饮水型砷暴露对幼鼠海马和皮质DNA甲基化/去甲基化过程的影响及干扰机制研究

Arsenic pollution has become a global problem, and the contaminated groundwater is one of the major sources of arsenic exposure. Epidemiological and in-vivo studies have proved the long term exposure to arsenic induced damage to central nervous system of children and adolescents. DNA epigenetic change is considered an important mechanism of chronic arsenic poisoning. However, so far the adverse effects and the specific molecular mechanism of arsenic exposure on DNA epigenetic modification in brain tissues remained unclear. Arsenic is proven to alter DNA methylation at both the genome-wide and gene-specific levels. Previous epigenetic studies focused on the liver and DNA methylation process. Few reports have addressed arsenic toxicity on DNA demethylation process in brain. Our preliminary study found that low-dose arsenic exposure via drinking water induced the tissue-specific changes of 5mC and 5hmC levels in rat brain, suggesting that arsenic would affect DNA epigenetic processes in brain and its toxicity may be tissue specific. On the basis of previous reports and our preliminary study, this project intends to investigate the influence of arsenic exposure on DNA epigenetic modification in hippocampus and cortex of young rats. Firstly, young rats are exposed to arsenic via drinking water (0.05 ppm, 0.5ppm, 5ppm and 50 ppm) for 24 or more weeks, and then evaluate their learning and memory ability using Morris water maze and slice electrophysiology method. The DNA methylation and hydroxymethylation of total genome and promoter regions of featured learning and memory –related genes were measured for hippocampus and cortex. Furthermore, we aims to study the DNA epigenetic mechanism of arsenic exposure on hippocampus and cortex from the levels of DNA methylation and demethylation processes: investigate the interaction of arsenic methylation and DNA methylation via the consumption of the same methyl donor SAM; investigate and verify the hypothesis that arsenic exposure may induce oxidative stress and consequently influence the DNA demethylation process; systematically investigate the interaction network of arsenic methylation, DNA methylation, DNA demethylation, oxidative stress and tricarboxylic acid cycle in brain tissues, finally reveal the DNA epigenetic molecular mechanism of arsenic exposure in hippocampus and cortex.

流行病学和动物模型均证明砷会对儿童中枢神经系统造成损害，DNA表观遗传的改变是慢性砷中毒的重要机制，但是砷对大脑DNA表观遗传的影响和具体分子机理尚不明确。我们前期研究发现，经饮水型砷暴露后，大鼠脑组织5mC和5hmC水平呈现组织特异性变化，提示砷会对DNA表观遗传过程产生影响。在前期研究基础上，本项目拟建立长期、低剂量饮水型砷暴露的幼鼠模型，用Morris水迷宫和电生理学方法检测幼鼠的学习记忆能力改变，研究砷对海马和皮质的全基因组和特征基因启动子区甲基化的影响，并进一步从DNA甲基化和去甲基化两个方面探讨其干扰机制：研究在低剂量下砷甲基化和DNA甲基化过程间的甲基供体耦合机制；研究和验证“砷暴露通过氧化应激影响DNA去甲基化过程”假说；揭示砷甲基化、DNA甲基化、DNA去甲基化、氧化应激和三羧酸循环的相互作用网络，从而初步阐明海马和皮质中砷暴露影响DNA表观遗传的分子机理。

砷的神经毒性是一个非常严重的健康问题。DNA表观遗传的改变可能是重要的致病机制，但其分子途径仍不清楚。本研究对断乳期雄性SD大鼠进行了6个月饮水砷暴露，来模拟儿童发育期的暴露情况。我们发现，脑皮质和海马组织中总砷浓度显著增加，并呈现明显的剂量依赖性，而且砷显著影响了大鼠的空间学习记忆能力，并改变了神经元活性调节基因的表达。5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC)水平的下降以及甲基化转移酶(DNMTs)和甲基胞嘧啶双加氧酶(TETs)表达的下调表明：DNA甲基化/去甲基化过程在脑组织中受到显著抑制。 S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)作为重要的甲基供体，同时参与砷甲基化过程和DNA 甲基化过程。传统理论认为，过量的砷甲基化可能会造成SAM 的缺乏从而导致全基因组的低甲基化。本研究中，我们发现砷暴露后脑组织SAM水平并没有发生显著变化，这是由于脑血屏障只允许少量的砷到达脑组织而无法引起SAM缺乏。脑组织中氧化应激和三羧酸循环中重要指标发生了显著改变，表明砷可以通过三羧酸循环和α-酮戊二酸(α-KG)途径破坏氧化/抗氧化平衡，进一步抑制TETs的表达，并最终影响正常的DNA甲基化/去甲基化过程，从而验证了我们所提出的 “砷暴露通过氧化应激影响DNA去甲基化过程”的假说。除了表观遗传学研究，我们还通过蛋白组和代谢组学技术对砷神经毒性进一步进行了研究。砷暴露对大鼠皮质蛋白质组轮廓造成显著影响，筛选出的差异表达蛋白质所涉及的生物学通路包括氧化磷酸化、帕金森症、阿兹海默症以及三羧酸循环等，这些分子功能或生物学通路的异常与神经系统疾病紧密联系。砷暴露还能显著干扰大鼠皮质中神经递质代谢相关氨基酸的代谢过程以及长链溶血磷脂的转化过程，导致皮质脑内微环境的改变，进而引发神经功能紊乱。