The Study On The Mechanism Of Immune Injury Induced By Cadmium And Chlorpyrifos Exposure In Common Carp Gills

Cadmium(Cd),a highly toxic heavy metal,is widely used in industrial production.Chlorpyrifos(CPF)is a broad-spectrum organophosphate insecticide.While applying Cd and CPF,it polluted the freshwater environment and harmed freshwater fish.Common Carp is one of the most important freshwater fishes in the world.Gill is an important respiratory and immune organ of fish.Gill plays a key role in physiological activities such as gas exchange,ion regulation and acid-base balance.At present,the toxicity mechanism of Cd and CPF combined to induce fish gill immune damage is still unclear at home and abroad.Therefore,this experiment established a single and combined poisoning model for Cd and CPF in common carp,which will provid a reference for the toxic effects of Cd and CPF single and combined in common carp,and will provide evidence for the toxic effects of water environmental pollutants on immune function and structural integrity in fish gills.One hundred twenty healthy common carps were randomly divided into four groups: the control group,the Cd group,the CPF group,and the Cd+CPF group,each group of 30 fish.The fish were fed with a commercial diet(containing protein 47.7% and lipid 10.7%).On the 15 th,30th,and 45 th days of the experiment,6 fish were randomly selected from each group to be executed and gill tissue samples were collected.The following examinations were carried out: microstructural and ultrastructural observation,metal element concentrations(Cu,Fe,Zn,Mn,Co,Cr,Cd,Pb,and Hg);oxidative stress indexes(SOD,GPx,GST,CAT,T-AOC,GSH,and MDA);apoptosis-related genes(P53,PUMA,Bax,Bcl-2,Apaf-1,Caspase-9,and Caspase-3);and cytokine(IFN-?,TNF-?,IL-2,IL-4,IL-6,and IL-10)in the gills of common carp.The results show:(1)Single and combined exposure of Cd and CPF caused morphological changes in common carp gills.Microstructural changes include lamellar epithelia hypertrophy,epithelial hyperplasia and lamellae fusion,mucinous cell hypertrophy and hyperplasia,mucinous cell vacuolation,and aneurysm.Ultrastructural changes include cytoplasmic,chromatin contraction;nuclear chromatin condensation;and mitochondrial swelling.TUNEL analysis showed that the number of TUNEL-positive apoptotic cells in the gill filament and lamellar epithelial cells were higher than that in the control group.The number of TUNEL-positive apoptotic cells in Cd+CPF group was higher than that in Cd group and CPF group,indicating that Cd and CPF caused damage and apoptosis of common carp gills.In addition,the degree of cell damage and apoptosis in the Cd+CPF group were stronger than those in Cd group and CPF group,indicating that Cd and CPF had a joint toxic effect.(2)Cd poisoning reduced the content of trace elements Cu,Fe,Zn and Mn,and increased the content of toxic elements Cd,Pb and Hg.Cd exposure increased the Cd content in the gills of common carp,indicating that the function of gills could be impacted by Cd.(3)Single and combined exposure of Cd and CPF reduced SOD,GPx,GST,CAT,and T-AOC activities;reduced GSH content;induced MDA content;led to oxidative stress;and affected the immune function of common carp gills.The oxidative stress intensity in Cd+CPF group was higher than that in Cd group and CPF group,indicating that Cd and CPF had a union poisoning effect.In addition,CAT,T-AOC,and MDA in the Cd+CPF group;GPx,T-AOC,GSH,and MDA in Cd group and CPF group showed a time-dependent effect.(4)Single and combined exposure of Cd and CPF down-regulated Bcl-2 m RNA expression;up-regulated P53,PUMA,Bax,Apaf-1,Caspase-9,and Caspase-3 m RNA expressions;and caused apoptosis and immune damage in common carp gills.The degree of apoptosis in the Cd+CPF group was higher than that in Cd group and CPF group,indicating that Cd and CPF had a union poisoning effect.P53,PUMA,Bcl-2,Caspase-9 and Caspase-3 m RNA expressions in the Cd+CPF group;Caspase-3 m RNA expression in Cd group;and Caspase-9 and Caspase-3 m RNA expression in CPF group had a time-dependent effect.(5)Single and combined exposure of Cd and CPF decreased IFN-?,IL-2,and IL-10 m RNA expressions;increased TNF-?,IL-4,and IL-6 m RNA expressions;and resulted in immune damage of common carp gills.The intensity of immune damage in the Cd+CPF group was higher than that in Cd group and CPF group,indicating that Cd and CPF had a joint toxic effect.IFN-?,TNF-?,IL-2,IL-4,IL-6,and IL-10 m RNA expressions in the Cd+CPF group;and IFN-?,IL-4,IL-6,and IL-10 m RNA expressions in Cd group and CPF group had a time-dependent effect..The results revealed that Cd and CPF exposure destroyed the structural integrity;changed the concentration of metal elements and the expression levels of cytokines;induced oxidative stress and apoptosis in common carp gills;and caused immune damage of common carp gills.Both Cd and CPF showed toxic effects in common carp gills,and the combined toxicity of Cd and C PF was greater than single toxicity.