【作者】 李铸衡；

【导师】 徐镜波；

【作者基本信息】 东北师范大学 ， 环境科学， 2014， 博士

【摘要】 一些重金属在环境介质中不能被分解，在环境因子变化下或与其他物质联合胁迫会产生不同的毒性效应。砷(As)为元素周期表中VA族中的一种类金属元素，在自然界中分布广泛，是环境中最常见和最严重的致癌污染物之一。因其理化性质和环境行为与重金属有许多相似之处，故在环境化学和毒理学领域将其归入重金属研究范畴。不同的环境条件下As的毒理作用会随之变化。秀丽隐杆线虫(C.elegans)被广泛用做模式生物进行生命科学研究，并作为指示生物应用于生态监测。秀丽隐杆线虫成为生态毒理学首选的模式生物之一，主要进行污染物标准化毒性测试。分子生物标志物（molecular biomarkers）可以直接反映外来理化因素与细胞靶分子的相关关系，作为分子伴侣的热休克蛋白因其在胁迫下的显著效应被广泛关注。本研究以C.elegans为活体模型，并通过细胞体外试验，探讨砷的毒理学机制。利用LC50、头摆行为、繁殖率等端点判定指标，研究了不同温度影响下As的毒性变化；揭示砷与一种典型农药草甘膦，及与一种典型重金属Cu的复合作用模式；基于线虫突变体和蛋白表达方法，探讨了Hsp90作为砷污染生物标志物的可行性及最优检测方法，探讨砷胁迫下线虫休眠（dauer)反应及hsp90响应特征。最后，通过As对不同细胞作用的体外试验，深入探究了砷的毒理学机制。研究结果表明在20℃正常培养温度下，As对N2成虫的LC50值为130.5mg/L。环境温度降低在一定程度上可增加线虫成虫抗As的能力，冷冲击与As对N2成虫的联合胁迫表现为拮抗效应，热极限温度通过削弱机体机能可大幅度协同增强重金属的毒性。强热与As对线虫的胁迫表现为协同增强作用。线虫头摆率与繁殖率随砷胁迫强度的增大而降低。草甘磷作对秀丽隐杆线虫的24h LC50为18.53mg/L，草甘磷对秀丽隐杆线虫表现出较强的生殖毒性。低浓度的砷与低浓度的草甘磷协同作用不显著，低浓度的砷与高浓度的草甘磷表现出极强的协同作用，草甘磷对较高浓度的砷毒性协同作用明显。与砷相比，低浓度的草甘膦对线虫行为的抑制作用较强。低浓度砷与草甘膦联合胁迫，对线虫头摆率表现出协同效应。而较高浓度的联合作用下，对线虫行为的协同作用不明显。低浓度胁迫强度下，砷与草甘膦的联合效应对线虫繁殖率表现为协同效应；高强度下二者对线虫繁殖率的联合效应表现为拮抗作用。与砷相比，铜对线虫繁殖率的影响较弱。作为典型重金属，砷与铜对线虫行为的毒性效应存在差异。砷与铜对线虫行为的抑制效应为协同作用。Cu浓度越低，与砷的这种协同作用越明显。低浓度胁迫下砷与铜联合作用下线虫繁殖率显著降低。高浓度胁迫下，铜与砷的联合作用对线虫繁殖率的影响表现为拮抗效应。环境温度变化会影响As对C.elegans的毒性。秀丽隐杆线虫进入休眠阶段增强了机体对砷的抗性，环境温度变化可影响其砷胁迫下的应激反应，进入休眠阶段的幼虫可作为环境温度变化下砷污染的指示生物。免疫印迹法和酶联免疫法均可以用于C.elegans的Hsp90表达水平半定量分析；但酶联免疫法的特异性优于免疫印迹。因此，与野外实际相符的污染强度下（一般为中低强度的污染），利用酶联免疫检测线虫蛋白标志物的精确度会好于免疫印迹法。此外，与免疫印迹法相比，酶联免疫法还具有操作简单、检测通量高及自动化等的特点，使其更适合环境污染物的高通量检测。砷胁迫下秀丽隐杆线虫Hsp90蛋白表达量有下调趋势，与休眠相关的基因daf-21在线虫对砷的应激耐受通路中具有负调控表现，应激蛋白Hsp90可作为高强度砷胁迫的生物标志物。体外毒性试验表明砷的细胞毒性与细胞种类有关，砷通过增加细胞中ROS (reactiveoxygen species)量而影响细胞功能。砷与细胞作用过程具有浓度和能量依赖性。结合活体试验（动物模型）与体外试验（细胞）进行砷毒理研究，对低浓度砷暴露的针对性防治，砷的生物毒性产生过程和致毒机理具有理论与应用意义。更多还原

【Abstract】 Heavy metals are ubiquitous pollutants worldwide, while herbicides are some of themost prevalently used agrochemicals and became more pollutant in environment. Theenvironmental factors such as temperatures and other chemicals could affect the toxicity ofcontaminations. Arsenic (As), an element of wide distribution and multiple purposes, isknown to be a toxic environmental pollutant and carcinogen. Environmental factors such astemperature could affect the toxicology of As stress to organisms. Nematodes are frequentlyused for toxicological studies in various exposures, and becomes a useful animal model forthe study of the ecotoxicological relevance of chemical-Caenorhabditis elegans, a free-livingsoil nematode, is an excellent model organism because of its short lifespan, ease ofmanipulation, and low cost. Because of its high quality and inexpensive advantage, C. eleganis widely used as a model organism in the field of life sciences. Both nematode dormancy(dauer) state and stress protein (Hsps) expression have the characteristic of outstanding stressresponse and could be environmental ecotoxicological biomarkers in potential. induced andbiochemical level responses. Classical heat shock responses in C. elegans were describedmore than three decade ago.In this study, we chose nematode C. Caenorhabditis elegans as the model organism invivo and mammal cell line in vitro to study toxicological effects of arsenic. We used theending-points in C. elegans including24-h median lethal concentration (LC50), head thrash,reproductive rates/brood size to observe the effects of temperature (cold shock and heat shock)on of As stress to adults and dauer larval nematodes, and detecting mortality and Hsp90protein of nematode expression under Joint effects of As and pesticides stresses. Bothnematode dormancy (dauer) state and Hsps expression have the characteristic of outstandingstress response and could be environmental ecotoxicological biomarkers in potential.24-hLC50of As on wild type adults at20°C was130. mg·L-1through Probit analysis. Cold shockenhanced resistance of nematodes under arsenic stress, and high temperature significantlyreduced nematode tolerance to arsenic exposure. The head thrash and brood size declinedunder the increasing As extends.We also used C. elegans to investigate24h acute toxicities and joint toxicity of arsenicwith glyphosate, dichlorvos and cooper. Results showed that the24h-LC50for glyphosate toC. elegans were18.5mg·L-1. Glyphosate showed a high toxic effect on the reproduction to C.elegans. Results of equivalent toxicity design indicated that the joint toxicity of arsenic andglyphosate on C. elegans followed synergic especially when the concentration of As is high.Lower concentration glyphosate had a strong depression on the nematodes head thrash. Jointtoxicity of As and glyphosate at low concentration showed synergic effects on head thrash andreproductive rate of C. elegans. However, high concentrations of As and glyphosate had aantagonistic effects on the reproductive rate of C. elegans. Compared to As, Cu as a classicalheavy metal especially at low concentrations had a different toxic effect on C. elegans. The joint toxicity of As and Cu on C. elegans showed to be synergic. Cu and low concentration ofAs made a significant decrease of reproductive rate in C. elegans, while at high concentration,the joint toxicity of As and Cu showed a antagonistic effects on reproductive rate of C.elegans.We observed temperature played an important role in determining LC50values of As. Weexamined the differences in lethal toxicity induced by As exposure in dauer larvae and adultsdaf-21mutant at various temperatures. Hsp90is an essential gene in C. elegans, and the geneencoding Hsp90was identified as daf-21which is involved in C. elegans dauer formation.The lethal values in daf-21adults increased with temperatures increasing which indicates thatdauer larval had strong resistance to combined heat and As effects. It also implicated thespecial role of daf-21in dauer formation and maintenance. We further confirm using ELISAto assay the changes in Hsp90expression under the combined effects of heat and As. In thepresent study, we integrated the application of Western blot and ELISA assay method. Therewere differences of Hsp90expressions by Western blots and ELISA in C. elegans underdifferent concentrations of arsenic stress. The consistency in the results of protein expressionobtained from both methods confirms that temperature and pollutants has joint effect onchange of heat shock proteins expression in the nematode, which could be effective detectedand characterized by ELISA. Our results showed decreased Hsp90accumulation followingexposure of C. elegans to As stress, which indicates daf-21regulatory function in the pathwayof heat stress and possible role in As toxic response pathway in C. elegans. The significantdecreases in Hsp90levels observed in wild type adults exposed to As at higher temperaturessuggested that Hsp90could be a biomarker for heavy As pollution.For As toxicity study in vitro, PC12cell line (rat pheochromo-cytoma cell line) has beenselected as a mode cell line for evaluating toxicological effects of As. The value (47μM) of24h median lethal concentration (LC50) of NaAsO2for PC12cells has been obtained byMTT assay. The cytotoxicity of As has been systematically studied by exposure of PC12cells to relatively low concentration (5and20μM) of As. We found that As is able tosignificantly decrease cell proliferation, generate strong genotoxicity and increase ROS(reactive oxygen species) level of PC12cells. The experimental results demonstrate that lowconcentration of (e.g.,5μM, about10%of its LC50) As can generate high cytotoxicitytowards PC12cell line.更多还原