Studies On Toxic Effect And Mechanism Of Heavy Metals On Zebrafish

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.