Effects Of Individual And Co-exposure Of Copper And Cadmium On Oxidative Stress And Apoptosis In Rare Minnow Gobiocypris Rarus

With the development of society,the impact of industrial pollution on human life is increasing,and the discharge of sewage leads to the accumulation of a large number of toxic substances in the water of aquatic life.Heavy metal ions through the gills and fish surface into the fish body in the water,and accumulation in the body,result in toxic effects.And this toxic effect will be affect human health by food chain.In order to explore the toxic effects of heavy metals on the oxidative stress and apoptosis of fish,in this study,Gobiocypris rarus exposed to copper,cadmium and copper and cadmium.The exposed solutions were CuSO4 · 5H2 O and CdCl2 · 2.5H2 O.By pre experiment,at 25 degrees Celsius under conditions of copper on Gobiocypris rarus 96 h semi lethal concentration was 0.196mg/L;cadmium on Gobiocypris rarus 96 h semi lethal concentration was 1.498mg/L.The ion concentration of the exposed group was set to 20% of its semi-lethal concentration,and the ion concentration in the combined exposure group was set to 10% of the two metal lethal concentrations.The control group was treated with clear water.The larvae of 4-month-old Gobiocypris rarus were exposed to 1 day and 7 days,and their gills,liver and ovaries were subsequently tested.This study examined the Rare Minnow gill,liver,ovary antioxidase SOD and CAT,GSH and MDA activity.The expression of oxidative stress related genes Cu-Zn-sod,Mn-sod,Cat,Gpx1,Nrf2,NF-kb was detected by real-time fluorescence quantitative PCR.In addition,AO-EB fluorescent staining was used to detect the apoptosis rate,The expression of apoptosis related genes p53,Bax,Bcl2,casp3,casp8,casp9,c-Jun,c-Fos,c-myc,p38 a,Jnk1 were detected by real-time fluorescence quantitative PCR.To study the toxic mechanism of copper,cadmium and their combined effects from two aspects of oxidative stress and apoptosis.The main conclusions are as follows:(1)The activities of antioxidant enzymes SOD,CAT and GSH were increased firstly and then decreased after exposure to heavy metals,.The content of MDA,the final product of lipid peroxidation induced by reactive oxygen species,increased with the exposure time.And the expression of Cu-Zn-sod,Mn-sod,Cat,Gpx1,Nrf2,and NF-kb of oxidative stress increased with time.This shows that copper and cadmium can cause the generation of oxidative stress in rare minnow,Antioxidant defense system is activated under low oxidative stress.And the activity of antioxidant enzymes is not synchronized with the expression of mRNA in the corresponding genes.(2)AO-EB double fluorescence staining showed that the apoptosis rate of the cells increased with the exposure time.The expression of apoptosis related genes p53,Bax,casp3,casp9,c-Jun,casp8,c-Fos,c-myc,p38 a,and Jnk1 was significantly increased with the exposure time.However,the expression of Bcl2 gene was firstly increased and then decreased.This shows that heavy metals copper,cadmium and the combined exposure can cause apoptosis of Rare Minnow,and the apoptotic rate is the time-effect relationship.The expression of apoptosis related genes casp3,casp8 and casp9 were up-regulated,These results indicate that Casp family has an important relationship with heavy metal induced apoptosis.The expression of MAPKs pathway related genes P38 a and Jnk1 were up-regulated,indicating that the apoptosis caused by copper and cadmium has the participation of MAPKs pathway.(3)The results indicated that the effect of the combined exposure group was significantly higher than single exposed groups,so it was speculated that the toxic effects between copper and cadmium were synergistic.In summary,This study investigated the toxic effects of copper and cadmium and the combined exposure of Rare Minnow from two aspects of oxidative stress and cell apoptosis,And its toxic mechanism was discussed.Which laid the foundation for the research of the subsequent heavy metal toxicity.