消毒副产物暴露对男性精子发育的影响及DNA甲基化的介导作用

Disinfection by-products (DBPs) are demonstrated to cause male reproductive toxicity in toxicological studies. Our previous studies suggest that exposure to DBPs is associated with decreased semen quality and altered DNA methylation in repetitive elements. Meanwhile, substantial evidence shows that exposure to DBPs adversely affect sperm development by changing sperm DNA methylation. To test the hypothesis, this project will recruit healthy reproductive-aged men screened as potential sperm donors and explore the effect of DBPs exposure during phases of spermatogenesis, meiotic division, development of sperm motility and epididymal storage on sperm development, using trihalomethanes in blood and haloaceticacids in repeated urine samples as internal exposure markers, and conventional semen quality, sperm motion parameters, sperm DNA integrity and spermatozoa apoptosis parameters as markers of sperm development. Additionally, we will detect sperm DNA methylation in repetitive sequences, and assess the mediating role of DNA methylation on the association of DBPs exposure with sperm development. This project will elucidate the male reproductive toxicity induced by DBPs on the principle of synchronously changes in exposure, epigenetic modification and health effect, which ultimately provides scientific basis for the risk estimation of adverse effect induced by DBP exposure on male reproductive health and for the screening of therapeutic target of sperm development impairment.

毒理学研究表明消毒副产物（disinfection by-products, DBPs）具有雄性生殖毒性。本团队前期研究发现DBPs暴露与男性精液质量降低、机体重复序列甲基化改变有关。已有证据表明DNA甲基化改变与精子发育异常密切相关，我们推测DBPs暴露可通过影响精子DNA甲基化损害精子发育。本课题拟测定捐精筛查普通男性血三卤甲烷和尿卤乙酸精准评估个体暴露，以精液质量常规参数、运动参数、DNA损伤、细胞凋亡为生殖效应指标，分析附睾存储期、获得前向运动能力期、减数分裂期和精原细胞发生期DBPs暴露对精子发育的影响；在此基础上，测定精子DNA重复序列甲基化水平，明确DNA甲基化在DBPs致精子发育损伤中的介导作用。本课题将有助于从“暴露-表观遗传修饰-健康效应”改变一致的原则上深入揭示DBPs男性生殖毒性，为饮用水DBPs暴露男性生殖健康风险的评估以及精子发育损伤治疗新靶点筛选提供科学依据。

毒理学研究表明消毒副产物具有雄性生殖毒性。本项目依托利湖北省人类精子库，2017年4月至12月共招募了1319名捐精筛查的普通育龄男性，在90天左右的捐精筛查或捐献过程中对研究对象进行问卷调查和生物样本的采集。问卷收集了研究人群基本人口学特征、健康状况（体力活动、握力、抑郁等）、职业暴露、吸烟饮酒习惯以及日常用水行为等信息。采集基线时的全血样本，并测定了4种三卤甲烷浓度。在90天左右的捐精筛查或捐献过程中，共采集3367次尿样，并测定了尿液中卤代乙酸、氧化应激标志物以及必需微量元素浓度。在基线时和捐精过程中的不同阶段共重复采集研究对象5000余次精液样本，并测定了精液质量参数（精液浓度、精子总数、前向运动活力和总活力）、精子线粒体拷贝数、精子端粒长度、精子DNA甲基化等指标。多元回归分析结果提示血三卤甲烷浓度与季节、洗澡频率和水温有关；尿三氯乙酸浓度与季节、年龄、体质指数、月收入、饮用水来源有关；精液质量参数与季节、年龄、文化程度、吸烟、饮酒和洗澡时间有关。发现血三卤甲烷浓度与尿8-羟基-2-脱氧鸟苷浓度升高有关，而与精子总数、总活力、前向运动活力、线粒体拷贝数、端粒长度降低有关；发现尿三氯乙酸浓度与精液质量参数无显著性关联，但在同济医院横断面人群中发现尿三氯乙酸浓度与精子DNA损伤呈正相关，与血清总睾酮、性激素结合蛋白浓度呈负相关。在精子库人群中还发现高强度的体力活动时长与精子活力呈正相关；握力与精子总数、活力、密度呈正相关；尿必需微量元素锌、钼浓度与精子活力以及总数呈正相关;贝克抑郁评分与精液体积、精子密度、总数和活力呈负相关。研究结果提示育龄男性精液质量不仅受体力活动、握力、抑郁、必需微量元素水平等因素影响，也与饮用水DBPs暴露相关；DBPs暴露可能通过影响精子线粒体拷贝数、精子端粒长度、精子DNA损伤、激素水平，进而损害男性精液质量。本项目研究成果为饮用水DBPs暴露男性生殖健康风险的评估提供了重要科学依据。