Regulation of zebrafish CYP3A65 transcription by AHR2.

CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5' flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter.

Ethanol inhibits retinal and CNS differentiation due to failure of cell cycle exit via an apoptosis-independent pathway.

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.