Intestinal Toxicity Effect Of Heavy Metal Cadmium On Procambarus Clarkii

Purpose:Cadmium ions in water bodies are absorbed by aquatic animals and will lead to serious tissue damage or even organismal death.There is evidence that the intestine is one of the main target organs for cadmium accumulation,and the massive accumulation of cadmium will eventually lead to intestinal damage.As an important aquatic food chain and aquaculture species,the cadmium toxicity effect of crustacean intestine still needs further study.Evolutionarily conserved signaling pathway molecules such as transcription factor Nrf2,NF-κB and protein kinase p38 MAPK play important roles in response to environmental stresses.Homologous genes of the above signaling pathway molecules have also been identified in crustaceans and share some homology with other species.Current studies on crustacean signaling pathway molecules are mostly focused on the molecular level;information on the protein level,especially on the tissue localization of proteins,is still lacking.Physical localization techniques such as IHC can visualize the location of proteins in tissues or cells and are important for studying gut function in crustaceans.In this study,we examined the oxidative stress state,histopathology,digestive and immune enzymes,and apoptosis in the intestine of Procambarus clarkii under cadmium exposure to provide a new perspective for the study of intestinal toxicity effects in invertebrates under environmental stress;and used IHC staining to detect protein localization and expression of signaling pathway molecules in the P.clarkii intestine to explore the sites in the intestine that exert response functions;finally,we provided a theoretical basis for the study of intestinal toxicity effects in crustaceans under environmental stress,and provided new ideas for healthy crustacean breeding.Methods:Procambarus clarkii were used as experimental animals,and control groups and experimental groups with Cd²⁺concentrations of 1,5 and 10 mg/L(about 1/40,1/8 and 1/4of the P.clarkii 96 h LC₅₀)were set up to establish a 96 h acute cadmium exposure model.Five aspects of intestinal oxidative stress status,histopathology,apoptosis,digestive enzymes and immune enzyme activities were assessed for intestinal toxicity under cadmium exposure;IHC staining was performed for protein localization and expression studies of signaling pathway molecules.Results:1.Cadmium exposure induced intestinal ROS and MDA production in P.clarkii:from48 h of Cd exposure,a significant increase in ROS content was observed（P<0.05）;late（96 h）of 5 and 10 mg/L Cd²⁺exposure,a significant increase in MDA production was observed（P<0.05）.Disturbance of antioxidant enzyme activity:at 96 h of Cd exposure,5and 10 mg/L Cd²⁺exposure significantly inhibited SOD activity and 1 and 5 mg/L Cd²⁺exposure significantly activated CAT activity（P<0.05）.2.Cadmium exposure(5,10 mg/L Cd²⁺)resulted in varying degrees of pathological damage to crayfish intestinal tissues,manifested by loosely arranged epithelial cells,blurred cell borders,cytoplasmic damage,and abnormal cell nuclear morphology.Induction of apoptosis:TUNEL staining showed apoptosis in cells located in the intestinal epithelium;10 mg/L Cd²⁺exposure for 48 h-96 h and 5 mg/L Cd²⁺exposure for 96 h induced a significant increase in the rate of apoptosis（P<0.05）.Inhibition of digestive enzymes and PO activity:1,5 mg/L Cd²⁺significantly inhibited lipase activity at all exposure times except for 48 h exposure,and 10 mg/L Cd²⁺significantly inhibited lipase activity throughout the exposure period;10 mg/L Cd²⁺significantly inhibitedα-amylase activity at 24 h-72 h cadmium exposure（P<0.05）;96 h cadmium exposure,all concentrations significantly inhibited PO activity（P<0.05）.3.IHC staining showed that all three signaling pathway molecules were specifically stained in the P.clarkii intestine;tissue localization showed that they were located in the intestinal epithelium,but the subcellular distribution was different:Nrf2 and phosphorylated p38 MAPK（p-p38 MAPK）were distributed in the nucleus of the intestinal epithelium,NF-κB was distributed in the cytoplasm of the intestinal epithelium,while p38 MAPK was distributed in the epithelial p38 MAPK was distributed in both nucleus and cytoplasm;cadmium induced the expression of signaling pathway molecules,especially at the late stage of 10 mg/L Cd²⁺exposure（96 h）（P<0.05）.Conclusion:The present study showed that cadmium exposure induced excessive ROS production in the intestine and disrupted the antioxidant system,eventually leading to oxidative stress;ROS not only triggered histopathology in the intestine and was also a trigger of apoptosis,but also cadmium affected digestive and immune enzyme activities,disrupting normal digestion and innate immune function;there were conserved signaling pathway molecules in the P.clarkii intestine and their expression was induced by cadmium.The results of correlation analysis suggested that there was a correlation between the three signaling pathway molecules;in addition,the damage sites shown in HE and TUNEL staining results and the localization of signaling pathway molecules in IHC staining confirmed that epithelial cells are highly susceptible to environmental stress compared to other sites and may play a role in response to environmental stress.This study provides a theoretical basis and experimental basis for the study of the toxic effects of environmental factors on the crustacean intestine,and also has some guidance for the healthy breeding of crustacean economic species.