三种高膳食暴露风险内分泌干扰类农药联合毒性作用机制研究

Humans are usually exposed to various pesticides simultaneously, resulting in higher toxicities to human health compared to individual pesticides. The mechanism of combined actions among pesticides need to be addressed in the risk assessments which is remain poorly understood in humans. Pesticides in a mixture could interact at various cellular levels, and it is important to take into account the multiplicity of possible sites of interaction. Prochloraz, cypermethrin and difenoconazole were selected due to their co-application in agriculture, high detection rate and co-occurrence in food, as well as high exposure levels through diet among Chinese consumers. They are also referred to as ‘endocrine disruptors’ (EDs) which can interrupt reproductive development in wildlife and humans by inhibiting the actions of estradiol or testosterone and may result in reproductive disorders in humans. Firstly, tests for individual and combined pesticides with their ability to regulate the mitochondrial respiratory chain associated with apoptosis and increased ROS production will be conducted in human HepG2 cells in vitro. Because toxicity of the selected pesticides may be modulated by induction of the metabolizing CYP3A4 enzymes, we will monitor the induction of CYP3A4 in human liver microsome. Changes in the transcription of the CYP3A4 genes, activity of the respective enzymes mediated by CYP3A4 will be examined after exposed to these pesticides alone and in combinations. This study is also designed to investigate the in vitro dynamic metabolism of among the selected pesticides and between pesticides and endogenous steroid hormones to examine possible metabolic interactions that pesticides may have with other substrates. Moreover, the selected pesticides are efficient activators of pregnane X receptor (PXR), so we will investigate their effects on induction of CYP3A4 mRNAs and catalytic activities in human HepG2 cells. The results will enable us to further understand the metabolic interactions affected by mixtures of prochloraz, cypermethrin and difenoconazole and will be of great significance to more comprehensive evaluation of combined effects for pesticide mixtures as well as the risk assessments for human health.

多种农药残留混合污染现象在食品中普遍存在。农药残留混合物在体内外毒性试验中表现出远超单一农药毒性的联合效应，对现阶段基于单一农药的风险评估模式提出了挑战。目前农药混合物联合作用的调控机制尚未明确。研究表明，细胞凋亡是农药产生毒性作用的重要毒性通路，而细胞色素酶CYP3A4是农药混合物代谢性联合效应的分子基础，能够改变混合物中其他组分的代谢特征，并诱导其基因和蛋白表达。本项目选择食品安全风险监测中多残留现象突出、膳食暴露风险高且具有内分泌干扰效应的氯氰菊酯、咪鲜胺、苯醚甲环唑等3种农药，研究农药单剂和混合物在体外细胞水平的毒性与凋亡作用及其对CYP3A4的毒性代谢动力学影响，探讨相关农药混合物对CYP3A4基因及蛋白表达的调控作用，以及由孕烷X受体介导的CYP3A4转录激活机制。项目有望揭示高风险农药混合物组分间相互作用机制，为农药混合物毒性评价和食品安全风险评估提供科学依据。

农药在单个样品中同时检出残留的现象较为突出，造成的膳食暴露风险较高，属于食品安全重点监管的农药种类。多种农药残留混合污染现象在食品中普遍存在，并在体内外毒性试验中表现出远超单一农药毒性的联合效应，对现阶段基于单一农药的风险评估模式提出了挑战。目前农药混合物联合作用的调控机制尚未明确。本项目选择食品安全风险监测中多残留现象突出、膳食暴露风险高且具有内分泌干扰效应的氯氰菊酯、咪鲜胺、苯醚甲环唑等3种农药，研究农药单剂和混合物在体外细胞水平的毒性与凋亡作用及其对CYP3A4的毒性代谢动力学影响，探讨相关农药混合物对CYP3A4基因及蛋白表达的调控作用机制。.本项目首先基于人肝癌HepG2细胞，以农产品中应用广泛、广谱性强的苯醚甲环唑、氯氰菊酯和三唑磷农药为研究对象开展联合暴露实验，利用浓度相加（CA）、独立作用（IA）和联合指数（CI）等预测模型评价农药混合物联合效应，建立了农药联合暴露毒性效应评价技术。同时，利用分子对接探究农药及其混合物在细胞色素酶代谢水平上的相互作用模式和可能的作用机制。本项目在细胞水平上分析了农药混合物组分对HepG2细胞增殖、细胞周期和细胞凋亡的影响，结果发现不同农药混合物组合与单一组分相比，表现出更加明显的增殖抑制活性、周期抑制作用和凋亡诱导作用，尤其是苯醚甲环唑+三唑磷组合在低浓度水平下便表现出明显的毒性效应，且随着药物浓度的增加，毒性效应更加明显。此外，本项目通过毒性代谢动力学分析，探明了细胞色素酶CYP3A4介导的农药混合物组分间相互作用机制。.本项目通过研究不同农药混合物组分对凋亡通路和CYP3A4酶活性的影响，阐明了不同农药组合之间存在协同的联合作用方式，为农药混合物毒性评价以及农药残留风险监测和人群暴露评估研究提供了基础数据和科学依据，有助于科学评价内分泌干扰类农药对人类健康影响的风险，研究成果对提升我国食品中农药残留风险管理水平、保障食品安全具有重要意义。