基于Nrf2通路的饮用水消毒副产物毒效应研究

Toxicity testing is fundamental to health risk assessment. Traditional toxicity testing, which extrapolates human health risk from high-dose animal studies, are struggling to meet the demand for the vast volume of chemicals coming into commerce. High-throughput screening assays using in vitro human cells and anchored on toxicity pathways have become the core framework of toxicity testing in the 21st century (TT21C). As a master regulator of the oxidative stress response, Nrf2 plays an integral role in maintaining cellular redox homeostasis and containing oxidative damage. The proposed study is focused on the Nrf2-mediated antioxidant response, which is one of the toxicity pathways recommended in the report “TT21C: a vision and a strategy”. The study will utilize HepG2 and LO-2 cell models to investigate the effects of drinking water disinfection by-products (DBPs) found in real-world exposure scenarios. Single DBPs, mixtures of DBPs of similar or different chemical classes, and mixtures of simulated combinations of DBPs will be tested for oxidative stress markers, antioxidant response element (ARE) activities, and gene and protein expression regulated in the Nrf2 pathway. Through validation against 8-OHdG and genotoxicity induced by DBPs for testing accuracy and reliability, the proposed suite of toxicity testing assays are expected to establish evaluation methods and prediction tools for mixed DBPs exposure. The research outcome will help lay the foundation for implementing the toxicity pathway based chemical testing vision and promote the development of TT21C in China.

毒性测试是健康风险评估的重要基础。基于高剂量、由实验动物外推至人的传统毒性测试已难以满足当代发展的需求。基于体外人源性细胞系高通量筛选和毒性通路已成为21世纪毒性测试（TT21C）的核心理念。鉴于氧化应激反应是机体基本防御机制，Nrf2在维持细胞氧化还原稳态和抑制氧化损伤方面有重要作用。本研究以TT21C愿景与战略推荐的Nrf2抗氧化应答通路为测试靶标，人源性细胞系HepG2和LO-2为模型，以饮水消毒副产物（DBPs）为对象，以现实暴露为基础，分析单一DBPs，同类DBPs的混合物、不同类别混合物和模拟混配的整体DBPs对氧化应激产物、ARE活性和Nrf2通路相关基因和蛋白表达及其调控网络的影响。以DBPs引起8羟基鸟苷和遗传毒性验证方法准确性和可靠性；建立基于Nrf2通路的DBPs混合暴露效应评价方法和预测工具。结果将为建立基于毒性通路的毒性测试和推动TT21C在中国的发展奠定基础。

本研究在国家自然基金重点项目的支持下，针对水中众多消毒副产物是强烈的应激原，而Nrf2通路在维持细胞氧化还原稳态和抑制氧化损伤和机体正常的防御反应中起核心作用，本研究开展了基于Nrf2通路的污染物混合暴露的毒效应测试和方法研究。围绕重点项目计划任务书的研究目标和内容，以长江水源水生产的饮用水出厂水为对象，采用GC-MS和/或LC-MS/MS分析饮用水中亚硝胺类（NAms）和卤代酰胺类（HAMs）等十类DBPs，基于现实饮用水中DBPs的种类和浓度，以人源性的细胞系HepG2和LO-2为细胞模型，开展了基于不同场景下DBPs混合暴露对Nrf2通路的影响及其毒效应研究。分析不同DBPs对HepG2和LO-2的细胞毒性特征及DBPs暴露与氧化应激相关的生物标志物ROS、GSH、MDA和SOD的浓度和时间效应关系；DBPs暴露与Nrf2/ARE活性与激活状态及消毒副产物对Nrf2/Keap1/ARE通路中的抗氧化酶和Ⅱ相解毒酶基因和蛋白的表达水平；利用氧化应激基因芯片分析Nrf2相关基因及其调控网路的变化；整合毒理学测试数据和从毒理学数据库采集的毒性数据构成毒性数据库，分析构建DBPs暴露与氧化应激反应和Nrf2通路扰动间的关系，建立了基于Nrf2通路的污染物混合暴露的毒性测试评价方法和基于暴露特征毒性通路的毒效应预测方法。为上海全面采用深度工艺生产饮水饮用水提供了科学依据。