Toxic Effects And Mechanisms Study Of Ammonia Nitrogen On Gill Mitochondria Of Ruditapes Philippinarum

In recent years,with the rapid development of industry and agriculture and the continuous improvement of human living standards,the discharge of pollutants,including ammonia nitrogen,has continued to increase.Finally,these pollutants flow into the ocean and cause serious pollution of the marine environment.Since the coastal area is the place where the rivers flow into the ocean,land-based pollutants first accumulate on the shore.So the coastal pollution is more serious than other places.Ammonia exists in water in two forms,NH₄⁺and NH₃,non-ionic ammonia(NH₃)is the main form that causes aquatic poisoning,because it is easier to diffuse into cells through cell membranes than NH₄⁺.Ammonia nitrogen can affect the respiration,food intake,metabolism,immunity,ion regulation and other physiological activities of organisms.In this study,the clams Ruditapes philippinarum were used as the experimental animals,combined with morphological structure observation,physiological and biochemical indicators detection and mitochondrial proteomics methods,to explore the mitochondria toxic effects and its mechanism in gill tissus of Ruditapes philippinarum induced two environmentally relevant concentration of ammonia nitrogen for 21 days,aiming to providing theoretical basis for biological monitoring of ammonia nitrogen pollutions.The results of the study are as follows.1.Observation of the mitochondrial morphology and structure in the gill tissues of Ruditapes philippinarum after ammonia exposureAt the subcellular level,the ultrastructure of mitochondria was observed by transmission electron microscopy.Compared with the control group,0.1 mg/L and 0.5 mg/L ammonia nitrogen was exposed for 3 days and 21 days average caused mitochondria to be damaged in different degrees,and the mitochondrial damage was more serious in the long-term exposure and high-concentration exposure groups.The results showed that higher concentration of ammonia nitrogen and longer exposure duration induced more severe damage to the mitochondria.It was mainly manifested by the fuzzy shedding of inner cristae,the generation of vacuoles,and the increase of lysosomes.In addition,the changes in absorbance were used to detect mitochondrial swelling,and the results confirmed that mitochondrial swelling was more serious in experiments with high concentrations and long exposure times.Therefore,exposure to ammonia nitrogen causes damage to the mitochondrial structure of Ruditapes philippinarum.2.Physiological toxicity of on mitochondria in gill tissue of Ruditapes philippinarum after ammonia exposureAfter 21 days of exposure to ammonia nitrogen,gill tissue of Ruditapes philippinarum was used as the experimental target to detect the physiological and biochemical levels of mitochondria,such as cell calcium ion concentration,caspase-3 activity,mitochondrial membrane potential,and mitochondrial specific COX I gene expression.The main results include several aspects:(1)Mitochondrial membrane potential decreased and succinate dehydrogenase activity decreased in the ammonia exposure group,which probably lead to the insufficient supply of mitochondrial ATP.In addition,the calcium ion concentration of cells in the ammonia exposure group decreased,which may be due to the damage of the mitochondrial membrane structure caused by ammonia exposure,resulting in change in the permeability of the membrane,which caused the outflow of calcium ions in the mitochondrial matrix.(2)Under acute exposure conditions,the redox reaction in the Ruditapes philippinarum was increased,but the response of low-concentration and low-concentration exposure was different.Exposure to low concentration of ammonia nitrogen resulted in a significant increase of active oxygen in the gill tissue,while the high concentration group showed a decrease in active oxygen.This may be due to cell damage caused by exposure to high concentrations of ammonia nitrogen,thereby inhibiting the redox reaction of mitochondria and causing its reactive oxygen species to decrease.Decreased mitochondrial membrane potential and abnormal reactive oxygen species may induce cell apoptosis.Caspase-3activity test results show that short-term ammonia nitrogen exposure leads to increase of Caspase-3 activity,while long-term exposure decreases its activity,and the high-concentration exposure group has a significant increase.It shows that short-term ammonia nitrogen exposure can induce cell apoptosis,but long-term exposure to Caspase-3activity is inhibited by ammonia nitrogen.(3)Finally,q RT-PCR was used to quantify the expression level of mitochondrial specific gene COX?to detect the stability of mitochondrial genome.The expression level of COX I gene was down-regulated on the 1^st,3^rd,and 7^th-days after exposure,but its gene expression was up-regulated on the 21^th-days.It implied that the stability of mitochondrial genome is affected by ammonia nitrogen exposure.3.The effect of on the differential expression of mitochondrial proteomics in the gill tissue of Ruditapes philippinarum after ammonia exposureAfter exposure to ammonia nitrogen,the gill tissues of the Ruditapes philippinarum were used as samples for 3 and 21 days.The overall response of the gill mitochondrial protein of the Ruditapes philippinarum under the condition of ammonia nitrogen exposure was analyzed using TMT technology at the protein level.GO annotation and KEGG pathway analysis found that ammonia nitrogen has a greater impact on metabolic activities and pathways involving molecular functions.In summary,exposure to ammonia nitrogen has a serious impact on the structure and physiological and biochemical reactions of mitochondria in the gill tissue of Ruditapes philippinarum.Ammonia nitrogen interferes with the oxidative phosphorylation process of mitochondria by affecting the Ca²⁺concentration and transmembrane potential of the gill tissue mitochondria,which not only reduces the production of ATP,but also leads to excessive ROS production and oxidative stress on the mitochondria,resulting in serious cell function damage,and finally leading to gill cell apoptosis and mitochondrial genome instability,mitochondrial proteins,especially the expression of a variety of proteins involved in molecular functions,have undergone significant changes.This thesis provides reliable physiological,biochemical and proteomic data to support the in-depth study of the toxic effects of ammonia nitrogen on the mitochondria of the gill tissue of Ruditapes philippinarum...