Research On Toxicity Mechanism Of Amantadine On Apostichopus Japonicus

Amantadine is an antiviral drug,which can improve the symptoms of Parkinson’s disease and prevent influenza virus.Because of its low price and good anti-influenza effect,it is often used to prevent and control avian influenza.However,excessive use of amantadine can produce residues in the environment and food,resulting in drug resistance of viruses,which can spread to human beings through the food chain,causing potential health hazards,and bringing certain ecological and food safety risks.Although countries have started to restrict the use of amantadine,the residues of amantadine can still be detected in some foods on the market.Furthermore,the detection of amantadine residues in food in China is focused on the poultry and egg industry,with less detection in seafood and the environment,and research on the safety of amantadine in China is still in its infancy.In this research,amantadine was selected as the exposed contaminant,and the antioxidant enzymes activity and tissue structure of the Apostichopus japonicus were investigated using liquid chromatography（LC-MS）based non-targeted metabolomics and isotope labeling quantitative techniques（TMT）based proteomics to investigate the toxic mechanism of 100μg/L amantadine on Apostichopus japonicus after 96 h exposure.This study will provide a theoretical basis for further study of the toxicity mechanism and a reference for the determination of amantadine residues in a wider range of plants and animals and the evaluation of the food and ecological safety risks they pose.The results of the study are as follows.（1）Effects of amantadine on the antioxidant system of Apostichopus japonicusThe antioxidant enzyme activities of the intestinal tissues,respiratory tree tissues,longitudinal muscle tissues and outer wall tissues of the Apostichopus japonicus were examined after amantadine stress.The results showed that the antioxidant enzymes in all tissues of the Apostichopus japonicus were changed after amantadine stress.Superoxide dismutase（SOD）and reduced glutathionase（GSH）were inhibited in the intestinal tissues,respiratory tree tissues,longitudinal muscle tissues and outer wall tissues of the Apostichopus japonicus,indicating that the Apostichopus japonicus produced too much superoxide anion（O₂^-）and accumulated too much reactive oxygen species（ROS）under the stimulation of high amantadine concentration,SOD activity and GSH content were inhibited.Catalase（CAT）and malondialdehyde enzyme（MDA）showed an overall state of induction followed by inhibition in intestinal and respiratory tree tissues,and in longitudinal muscle and outer wall tissues,the experimental group showed an overall state of inhibition,it indicates that there are differences in the antioxidant enzyme systems or resistance of different tissues when the organism is subjected to the same exogenous stress.The intestinal tissues,respiratory tree tissues and external wall tissues of the Apostichopus japonicus were damaged to different degrees after high concentration of amantadine stress.The intestinal connective tissue of Apostichopus japonicus was thinned,the gap between folds was enlarged and the epithelial cells were necrotic;the respiratory tree of Apostichopus japonicus showed disorganized cell arrangement in the upper layer of body cavity,thinning of muscle fibers,disappearance of connective tissue and obvious enlargement of the central cavity;the microstructure of outer wall of Apostichopus japonicus after stress was fractured and empty spots appeared at the edges of cuticle and epidermis,and obvious cavities appeared in the dermis.（2）Metabolomic effects of Amantadine stress on Apostichopus japonicusThe metabolomic results showed that 115 differential metabolites were screened after amantadine stress,72 differential metabolites were up-regulated and 43 differential metabolites were down-regulated.The screened differential metabolites were subjected to KEGG functional pathway annotation analysis,and the enrichment results showed that biological processes such as amino acid metabolism,lipid metabolism,signal transduction,nucleotide metabolism and endocrine system were significantly affected.According to the physiological functions of the screened differential metabolites and the analysis of their involved metabolic pathways,adenosine and adenosine monophosphate（AMP）contents in the intestine of Apostichopus japonicus decreased after amantadine stress,which led to the disruption of energy metabolism in Apostichopus japonicus by affecting the c GMP-PKG and AMPK signal pathway;the decreased contents of testosterone and taurodeoxycholic acid（TCDCA）might have an effect on sex differentiation and cholesterol metabolism,and when the cholesterol content is reduced,it will cause damage to the intestinal cell membrane of Apostichopus japonicus;glycine and proline can synthesize collagen,and after the deformation of cell membrane,the decrease of external intake of glycine and proline and the stress of amantadine may lead to the degradation of collagen inside Apostichopus japonicus.（3）Proteomics effects of amantadine stress on Apostichopus japonicusProteomics results showed that 111 differentially expressed proteins were altered in the intestinal tract of Apostichopus japonicus under amantadine stress,with 42differentially expressed proteins up-regulated and 69 differentially expressed proteins down-regulated.The analysis of metabolic pathways showed that the glycolysis/glycogenesis pathway might enhance the energy production and conversion in the Apostichopus japonicus after amantadine stress;in signal transduction,amantadine stress might induce NF-κB,TNF and IL-17 signaling pathways to activate NF-κB,thus triggering intestinal inflammation and apoptosis in Apostichopus japonicus;amino acid metabolism showed that valine,leucine and isoleucine degradation pathways and phenylalanine metabolism pathway could inhibit protein synthesis and growth in Apostichopus japonicus.