二维纳米材料对细胞膜外排泵活性的影响及其毒性增敏效应研究

Two-dimensional (2D) nanomaterials such as graphene and molybdenum disulfide (MoS2) have wide application prospects in many fields. But their environmental risk also receive great attention. The layered structure of 2D nanomaterials makes them easily absorb on or insert into cell membrane and change the structure and functions of cell membrane. This change might inhibit activity of carrier proteins in cell membrane, which can pump xenobiotics out of cell, and increase toxicity of the xenobiotics. The previous research on graphene conducted by proposer verified the hypotheses. However, information on different inhibiting effect of 2D nanomaterials and their mechanism of action are not available. Thus, this project will choose graphene, graphene oxide, MoS2, tungsten disulfide (WS2) and Boron Nitride (BN) as target 2D nanomaterials. The human cell lines Caco-2 and HepG2 will be applied to build model, which could stimulate intestinal absorption and hepatotoxicity, respectively. Based on high throughput screening and biomics technologies, we will use the cell model to study inhibiting effect of five 2D nanomaterials on efflux pump in intestinal and hepatic cells and mechanism of their actions. Then, we will analyze how the inhibition of efflux pump activity cause the sensitizing effect on toxicity of toxic pollutants (paraquat, benzo(a)pyrene, arsenic and cadmium) which have been proved to be pumped out of cell by membrane efflux proteins. We will also use mouse as animal model to identify the results from in vitro tests. This project will provide new insight for risk assessment of 2D nanomaterials, and also provide basic data for development and application of 2D nanomaterials.

以石墨烯、二硫化钼为代表的二维纳米材料具有广阔的应用前景，其环境风险也受到广泛关注。二维纳米材料的层状结构使其较易吸附在或插入细胞膜，改变细胞膜结构和功能。这种改变可能会抑制细胞膜上外排外源化合物的载体蛋白的活性，从而对外源化合物产生毒性增敏效应，申请人前期研究虽初步验证了这一推断，但不同二维纳米材料的效应差异性及其相关机制尚不明确。本项目拟选择石墨烯、氧化石墨烯、二硫化钼、二硫化钨和氮化硼等二维纳米材料，借助Caco-2和HepG2细胞建立模拟肠道吸收和肝毒性的复合评价模型，结合高通量/高内涵筛选和生物组学技术，研究二维纳米材料对肠道和肝脏细胞膜外排泵活性的抑制效应，探讨外排泵活性抑制对依赖外排泵的环境污染物毒性的增敏效应，阐明其分子机制，同时采用小鼠试验验证体外细胞试验结果。本项目将为二维纳米材料环境风险评价提供新的切入点和科学基础，也将为二维纳米材料的研发和应用提供基础信息。

随着二维纳米材料在多领域的广泛应用，它们的环境风险也受到越来越多的关注。细胞膜损伤已被证实为二维纳米材料的主要致毒机制之一。细胞膜损伤可能会影响外排外源化合物的跨膜蛋白活性，进而改变外源化合物的细胞毒性，目前对二维纳米材料这种毒性效应的认识仍不明确。本项目以石墨烯、氧化石墨烯、二硫化钼、二硫化钨和氮化硼等5种二维纳米材料为研究对象，采用高内涵筛选和生物组学技术系统研究了二维纳米材料对肠道、肝脏细胞膜外排泵活性抑制及其对环境污染物的毒性增敏效应。发现石墨烯、氧化石墨烯、二硫化钼和氮化硼可显著抑制Caco-2和HepG2细胞增殖，提高细胞内ROS含量，诱导线粒体损伤；二硫化钨细胞毒性相对较低。5种二维纳米材料通过损害细胞膜完整性和流动性、降低能量代谢等机制抑制细胞膜ABC转运蛋白活性，并对砷、镉、苯并a芘和百草枯等污染物的细胞毒性产生增敏效应。与体外细胞实验结果相反，活体动物实验发现二维纳米材料显著降低了砷在小鼠小肠和肝脏中的生物有效性和毒性。上述结果表明，在研究二维纳米材料与污染物的联合毒性时，要充分考虑它们在体内吸收代谢的影响。本项目成果提升了对低浓度二维纳米材料毒性的认识，为二维纳米材料的环境风险评价及安全应用提供了理论支撑。