重金属对斑马鱼的毒性效应及作用机制研究

发布时间：2018-07-29 11:17

【摘要】：全球的污染物排放增加,带来了巨大的健康和环境问题。重金属如镉(Cd)、铅(Pb)、铜(Cu)、铬(Cr),具有高毒性,是重要的环境污染物,通过工业废水、城市和矿业资源等排放到环境中,到达水生环境,在水生生物体内积累,经由食物链对人类造成危害。斑马鱼是一种对环境污染物较为敏感的模式生物,已被广泛应用于胚胎发育毒理学、环境毒理学、病理毒理学、药物毒理学等多个领域,并展现出其特有的优势。本研究旨在全面评价重金属(镉,铅,铜,铬)对斑马鱼的毒性效应,了解重金属毒性作用的特点、性质、程度及毒性作用机制,明确重金属(镉,铅,铜,铬)对斑马鱼抗氧化防御系统,细胞凋亡及免疫系统的毒性作用及其机制,为阐明重金属毒性作用和重金属污染的环境风险评估提供依据。主要研究内容包括：重金属(镉,铅,铜,铬)对斑马鱼抗氧化能力指数的影响、重金属镉(Cd)对斑马鱼成鱼和胚胎的毒性作用及机制研究、重金属铅(Pb)对斑马鱼成鱼和胚胎的毒性作用及机制研究、重金属铜(Cu)对斑马鱼成鱼的毒性作用研究、重金属铬(Cr)对斑马鱼成鱼的毒性作用研究。主要研究结果如下： 1)本研究以抗氧化能力指数(Oxygen radical absorbance capacity, ORAC)检测方法为基础,建立一种机体清除自由基能力的检测方法,并应用于生物抗氧化能力的的评价。研究选用国际标准试验用鱼-斑马鱼作为试验生物,针对过氧自由基(ROO·)进行检测,以偶氮化合物AAPH作为氧自由基来源,荧光素钠(FL)为荧光指示剂,并以维生素E类似物Trolox为定量标准,观察自由基与荧光素钠作用后荧光强度的衰退过程,检测各种物质加入反应体系后延缓荧光素钠荧光强度衰退的能力,以此评价反应体系中抗氧化剂的抗氧化能力。本研究所建立的定量分析方法具有特异性强；精密度高,变异系数小于5%；准确度在97%-108%之间；重现性佳(变异系数是2.92%)；定量检出限和最低检测限分别为3.03和1.00μmol Trolox/mg protein,可接受相关系数(R2)≥0.99。依据所建立的定量分析方法对斑马鱼ORAC生理正常值进行检测,正常值范围为0-50μmol Trolox/mg protein,并根据重金属对斑马鱼ORAC的影响,对重金属的污染程度和毒性效应进行了评级和分类,提高了试验数据互认程度,推动了抗氧化防御系统生物标志物在环境污染监测中的应用。 2)重金属镉(Cd)对斑马鱼成鱼和胚胎的毒性作用及机制研究结果表明：重金属Cd对斑马鱼成鱼和胚胎的96h-LCso分别为24.341mg/L和46.67mg/L,胚胎孵化抑制作用的96h-EC50为42.499mg/L。重金属Cd引起斑马鱼成鱼SOD活性升高,MDA含量增加,ROS水平明显升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD, Nrf2,HO-1mRNA表达水平上调；细胞凋亡相关基因bc1-2mRNA表达水平下调,而p53mRNA表达水平上调；免疫系统相关基因-α,IL-1β,INF-γ和IL-8mRNA表达水平均显著性升高；MAPKs信号转导通路相关基因p38a,p38b,ERK2,ERK3和核转录因子AP-1亚家族c-jun mRNA表达水平均上升。重金属Cd引起斑马鱼胚胎心率减慢,体长减小；ROS水平显著升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD,CAT,Nrf2,HO-1mRNA表达水平上调；细胞凋亡相关基因bc1-2和p53mRNA表达水平均上调；免疫系统相关基因TNF-a,IL-1β,INF-y mRNA表达水平均显著性升高；MAPKs信号转导通路相关基因p38a,p38b,ERK2,ERK3,JNK1和核转录因子AP-1亚家族c-jun,c-fos以及NF-κB亚家族relA mRNA表达水平均升高。以上结果提示Cd对斑马鱼成鱼和胚胎均具有明显的毒性作用,能够诱导氧化应激,细胞凋亡及免疫毒性的发生,毒性作用机制与MAPKs信号转导通路相关。 3)重金属铅(Pb)对斑马鱼成鱼和胚胎的毒性作用及机制研究结果表明：重金属Pb对斑马鱼成鱼和胚胎的96h-LC50分别为0819mg/L口22.574mg/L,胚胎孵化抑制作用的96h-ECso为19.594mg/Lo重金属Pb引起斑马鱼成鱼SOD活性升高,MDA含量增加,ROS水平明显升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD,Nrf2,HO-1mRNA表达水平上调；细胞凋亡相关基因bcl-2mRNA表达水平下调,而p53mRNA表达水平上调；免疫系统相关基因TNF-a,IL-1β, INF-γ和IL-8mRNA表达水平均显著性升高：MAPKs信号转导通路相关基因p38a,p38b, ERK2,JNK1和核转录因子AP-1亚家族c-jun, c-fos以及NF-κB亚家族nfkb2mRNA表达水平均上升。重金属Pb引起斑马鱼胚胎心率减慢,体长减小；ROS水平显著升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD,CAT,Nrf2,HO-1mRNA表达水平上调；细胞凋亡相关基因bc1-2和p53mRNA表达水平均上调；免疫系统相关基因TNF-α,IL-1βINF-γ mRNA表达水平均显著性升高；MAPKs信号转导通路相关基因p38a,p38b,ERK2,ERK3,JNK1和核转录因子AP-1亚家族c-jun,c-fos以及NF-κB相关基因relA mRNA表达水平均上升。以上结果提示Pb对斑马鱼成鱼和胚胎均具有明显的毒性作用,能够诱导氧化应激,细胞凋亡及免疫毒性的发生,毒性作用机制与MAPKs信号转导通路相关。 4)重金属铜(Cu)对斑马鱼成鱼的毒性作用研究结果表明：重金属Cu对斑马鱼成鱼的96h-LC5o0为0.462mg/L;引起SOD活性升高,MDA含量增加,ROS水平明显升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD,Nrf2,mRNA表达水平上调；细胞凋亡相关基因p53mRNA表达水平上调；免疫系统相关基因TNF-a, IL-1β, INF-γ(?)IL-8mRNA表达水平均显著性升高。以上结果提示Cu对斑马鱼成鱼具有明显的毒性作用,能够诱导氧化应激,细胞凋亡及免疫毒性的发生。 5)重金属铬(Cr)对斑马鱼成鱼的毒性作用研究结果表明：重金属Cr对斑马鱼成鱼的96h-LC5o为113.808mg/L；引起SOD活性升高,MDA含量增加,ROS水平明显升高；抗氧化防御系统相关基因Mn-SOD,Cu/Zn-SOD,Nrf2,mRNA表达水平上调；细胞凋亡相关基因p53mRNA表达水平上调；免疫系统相关基因TNF-a, IL-1β, INF-γ和IL-8mRNA表达水平均显著性升高。以上结果提示Cr对斑马鱼成鱼具有明显的毒性作用,能够诱导氧化应激,细胞凋亡及免疫毒性的发生。本研究对传统的ORAC方法进行了一定程度上的改良,并将此法应用于环境污染监测和毒理学研究,研究了重金属(镉,铅,铜,铬)对斑马鱼抗氧化防御系统的影响,并全面评价了重金属(镉,铅,铜,铬)对斑马鱼成鱼和胚胎的毒性效应及作用机制,发现氧化应激,细胞凋亡和免疫毒性之间存在着紧密的联系。
[Abstract]:Heavy metals, such as cadmium (Cd), lead (Pb), copper (Cu), and chromium (Cr), are highly toxic and are important environmental pollutants. They are discharged into the environment through industrial waste water, urban and mining resources, and accumulate in aquatic organisms and are caused by food chains. Zebrafish is a more sensitive model organism for environmental pollutants. It has been widely used in many fields, such as embryonic development toxicology, environmental toxicology, pathological toxicology, drug toxicology and so on, and has shown its unique advantages.
The purpose of this study is to evaluate the toxic effects of heavy metals (cadmium, lead, copper and chromium) on zebrafish, to understand the characteristics, properties, degree and mechanism of toxic effects of heavy metals, and to clarify the toxic effects and mechanisms of heavy metals (cadmium, lead, copper, chromium) on zebrafish antioxidant defense system, cell apoptosis and immune system, in order to clarify the toxicity of heavy metals. The effects of heavy metals (cadmium, lead, copper, chromium) on the antioxidant capacity index of zebrafish, the toxic effects and mechanisms of heavy metal cadmium (Cd) on zebrafish adult and embryos, the toxicity and mechanism of heavy metal lead (Pb) on the adult and embryo of zebrafish, are the main contents of the study. The toxic effects of heavy metal copper (Cu) on adult zebrafish were studied. The toxic effects of heavy metal chromium (Cr) on adult zebrafish were studied.
1) based on the Oxygen radical absorbance capacity (ORAC) detection method, a method for detecting the free radical scavenging capacity of the body was established and applied to the evaluation of biological antioxidant capacity. The study selected the international standard test fish zebra fish as experimental creature and the peroxy free radical (ROO). By using azo compound AAPH as the source of oxygen free radicals, fluorescein sodium (FL) as a fluorescent indicator, and using the vitamin E analogue Trolox as the quantitative standard, the fluorescence intensity decline process after the action of free radicals and fluorescein sodium was observed, and the ability to delay the fluorescence intensity decline of fluorescein sodium after various substances were added to the reaction system was evaluated. The anti oxidant ability of the antioxidant in the system of valence reaction. The quantitative analysis method established in this study is specific, high precision, the coefficient of variation is less than 5%; the accuracy is between 97%-108%; the reproducibility is good (the coefficient of variation is 2.92%); the quantitative detection limit and the minimum detection limit are 3.03 and 1 mu mol Trolox/mg protein, acceptable The correlation coefficient (R2) > 0.99. was measured on the normal value of zebrafish ORAC according to the quantitative analysis method established. The normal range was 0-50 Mu mol Trolox/mg protein. According to the influence of heavy metal on zebrafish ORAC, the pollution degree and toxic effect of heavy metals were rated and classified, and the degree of mutual recognition of the test data was improved. The application of biomarkers of antioxidant defense system in environmental pollution monitoring has been activated.
2) the toxic effect and mechanism of heavy metal cadmium (Cd) on the adult and embryo of zebrafish showed that the 96h-LCso of heavy metal Cd to zebrafish adult and embryo was 24.341mg/L and 46.67mg/L respectively. The 96h-EC50 of the inhibition of embryo hatching was 42.499mg/L. heavy metal Cd that caused the increase of SOD activity of the zebrafish fish, the MDA content increased and the ROS level was obvious. The expression level of Mn-SOD, Cu/Zn-SOD, Nrf2 and HO-1mRNA was up regulation, the expression level of bc1-2mRNA was down, and the expression level of p53mRNA was up regulated, and the expression level of the immune system related genes - alpha, IL-1 beta, INF- - y and IL-8mRNA were all significantly increased; the MAPKs signal transduction pathway related genes P3 The expression level of c-jun mRNA in 8a, p38b, ERK2, ERK3 and nuclear transcription factor AP-1 subfamily all increased. Heavy metal Cd caused the heart rate of zebrafish embryos to slow down, the body length decreased, and the ROS level increased significantly; the expression level of anti oxidant defense system related genes Mn-SOD, Cu/Zn-SOD, CAT, and expressions were up regulated; apoptosis related genes and expressions and expressions The levels of the immune system related genes TNF-a, IL-1 beta, and INF-y mRNA increased significantly, and the MAPKs signal transduction pathway related gene p38a, p38b, ERK2, ERK3, JNK1 and nuclear factor AP-1 subfamily c-Jun, as well as the nuclear factor kappa family were both elevated. Both have obvious toxic effects, which can induce oxidative stress, apoptosis and immune toxicity. The mechanism of toxicity is related to MAPKs signal transduction pathway.
3) the toxic effect and mechanism of heavy metal lead (Pb) on zebrafish adult and embryo showed that the 96h-LC50 of heavy metal Pb to zebrafish adult and embryo was 0819mg/L 22.574mg/L respectively. The 96h-ECso of inhibition of embryo hatching was 19.594mg/Lo heavy metal Pb causing the increase of SOD activity of zebrafish adult fish, MDA content increased, ROS level was obvious. The expression level of Mn-SOD, Cu/Zn-SOD, Nrf2, HO-1mRNA was up regulated, the expression level of bcl-2mRNA was down, and the expression level of p53mRNA was up regulated, and the expression levels of TNF-a, IL-1 beta, INF- gamma and IL-8mRNA in the immune system related genes were significantly increased: MAPKs signal transduction pathway related genes. P38a, p38b, ERK2, JNK1 and nuclear transcription factor AP-1 subfamily c-Jun, c-fos and NF- kappa B subfamily nfkb2mRNA expression level increased. Heavy metal Pb caused zebrafish embryo heart rate slowing, body length decreased; ROS level increased significantly; antioxidant defense system related genes up regulation; cell apoptosis The expression levels of related genes bc1-2 and p53mRNA were all up-regulated, the expression level of TNF- alpha and IL-1 beta INF- gamma mRNA in the immune system related genes increased significantly, and the expression level of MAPKs signal transduction pathway related genes p38a, p38b, ERK2, ERK3, JNK1 and nuclear transcription factor related genes increased. The results suggest that Pb has obvious toxic effects on the adult and embryo of zebrafish, and can induce oxidative stress, cell apoptosis and the occurrence of immuno toxicity, and the mechanism of toxicity is related to the MAPKs signal transduction pathway.
4) the toxicity of heavy metal copper (Cu) to zebrafish adult fish showed that the 96h-LC5o0 of heavy metal Cu to zebrafish was 0.462mg/L, the activity of SOD increased, the content of MDA increased, the level of ROS increased obviously; the levels of Mn-SOD, Cu/Zn-SOD, Nrf2, mRNA expression of the antioxidant defense system related genes were up regulated; apoptosis related genes p53mRNA The expression level of the immune system related genes TNF-a, IL-1 beta, INF- gamma (?) IL-8mRNA increased significantly. The above results suggest that Cu has obvious toxic effects on zebrafish adult fish, and can induce oxidative stress, cell apoptosis and immuno toxicity.
5) the toxic effect of heavy metal chromium (Cr) on zebrafish adult fish showed that the 96h-LC5o of heavy metal Cr to zebrafish was 113.808mg/L, the activity of SOD increased, the content of MDA increased, the level of ROS increased obviously; the expression level of the genes related to antioxidant defense system Mn-SOD, Cu/Zn-SOD, Nrf2, mRNA increased; apoptosis related genes p53m The expression level of RNA was up and the expression level of immune system related genes TNF-a, IL-1 beta, INF- gamma and IL-8mRNA increased significantly. The above results suggest that Cr has obvious toxic effect on zebrafish adult fish, and can induce oxidative stress, cell apoptosis and immuno toxicity.
In this study, the traditional ORAC method was improved to a certain extent. This method was applied to environmental pollution monitoring and toxicology research, and the effects of heavy metals (cadmium, lead, copper and chromium) on the antioxidant defense system of zebrafish were studied. The toxic effects of heavy metals (cadmium, lead, copper, chromium) on the adult and embryo of zebrafish were evaluated. It is found that there is a close relationship between oxidative stress, apoptosis and immune toxicity.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R994

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