重金属对斑马鱼的毒性效应及作用机制研究
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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。
The pollutant emissions increasing worldwide and bringing huge health and environmental problems. Heavy metals, such as cadmium (Cd), lead (Pb), copper (Cu) and chromium (Cr), are highly toxic metal and important environmental contaminant, which can reach aquatic systems derived from effluents of industrial, urban and mining sources.They are discharged into the environment and become accumulated from water into aquatic organisms, or from soil to plants, causing harmful effects to humans via food chains. Zebrafish is a sensitive model organism, which highly vulnerable to toxic contaminants, has been widely used in embryonic developmental toxicology, environmental toxicology, pathology toxicology, drug toxicology and other areas of toxicology due to their advantages.
The purpose of this study was to comprehensive evaluate heavy metals (Cd, Pb, Cu, Cr) toxicities on zebrafish, understanding toxicity characteristics, nature, extend and mechanism, elucidating the effect and mechanism of heavy metal on antioxidant system, apoptosis and immune system of zebrafish. The information presented in this study will help with elucidating the action mechanisms and environmental risk of heavy metal-induced toxicity in aquatic organisms. The main contents include:the effect of heavy metal (Cd, Pb, Cu, Cr) on Oxygen Radical Absorbance Capacity (ORAC) of zebrafish; the toxicity and its mechanism of heavy metal cadmium (Cd) on adult zebrafish and embryos; the toxicity and its mechanism of heavy metal lead (Pb) on adult zebrafish and embryos; the toxicity of heavy metal copper (Cu) on adult zebrafish; the toxicity of heavy metal chromium (Cr) on adult zebrafish. The results are listed as follows:
1) This study develop a detecting method of the free radical scavenging capacity for biological sample, based on the Oxygen Radical Absorbance Capacity (ORAC) methods. We chose international standard test fish-zebrafish as the experimental animals, and mainly tested the hydroperoxyl free radical (ROO·). The azo compound AAPH was chosen as the oxygen free radical source, Fluorescein (FL) as the fluorescence indicator, Trolox as the quantitative standard. Then we observed the fluorescence intensity decay process after free radicals acted with FL, and detected the ability of antioxidant delay the fluorescence intensity recession, to evaluate the effect of chemicals on ORAC. The established method has high specificity and good accuracy (between97%and108%) and precision (%CV less than5%); this method also has a good reproducibility (%CV is2.92%). The limit of quantitation (LOQ) and the limit of detection (LOD) are3.03and 1.00μmol Trolox/mg protein, respectively, the correlation coefficient (R2) is≥0.99. According to the new established quantitative analysis methods, we have determined the physiological normal range (0-50μmol Trolox/mg protein) and classified the heavy metal contamination degree and toxic effects based on the effect of metals on ORAC of zebrafish. Therefore, the study is effective to enhance the inter-accreditation of laboratory results and promoted the application of biomarkers for antioxidant defense in environmental pollution monitor.
2)The results showed that the estimated96h-LCso value of heavy metal Cd for adult zebrafish and embryos were24.341mg/L and46.67mg/L, respectively,50%inhibitory concentration of Cd on the hatching rate of embryos was42.499mg/L. Adult zebrafish exposed to Cd increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2, HO-1mRNA expression levels, down-regulated apoptosis related gene bcl-2mRNA expression level, but increased p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun gene mRNA expression level. Zebrafish embryos exposed to Cd slowed embryos heart rate, decreased embryos body length, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, CAT, Nrf2, HO-1mRNA expression levels, up-regulated apoptosis related gene bcl-2and p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily relA mRNA expression level. These results indicated that Cd has obviously toxicity on adult zebrafish and embryos, it could induced oxidative stress, apoptosis and immunotoxicity, and MAPKs signal pathway may involve in the toxicity mechanism of Cd.
3) The results showed that the estimated96h-LC50value of heavy metal Pb for adult zebrafish and embryos were0.819mg/L and22.574mg/L, respectively,50%inhibitory concentration of Pb on the hatching rate of embryos was19.594mg/L. Adult zebrafish exposed to Pb increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2, HO-1mRNA expression levels, down-regulated apoptosis related gene bcl-2mRNA expression level, but increased p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily nfkb2gene mRNA expression level. Zebrafish embryos exposed to Pb slowed embryos heart rate, decreased embryos body length, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, CAT, Nrf2, HO-1mRNA expression levels, up-regulated apoptosis related gene bcl-2and p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily relA mRNA expression level. These results indicated that Pb has obviously toxicity on adult zebrafish and embryos, it could induced oxidative stress, apoptosis and immunotoxicity, and MAPKs signal pathway may involve in the toxicity mechanism of Pb.
4) The results showed that the estimated96h-LCso value of heavy metal Cu for adult zebrafish was0.462mg/L. Adult zebrafish exposed to Cu increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2mRNA expression levels, up-regulated apoptosis related gene p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level. These results indicated that Cu has obviously toxicity on adult zebrafish, it could induced oxidative stress, apoptosis and immunotoxicity.
5) The results showed that the estimated96h-LC50value of heavy metal Cr for adult zebrafish was113.808mg/L. Adult zebrafish exposed to Cr increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2mRNA expression levels, up-regulated apoptosis related gene p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level. These results indicated that Cr has obviously toxicity on adult zebrafish, it could induced oxidative stress, apoptosis and immunotoxicity.
In this study, we improved the traditional ORAC method, and applied it in environmental pollution monitoring and toxicology studies. Furthermore, we use this method to study the effects of heavy metals (cadmium, lead, copper and chromium) on antioxidant system of zebrafish. And we comprehensively evaluated the toxic effects and its mechanism of heavy metals (cadmium, lead, copper and chromium) on adult zebrafish and embryos, suggesting that a close relationship between oxidant stress, apoptosis and immunotoxicity.
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。
The pollutant emissions increasing worldwide and bringing huge health and environmental problems. Heavy metals, such as cadmium (Cd), lead (Pb), copper (Cu) and chromium (Cr), are highly toxic metal and important environmental contaminant, which can reach aquatic systems derived from effluents of industrial, urban and mining sources.They are discharged into the environment and become accumulated from water into aquatic organisms, or from soil to plants, causing harmful effects to humans via food chains. Zebrafish is a sensitive model organism, which highly vulnerable to toxic contaminants, has been widely used in embryonic developmental toxicology, environmental toxicology, pathology toxicology, drug toxicology and other areas of toxicology due to their advantages.
The purpose of this study was to comprehensive evaluate heavy metals (Cd, Pb, Cu, Cr) toxicities on zebrafish, understanding toxicity characteristics, nature, extend and mechanism, elucidating the effect and mechanism of heavy metal on antioxidant system, apoptosis and immune system of zebrafish. The information presented in this study will help with elucidating the action mechanisms and environmental risk of heavy metal-induced toxicity in aquatic organisms. The main contents include:the effect of heavy metal (Cd, Pb, Cu, Cr) on Oxygen Radical Absorbance Capacity (ORAC) of zebrafish; the toxicity and its mechanism of heavy metal cadmium (Cd) on adult zebrafish and embryos; the toxicity and its mechanism of heavy metal lead (Pb) on adult zebrafish and embryos; the toxicity of heavy metal copper (Cu) on adult zebrafish; the toxicity of heavy metal chromium (Cr) on adult zebrafish. The results are listed as follows:
1) This study develop a detecting method of the free radical scavenging capacity for biological sample, based on the Oxygen Radical Absorbance Capacity (ORAC) methods. We chose international standard test fish-zebrafish as the experimental animals, and mainly tested the hydroperoxyl free radical (ROO·). The azo compound AAPH was chosen as the oxygen free radical source, Fluorescein (FL) as the fluorescence indicator, Trolox as the quantitative standard. Then we observed the fluorescence intensity decay process after free radicals acted with FL, and detected the ability of antioxidant delay the fluorescence intensity recession, to evaluate the effect of chemicals on ORAC. The established method has high specificity and good accuracy (between97%and108%) and precision (%CV less than5%); this method also has a good reproducibility (%CV is2.92%). The limit of quantitation (LOQ) and the limit of detection (LOD) are3.03and 1.00μmol Trolox/mg protein, respectively, the correlation coefficient (R2) is≥0.99. According to the new established quantitative analysis methods, we have determined the physiological normal range (0-50μmol Trolox/mg protein) and classified the heavy metal contamination degree and toxic effects based on the effect of metals on ORAC of zebrafish. Therefore, the study is effective to enhance the inter-accreditation of laboratory results and promoted the application of biomarkers for antioxidant defense in environmental pollution monitor.
2)The results showed that the estimated96h-LCso value of heavy metal Cd for adult zebrafish and embryos were24.341mg/L and46.67mg/L, respectively,50%inhibitory concentration of Cd on the hatching rate of embryos was42.499mg/L. Adult zebrafish exposed to Cd increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2, HO-1mRNA expression levels, down-regulated apoptosis related gene bcl-2mRNA expression level, but increased p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun gene mRNA expression level. Zebrafish embryos exposed to Cd slowed embryos heart rate, decreased embryos body length, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, CAT, Nrf2, HO-1mRNA expression levels, up-regulated apoptosis related gene bcl-2and p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily relA mRNA expression level. These results indicated that Cd has obviously toxicity on adult zebrafish and embryos, it could induced oxidative stress, apoptosis and immunotoxicity, and MAPKs signal pathway may involve in the toxicity mechanism of Cd.
3) The results showed that the estimated96h-LC50value of heavy metal Pb for adult zebrafish and embryos were0.819mg/L and22.574mg/L, respectively,50%inhibitory concentration of Pb on the hatching rate of embryos was19.594mg/L. Adult zebrafish exposed to Pb increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2, HO-1mRNA expression levels, down-regulated apoptosis related gene bcl-2mRNA expression level, but increased p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily nfkb2gene mRNA expression level. Zebrafish embryos exposed to Pb slowed embryos heart rate, decreased embryos body length, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, CAT, Nrf2, HO-1mRNA expression levels, up-regulated apoptosis related gene bcl-2and p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y mRNA expression level, increased MAPKs signal pathway related gene p38a, p38b, ERK2, ERK3, JNK1mRNA expression level, also up-regulated nuclear transcription factor AP-1subfamily c-jun, c-fos and NF-κB subfamily relA mRNA expression level. These results indicated that Pb has obviously toxicity on adult zebrafish and embryos, it could induced oxidative stress, apoptosis and immunotoxicity, and MAPKs signal pathway may involve in the toxicity mechanism of Pb.
4) The results showed that the estimated96h-LCso value of heavy metal Cu for adult zebrafish was0.462mg/L. Adult zebrafish exposed to Cu increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2mRNA expression levels, up-regulated apoptosis related gene p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level. These results indicated that Cu has obviously toxicity on adult zebrafish, it could induced oxidative stress, apoptosis and immunotoxicity.
5) The results showed that the estimated96h-LC50value of heavy metal Cr for adult zebrafish was113.808mg/L. Adult zebrafish exposed to Cr increased SOD activity and MDA level, elevated ROS level, up-regulated antioxidant system related gene Mn-SOD, Cu/Zn-SOD, Nrf2mRNA expression levels, up-regulated apoptosis related gene p53mRNA expression level, up-regulated immune system related gene TNF-a, IL-1β, INF-y and IL-8mRNA expression level. These results indicated that Cr has obviously toxicity on adult zebrafish, it could induced oxidative stress, apoptosis and immunotoxicity.
In this study, we improved the traditional ORAC method, and applied it in environmental pollution monitoring and toxicology studies. Furthermore, we use this method to study the effects of heavy metals (cadmium, lead, copper and chromium) on antioxidant system of zebrafish. And we comprehensively evaluated the toxic effects and its mechanism of heavy metals (cadmium, lead, copper and chromium) on adult zebrafish and embryos, suggesting that a close relationship between oxidant stress, apoptosis and immunotoxicity.
引文
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