纤蛇纹石石棉纳米线型残存物的细胞毒性作用研究

Inevitable damage to the environment and human health have been induced by the widely utilization of chrysotile asbestos. Although the toxicity of chrysotile asbestos is controversial, there is no doubt that longtime retention in body or tissues was accompanied by the emergence of linear nano residues. However, rare research had been made on their biological endurance, and interference effects on cellular homeostasis. In this project, the physico-chemical properties, biological activity and toxic mechanism of etched-chrysotile asbestos and sphericalnano-SiO2 will be studied by techniques of electron spin resonance (ESR), synchrotron radiation-X-ray fluorescence spectrum (SR-XRF), attenuated total reflection infrared spectroscopy (ATR-FTIR), confocal scanning laser microscope (CLSM), and non-invasive micro-test technique (NMT). The relationship between surface functional groups, dissolved elements, release of reactive oxygen species and bioactivity of the chrysotile asbestos and its leached products will be discussed. The migration and transformation of insoluble elements such as Si and Fe in cell membrane, cytoplasm and its irritating effect in the ROS formation and cell damage will be clarified through in vivo experiment. This project tries to indicate the essence of surface activity - biological activity – biological persistence of chrysotile fibers, its linear nano residues of etched-chrysotile asbestos. Finally, we will evaluate the biology activity and safety of chrysotile asbestos, etched-chrysotile asbestos as well as linear nano residues of etched-chrysotile asbestos, which would lay the groundwork for the establishing of the safety evaluation system and standard of chrysotile asbestos and their extended products.

纤蛇纹石石棉的大量使用对环境和人体健康产生不可避免的危害，然其毒性大小及毒性作用机制备受争议，对纤蛇纹石石棉体内残存物的生物活性也少有研究。本项目以纤蛇纹石石棉经人工浸蚀后形成的纳米线型残存物及球状纳米SiO2为研究对象，利用ESR、SR-XRF、ATR-FTIR、CLSM、NMT等测试手段，从矿物学、环境学和医学相结合角度对纤蛇纹石石棉及其浸蚀产物物化性质、自由基释放特性，细胞毒性及作用机理进行研究，分析不同形态和掺杂的SiO2载体表面基团、元素对生物活性的影响；探讨Si、Fe元素在细胞膜及胞质内的累积、溶解、键合、迁移及转化，阐明蛇纹石石棉溶出元素对自由基释放和细胞损伤的贡献。其意义在于，揭示纤蛇纹石石棉表面活性-生物活性-生物持久性之间的本质关系，寻求矿物学、环境科学和医学科学的综合评估方法，为纤蛇纹石石棉纤维及其纳米产物安全评估体系的建立及标准的制定奠定基础。

以纤蛇纹石石棉、浸蚀纳米线型残存物、合成掺铁蛇纹石石棉为研究对象，开展了其物化性质、自由基释放特征、细胞活性、毒性机制，特别是Fe在胞膜胞质内的累积迁移转化等研究。结果表明：3类石棉矿物相、表露基团基本一致，但八面体层Mg-OH含量、[Si-O]面网的振动、纤维表面的羟基、电位、液相溶出离子和·OH、·O2-、H2O2等自由基释放均不同。自由基释放40℃chry＞70℃chry＞90℃chry，1.5%Fesynchry＞0.3%Fesynchry＞synchry，释放量受Fe3+含量影响。表面官能团和电性不同使纤维与细胞膜吸附位点不同，引起氨基酸残基离子化羧基-COO-的-C(-O)2对称伸缩、磷酸二酯基团PO2-的对称伸缩、P-O-P的不对称伸缩振动增强，蛋白质分子二级结构改变，对GUV+的破坏更为剧烈，造成细胞膜通透性变化、胞内空泡、细胞骨架碎片化、胞核肿大浓缩等现象。纤维释放ROS和胞内自由基诱导的氧化损伤是引起细胞凋亡坏死的主要方式。胞内ROS产生与纤维所含SiO2、Fe2O3含量成正比，受主要溶出元素影响。掺铁石棉引起胞内ROS高于合成石棉，显著高于蛇纹石石棉，呈现1.5%Fesynchry＞（synchry、0.3%Fesynchry），chry＞40℃chry＞90℃chry。与Fe2+/Fe3+共同作用增强了细胞损伤，Fe2+作用强于Fe3+。Fe2+/Fe3+可与膜分子键合破坏细胞膜，但跨膜方式不同，约51.66% Fe3+通过胞外转化进入胞内并累积转化。Fe2+主要分布于胞质溶酶体、高尔基体内，其次富集在胞膜，细胞核及核仁上的浓度极低。强自由基可引发细胞核膜通透性改变，使Fe2+在核仁内聚集增多。Fe2+作用致使大量次级溶酶体和自噬体形成，通过胞吐释放降解后的离子，甚者崩解。纤维刺激胞内ROS升高、膜电位升高，通过异常表达Keap1、JNK1/2、c-caspase-3、Bcl-2、Nrf2、CHOP等引发氧化损伤、内质网应激和凋亡程序。验证了蛇纹石石棉溶出元素、表面基团对自由基释放和细胞损伤的贡献，初步揭示了纤蛇纹石石棉表面活性-生物活性之间的联系。联合发表SCI论文6篇、EI论文1篇，参加国内会议9次，作报告5次。研究成果可为纤蛇纹石石棉纤维及其纳米产物安全评估体系的建立，为寻求矿物学、环境科学和医学科学的综合评估方法作数据支撑