【作者】 魏巍；

【导师】 鲁文清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 多氯联苯(polychlorinated biphenyls, PCBs)与苯并(a)芘(benzo(a)pyrene,BaP)作为两种重要的污染物,因其在环境中广泛存在并可对人体造成包括致癌、致突变在内的多种有害效应而备受关注。现有的研究证实,PCBs在体内可有效的促进BaP的代谢活化,诱导后者转化生成其终致癌物,从而在联合作用时显著增强BaP的遗传毒性效应。由于上述遗传毒性终点效应发生在机体中往往要经过数十年的时间,且二者在环境中浓度较低,因此目前经毒理学研究发现的PCBs与BaP的有害效应在普通人群中得到证实的报道极为少见。鉴于以上原因,探寻机体接触PCBs与BaP后产生的早期应答事件,对于评价和预测这两种重要环境污染物暴露所导致的健康危害意义重大。目的：各种环境有害因素经过不同途径作用于人体,在绝大多数情况下都会直接或间接地引起相应基因的表达发生变化。和传统的毒理学指标相比,针对毒性相关的基因表达变化的研究能更敏感的反映环境因素的毒性特征。本研究拟以基因表达谱芯片技术探讨在PCBs与BaP联合作用下重要基因功能通路的表达变化,以期发现对二者联合暴露有重要意义的早期应答基因。并以此为线索,对涉及DNA损伤修复、细胞周期以及凋亡状态的部分重要功能通路进行深入的研究,从分子水平上揭示PCBs与BaP联合作用的毒性作用机制,最终为二者联合暴露所致健康危害的早期评价提供实验依据。方法：本文以HepG2细胞为靶细胞,选择PCBs的代表性同系物PCB126与PCB153为研究对象,设PCB126剂量为1nmol/L, PCB153剂量为10μmol/L, BaP剂量为50μmol/L,以10ml／L二甲基亚砜(DMSO)为溶剂对照组,进行各物质的单独染毒。同时以PCB126、PCB153预处理HepG2细胞48小时后,再与BaP联合染毒24小时。通过表达谱芯片技术检测染毒后细胞相关功能基因的表达变化；根据芯片结果的提示,选择PCB126为PCBs受试物,设PCB126四个剂量组(0.01、0.1、1、10nmol/L)、BaP一个剂量组(50μmol/L),以DNA损伤修复功能、细胞周期及凋亡状态的变化为研究重点,探讨二者联合作用对重要功能通路的影响。以蛋白质免疫印迹技术检测染毒后细胞核苷酸切除修复通路上关键修复因子XPA、XPB、XPC的蛋白表达变化,同时引入BaP代谢活化抑制剂ANF,在降低PCB126与BaP二者所导致的遗传损伤后,进一步探讨联合作用时DNA损伤程度与修复通路功能状态的联系；以流式细胞术检测染毒后细胞周期及凋亡状态的变化,借此寻找PCB与BaP联合暴露的早期效应。为更深入认识二者联合暴露的毒作用终点和毒作用机制,以RNA干扰技术建立芳烃受体低表达的细胞株,为后期研究芳烃受体在二者联合毒作用中所发挥的关键作用提供技术支撑。结果：BaP单独作用时主要诱导DNA损伤修复基因、细胞周期以及凋亡调控相关基因的表达发生变化,上述功能通路基因的表达变化总体上将有利于细胞维持自身的遗传稳定。PCB126与PCB153单独作用时可有效的诱导代谢酶相关基因的表达,且PCB126的诱导效应明显强于PCB 153。PCBs与BaP联合作用后对DNA损伤修复基因、周期以及凋亡调控基因的表达可产生复杂的不良影响,其诱导模式与BaP单独作用相比有较大的区别。PCB126和BaP单独作用均能有效的诱导修复因子XPA的表达上调,与溶剂对照比较,差异有显著性(P<0.01)。与BaP单独作用相比,除最低剂量以外,PCB126和BaP联合作用可明显抑制修复因子XPA的蛋白表达(P<0.01)。加入抑制剂ANF后,除PCB126最高剂量与BaP联合作用可显著抑制XPA的表达外(P<0.05),其余所有剂量组的XPA表达量均恢复至溶剂对照DMSO所诱导的水平。BaP单独作用可有效的抑制修复因子XPB的表达,与溶剂对照比较,差异有显著性(P<0.01);PCB126单独作用可有效诱导修复因子XPB的表达量升高,在浓度为1nmol/l和10nmol/l时差异有统计学意义。与BaP单独作用相比,PCB126和BaP联合作用可有效的诱导修复因子XPB的蛋白表达(P<0.01)。加入抑制剂ANF后,除PCB126单独作用在浓度为1nmol/l和10nmol/l的条件下仍可诱导修复因子XPB表达上调(P<0.01)外,其余所有剂量组的XPB表达量均恢复至溶剂对照DMSO所诱导的水平。PCB126和BaP单独作用时对修复因子XPC的表达量均没有明显影响。与BaP单独作用相比,除最低剂量以外,PCB126和BaP联合作用可明显抑制修复因子XPC的蛋白表达(P<0.01)。加入抑制剂ANF后,除PCB126最高剂量与BaP联合作用可显著抑制XPC的表达外(P<0.05),其余所有剂量组的XPC表达量均恢复至溶剂对照DMSO所诱导的水平。BaP单独作用可使细胞G1期比例上升,同时使其S期比例下降,与溶剂对照比较,差异有显著性(P<0.01); PCB126单独作用可使细胞G1期比例下降,而同时使G2期比例上升,与溶剂对照比较,差异有显著性(P<0.01)；与BaP单独作用比较,PCB126和BaP联合作用可使细胞G1期比例下降,二者联合作用可使细胞S期比例上升(P<0.01)。BaP单独作用能有效的诱导HepG2细胞发生凋亡,与溶剂对照比较,差异有显著性(P<0.01); PCB126单独作用各剂量组均可有效的诱导HepG2细胞发生凋亡,与溶剂对照比较,差异有显著性(0.01 nmol/l时P<0.05,其余各组P<0.01)；与BaP单独作用比较,PCB和BaP联合作用可使HepG2细胞凋亡率显著上升(P<0.01)。RNA干扰质粒转染成功的细胞(AhR(-))的AhR蛋白的表达明显低于正常细胞以及阴性对照细胞中的表达水平(P<0.05),与正常细胞比较AhR蛋白下降约18.3%。结论：(1) PCBs的存在干扰了机体针对BaP所致损伤的有序应答,可能涉及的机制包括抑制机体损伤修复能力、干扰细胞周期以及凋亡的调控。(2)上述三条机制的功能状态均是影响BaP暴露结局的关键早期因素,可为PCBs与BaP的健康危害监测提供一定的线索。(3)作为共面结构的PCBs, PCB126可通过抑制机体DNA损伤修复能力,影响细胞正常周期及凋亡状态的途径,增强BaP的遗传毒性。更多还原

【Abstract】 Both of polychlorinated biphenyls (PCBs) and benzoapyrene (BaP) are ubiquitous and important environmental pollutants, can lead to various adverse effects to human being which including carcinogenesis and mutagenesis. Previous studies confirmed that PCBs can effectively induce the bioactivation of BaP to its ultimate carinogenic, and finally enhance the genotoxicity of BaP exposure. But, the adverse effects discovered by toxicology research were few confirmed by epidemiology studies in normal population. For reasons that these end point genotoxic effects can only happen in human after a long period of PCBs and BaP exposure. Base on the fact, to evaluate and predict the adverse effects of exposure of these two environmental pollutants on human health, we need to explore the early effects of damage leading by PCBs and BaP.ObjectAlthough, environmental hazards react with human being in different approach, in most case they can directly or indirectly lead express change of related genes. Compared with the traditional toxicological methods, the research of expression changes of toxicity related genes can be a more sensitive indicator to reflect the characteristics of environmental factors. In the present study, to find out the early response genes, we employed expression microarray to investigate the gene expression changes on several important pathways after co-exposure to PCBs and BaP. Then base on the result of microarray, we focus our research on three important pathways:DNA damage repair, cell cycle and apoptosis. By. these studies we try to explain the toxic mechanism of PCBs and BaP co-exposure, and finally provide technological support for early evaluation of the health hazard leading by PCBs and BaP.MethodsIn present study, HepG2 was used as the target cells, PCB126 and PCB153 were chosen as the representative congers of PCBs. Cells were treated with PCB126 (lnmol/L), PCB153 (10μmol/L) and BaP (50μmol/L) for 72h alone, or pretreated with PCB126 and PCB153 for 48h then co-treated with BaP and PCBs, DMSO (10ml/L) was used as solvent control. Gene expression changes on important pathways were evaluated by gene expression microarray. Base on the results of microarray, we chose PCB126 (0.01,0.1,1, and 10nmol/L)and BaP (50μmol/L) as research compounds, to evaluate their effect on DNA damage repair, cell cycle and apoptosis. The protein expression levels of nucleotide excision repair (NER) factors XPA, XPB and XPC were detected; this experiments were carried out again with the BaP bioactivation inhibitor ANF, to evaluate the relationship between DNA damage and repair capability. Cell cycle and apoptosis change were detected by flow cytometry. For following studies to explore the role of aryl hydrocarbon receptor (AhR) in PCBs and BaP synergism, we developed stably transfected HepG2 cell line with low expression of AhR.ResultExposure to BaP mainly lead to expression change of DNA damage repair genes, cell cycle and apoptosis regulation genes, all of these changes will benefit the cells to maintain their genetic integrity. Exposure to PCB126 or PCB153 alone could induce the expression of genes related with metabolism, and meanwhile the inductive capability of PCB126 is higher than PCB153. The expression profiling of PCB126 and BaP co-exposure was quite different with BaP alone, it led a complex adverse expression effect on damage repair genes, cell cycle and apoptosis regulation genes.Exposure to BaP or PCB126 alone induced the expression of XPA, compared with solvent control respectively (P<0.01). Compared with BaP alone, co-exposure to PCB126 and BaP inhibited the expression of XPA (P<0.01) except for the co-exposure to 0.01-nM PCB126 and BaP (P>0.05). After add ANF, except for the co-exposure to 10-nM PCB126 and BaP could inhibited the expression of XPA, other groups were recovery to level of solvent control.Exposure to BaP alone inhibited the expression of XPB, compare with solvent control (P<0.01). Exposure to PCB126 alone induce the expression of XPB, at the concentration of 1-nM and 10-nM (P<0.01). Compared with BaP alone, co-exposure to PCB126 and BaP induced the expression of XPB (P<0.01). After add ANF, except for the exposure to 1-nM and 10-nM PCB126 alone could induce the expression of XPB, other groups were recovery to level of solvent control.No statistically significant change of XPC expression was observed in cells exposed to BaP or PCB126 alone, compared with solvent control respectively (P>0.05). Compared with BaP alone, co-exposure to PCB126 and BaP inhibited the expression of XPC (P<0.05), except for the co-exposure to 0.01-nM PCB126 and BaP (P>0.05). After add ANF, except for the co-exposure to 10-nM PCB126 and BaP could inhibited the expression of XPC, other groups were recovery to level of solvent control.Exposure to BaP alone caused a significant increase of the percentage of cells at G1 phase but decrease it at S, compared with DMSO solvent control (P<0.01). Exposure to PCB126 alone caused a significant decrease of the percentage of cells at G1 but increase it at G2, compared with DMSO solvent control (P<0.01). Compared with BaP alone, co-exposure to PCB126 and BaP could effectively decrease the percentage of cells at G0/G1 phase, but increase the percentage of cells at S phase (P<0.01).Exposure to BaP or PCB126 alone caused a significant increase of apoptosis, compared with DMSO solvent control (P<0.05). Compared with BaP alone., co-exposure to PCB126 and BaP could effectively increase the apoptosis of HepG2 (P<0.01).Compare with the normal cell, the protein expression of AhR in RNA inference cells was decreased by 18.3%.Conclusion(1) PCBs could affect the ordered response of organism to the damage leading by BaP exposure, which may be caused by inhibit the damage repair capability, interfere the cell cycle and apoptosis process.(2) All of mechanisms mentioned above are important factors, which can interfere the outcome of BaP exposure and provide technological support for evaluation of the health hazard leading by PCBs and BaP.(3) As the represent of coplanar PCBs, PCB126 can enhance the genotoxicity of BaP, which caused by inhibiting the DNA damage repair capability, interfering the normal cell cycle and apoptosis process.更多还原