CYP3A65在代谢BDE47及其所致斑马鱼发育异常中的作用及机制研究

2,2',4,4'-tetrabromodiphenyl ether (BDE47) is one of the most important dominant PBDE congeners, the structures of its metabolites are much similar to thyroid hormone, possibly inducing the developmental toxicity through disrupting thyroid hormone homeostasis. Besides, DNA methylation is one of the most important modifications involved in environmental exposure in epigenetics. Cytochrome P450 3A4 (CYP3A4) plays a very important role in the metabolism of xenobiotics. Our previous study had shown that BDE47 significantly increased the expression and activity of CYP3A1 in rat live whilst CYP3A1 mediated metabolic activation of BDE47. To explore the role of CYP3A65 (homologous to human CYP3A4) in BDE47-induced the developmental toxicity, CYP3A65 knockout zebrafish will be constructed. The spatial and temporal expression of CYP3A65, which may mediate the metabolism of BDE47 in situ, will be investigated using this model. In addition, the relationship between dysplasia phenotype in zebrafish and window period of BDE47 exposure will be explored. The methylation of thyroid hormone and related genes will be analyzed to reveal the underlying molecular mechanism. The purpose of this study is to explore expression and regulation of metabolic enzymes, biotransformation, dysplasia and possible epigenetic mechanisms. The results will help provide insights into the metabolism of BDE47 and the subsequent dysplasia, and give novel clues to the prevention and control of environmental pollutant-induced biological damages, supply a new strategy for toxicity evaluation and risk assessment as well.

四溴联苯醚BDE47是最重要的PBDEs之一，其代谢产物与甲状腺素结构非常相似，可能通过甲状腺素干扰效应导致发育毒性，而DNA甲基化被认为与环境因素密切相关的表观遗传学机制。CYP3A4是人体代谢环境污染物最主要的代谢酶，我们发现：BDE47可通过诱导CYP3A表达并增加其活性代谢产物而使其毒效应增强。为此，本研究在构建斑马鱼CYP3A65(与人CYP3A4同源)敲除模型的基础上，通过研究CYP3A65的时空表达谱、CYP3A65与BDE47原位代谢的相关性、BDE47暴露窗口期与斑马鱼发育异常表型、甲状腺素稳态及功能相关基因甲基化状态及调控靶点，从代谢酶调控与表达、生物转化、发育异常及表观遗传学机制等环节，以新的视角系统审视BDE47的生物转化及其引起发育异常的过程及机制，为预防PBDEs的生殖发育危害、保护人群健康提供重要参考，也为化学物的毒性评价与风险评估提供新的研究策略。

本研究以斑马鱼为研究模型，围绕项目的研究目标和任务，从BDE47对斑马鱼CYP3A65表达的影响及其调控机制、CYP3A65对BDE47诱导斑马鱼发育异常的影响及其分子机制、精子传递效应在BDE47所致斑马鱼子代心脏发育异常中的作用及其表观异常机制等三个方面系统阐明了CYP3A65（与人CYP3A4同源）代谢活化在BDE47引发斑马鱼发育异常中的重要作用及其分子机制。主要发现如下：①CYP3A65主要表达在肝脏和肠道，在24-144hpf其表达随发育时间推移而增加，BDE47可呈剂量依赖性地诱导CYP3A65表达，dre-miR-142a-3p可能在BDE47诱导CYP3A65表达中发挥了重要作用；②BDE47可引起斑马鱼背部弯曲和心包水肿等畸形，CYP3A65的表达可加重该效应，120-168 hpf可能是BDE47作用的窗口期；③氧化应激和甲状腺素稳态紊乱是BDE47引发斑马鱼发育异常的主要机制，其作用皆与CYP3A65代谢BDE47生成的羟基化和甲氧基等氧化代谢产物有关；④精子传递效应在BDE47所致斑马鱼子代心脏发育异常中发挥了重要作用，而BDE47导致的父系精子miR-142-3p下降并通过增加子代trib3表达可能是其引发斑马鱼子代心脏发育异常的分子机制。研究结果为系统阐明CYP酶介导的生物转化在溴代阻燃剂所致生殖发育异常中的作用及其机制提供了理论和实践依据。已圆满完成各项任务，现实了预期目标。研究期间，发表SCI论文14篇，已被引用60余次，应邀在国际国内会议上做学术报告8次，受到同行的广泛关注。此外，建立了斑马鱼研究平台，为校内外10余个课题组提供了技术服务；构建了cyp3a65和trib3敲除斑马鱼并制备了CYP3A65多克隆抗体，这些都将为后续系统研究环境化学物的发育毒效应及风险评估提供良好的基础条件和技术支撑。