Dasmahapatra Asok K, Khan Ikhlas A
National Center for Natural Product Research, University of Mississippi, University, MS, USA; Department of BioMolecular Sciences, Division of Pharmacology, University of Mississippi, University, MS, USA.
National Center for Natural Product Research, University of Mississippi, University, MS, USA.
Comp Biochem Physiol C Toxicol Pharmacol. 2015 Jan;167:90-100. doi: 10.1016/j.cbpc.2014.09.005. Epub 2014 Sep 20.
The present study was designed to investigate the probable roles played by cannabinoid (CB) receptors in fetal alcohol spectrum disorder (FASD) induction in Japanese rice fish (Oryzias latipes). Searching of public databases (GenBank, Ensembl) indicated that the Japanese rice fish genome includes three human ortholog CB receptor genes (cnr1a, cnr1b and cnr2). Quantitative real-time PCR (qPCR) and whole mount in situ hybridization (WMISH) techniques were used to analyze the expression of these cnr genes during Japanese rice fish embryogenesis and also in response to developmental ethanol exposure. qPCR analyses showed that the expression of all three CB receptor genes were developmentally regulated and only cnr2 showed maternal expression. The mRNA concentrations of these genes were found to be enhanced after 3 dpf and attained maximal levels either prior to or after hatching. WMISH technique indicated that all three cnr genes were expressed in the head region of hatchlings. During development, ethanol selectively attenuated the expression of cnr1a mRNA only. Blocking of cnr1a mRNA by CB1 receptor antagonists rimonabant (10-20 μM) or AM251 (0.2-1 μM) 0-2 dpf were unable to induce any FASD-related phenotypic features in embryos or in hatchlings. However, continuous exposure of the embryos (0-6 dpf) to AM251 (1 μM) was able to reduce the hatching efficiency of the embryos. Our data indicated that in Japanese rice fish, ethanol disrupted the expression of only cnr1a in a concentration-dependent manner that induced delay in hatching and might be responsible for the development of FASD phenotypes.
本研究旨在调查大麻素（CB）受体在日本青鳉（Oryzias latipes）胎儿酒精谱系障碍（FASD）诱导中可能发挥的作用。对公共数据库（GenBank、Ensembl）的检索表明，日本青鳉基因组包含三个人类同源CB受体基因（cnr1a、cnr1b和cnr2）。采用定量实时PCR（qPCR）和全胚胎原位杂交（WMISH）技术分析这些cnr基因在日本青鳉胚胎发育过程中的表达，以及对发育性乙醇暴露的反应。qPCR分析表明，所有三个CB受体基因的表达均受发育调控，只有cnr2显示母源表达。发现这些基因的mRNA浓度在受精后3天（dpf）后升高，并在孵化前或孵化后达到最高水平。WMISH技术表明，所有三个cnr基因均在幼体的头部区域表达。在发育过程中，乙醇仅选择性地减弱cnr1a mRNA的表达。在0-2 dpf时，用CB1受体拮抗剂利莫那班（10-20 μM）或AM251（0.2-1 μM）阻断cnr1a mRNA，未能在胚胎或幼体中诱导任何FASD相关的表型特征。然而，胚胎（0-6 dpf）持续暴露于AM251（1 μM）能够降低胚胎的孵化效率。我们的数据表明，在日本青鳉中，乙醇以浓度依赖的方式仅破坏cnr1a的表达，导致孵化延迟，这可能是FASD表型发展的原因。
