iro3. The lanes of A-F marked 1-6 represent zero h samples; Lane 7: control 2 dpf, lane 8:100 mM 2dpf, lane 9: 400 mM 2 dpf, lane 10: control 4 dpf, lane 11: 100 mM 4 dpf, lane 12: 400 mM 4 dpf, lane 13: control 6 dpf, lane 14: 100 mM 6dpf, lane 15: 400 mM 6 dpf. The lanes of G marked as 1-8 represent as zero h samples; lane 9: control 2 dpf, lane 10: 100 mM 2 dpf, lane 11: 400 mM 2dpf, lane 12: control 4 dpf, lane 13, 100mM 4 dpf, lane 14, 400 mM 4 dpf.. The unmarked lane extreme left is 100 bp ladder. The band at 542 bp is β actin (internal control). Some of the samples of zero dpf (except emx2 and zic5) showed bands of target gene products which indicate that the expression of these mRNAs are very rapid during embryogenesis.