title: Ethanol inhibits retinal and CNS differentiation due to failure of cell cycle exit via an apoptosis-independent pathway.
abstract: Alcohol exposure during embryogenesis results in a variety of developmental disorders. Here, we demonstrate that continuous exposure to 1.5% ethanol causes substantial apoptosis and abrogated retinal and CNS development in zebrafish embryos. Chronic exposure to ethanol for 24h before hatching also induces apoptosis and retinal disorder. After the 2-day post-fertilization (dpf) stage, chronic exposure to ethanol continued to induce apoptosis, but did not block retinal differentiation. Although continuous ethanol exposure induces substantial accumulation of reactive oxygen species (ROS) and increases p53 expression, depletion of p53 did not eliminate ethanol-induced apoptosis. On the other hand, sequestering ROS with the antioxidant reagent N-acetylcysteine (NAC) successfully inhibited ethanol-associated apoptosis, suggesting that the ethanol-induced cell death primarily results from ROS accumulation. Continuous ethanol treatment of embryos reduced expression of the mature neural and photoreceptor markers elavl3/huC, rho, and crx; in addition, expression of the neural and retinal progenitor markers ascl1b and pax6b was maintained at the undifferentiated stage, indicating that retinal and CNS neural progenitor cells failed to undergo further differentiation. Moreover, ethanol treatment enhanced BrdU incorporation, histone H3 phosphorylation, and pcna expression in neural progenitor cells, thereby maintaining a high rate of proliferation. Ethanol treatment also resulted in sustained transcription of ccnd1/cyclin D1 and ccne/cyclin E throughout development in neural progenitor cells, without an appropriate increase of cdkn1b/p27 and cdkn1c/p57 expression, suggesting that these cells failed to exit from the cell cycle. Although NAC was able to mitigate ethanol-mediated apoptosis, it was unable to ameliorate the defects in visual and CNS neural differentiation, suggesting that abrogated neural development in ethanol-exposed embryos is unlikely to arise from excessive apoptosis. In conclusion, we demonstrate that the pathological effect of ethanol on zebrafish embryos is partially attributable to cell death and inhibition of visual and CNS neuron differentiation. Excessive apoptosis largely results from the accumulation of ROS, whereas abrogated neural development is caused by failure of cell cycle arrest, which in turn prevents a successful transition from proliferation to differentiation.
authors.affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Hsin-Yu
authors.initials: HY
authors.lastname: Chung
authors.forename: Chin-Teng
authors.initials: CT
authors.lastname: Chang
authors.forename: Huay-Win
authors.initials: HW
authors.lastname: Young
authors.forename: Shing P
authors.initials: SP
authors.lastname: Hu
authors.forename: Wen-Shyong
authors.initials: WS
authors.lastname: Tzou
authors.forename: Chin-Hwa
authors.initials: CH
authors.lastname: Hu
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002490:Central Nervous System / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D004305:Dose-Response Relationship, Drug; D047108:Embryonic Development / Q000187:drug effects*; D000431:Ethanol / Q000037:antagonists & inhibitors / Q000633:toxicity*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D058953:Neural Stem Cells / Q000187:drug effects / Q000378:metabolism; D055495:Neurogenesis / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000254:growth & development; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D015027:Zebrafish
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D000431:Ethanol; D000111:Acetylcysteine
keywords: Apoptosis; Ethanol; Neural differentiation; ROS; Zebrafish