Study On The Physiological And Molecular Mechanism Of Crucian Carp (Carassius Auratus) Gill In Response To Di (2-ethyl) Hexyl Phthalate Stress

Phthalic acid esters（PAEs）are a kind of colorless,low water-soluble,and low-volatile synthetic organic compounds and often used as plasticizers and softeners of polyvinyl chloride（PVC）to increase the elasticity and durability of plastic products.Because of its low cost and good use effect,PAEs are widely used in personal care products,food packaging,medical supplies,building materials,and other fields.Among them,di（2-ethyl）hexyl phthalate（DEHP）is the most widely used PAEs compound in plastic production.In recent years,investigations and studies have shown that DEHP has been detected in many environmental media worldwide,including air,rain water,surface water,sediment,soil and so on.DEHP will show stable chemical properties in the water environment because of its low volatility,low water solubility,and high lipophilicity.Existing studies have shown that DEHP exposure will produce a series of toxicological effects on aquatic animals,such as oxidative stress,reproductive toxicity,developmental toxicity,immunotoxicity,endocrine disorders and so on,which seriously affect the growth development,and reproduction of aquatic organisms.However,at present,the understanding of DEHP on fish toxicity at the molecular level is still insufficient,especially the systematic research under different exposure times and concentrations has not been fully carried out.Therefore,further evaluation of the physiological and molecular mechanisms of aquatic organisms in response to DEHP stress is critical to reducing the harm of PAEs to aquatic organisms and promoting the healthy development of the aquatic environment.In this study,crucian carp gill tissue was taken as the research object,and crucian carp was exposed to 20,100,and 500μg/L DEHP,and a blank control group was set up to carry out 96 h acute and 30 d chronic toxicological exposure experiments with different environmental concentrations of DEHP.Metabolomics and transcriptomics techniques were used to explore the molecular response mechanism of crucian carp gill to DEHP exposure from metabolite level and gene level,at the same time,combined with biochemical analysis and histopathological observation,in order to evaluate the ecotoxicological effects of DEHP on aquatic organisms,reduce water pollution and harm to aquatic organisms,and provide new ideas and methods for exploring the mechanism of toxic effects of DEHP exposure on aquatic organisms.The main results of this study are as follows:（1）In the DEHP acute exposure experiment,at first,the effects of 20,100,500μg/L for 96 h of acute exposure to DEHP on antioxidant indicators,including glutathione peroxidase（GSH-Px）,catalase（CAT）,superoxide dismutase（SOD）,reduced glutathione（GSH）and malondialdehyde（MDA）,as well as on immune and indicators,including acid phosphatase（ACP）and alkaline phosphatase（AKP）,were measured in the gills of crucian carp.The results showed that SOD and GSH-Px increased significantly in a dose-dependent manner under the exposure of 100μg/L and500μg/L DEHP（p<0.05）,and the level of CAT increased significantly under the medium concentration of DEHP exposure.However,there was no significant difference between the high concentration and control groups,but there was no significant difference between the high concentration and control groups.The level of GSH in all DEHP exposure groups decreased significantly in a dose-dependent manner（p<0.05,p<0.01,p<0.001）.The level of MDA in the group exposed to the high concentration of DEHP was significantly higher than that in the group exposed to the high concentration of DEHP（p<0.01）,and the level of ACP in all groups exposed to DEHP was significantly higher than that in the group exposed to the high concentration of DEHP（p<0.01）.It is suggested that acute exposure to DEHP can induce obvious oxidative stress and immune response in the gills of crucian carp.Secondly,the effects of acute exposure to DEHP on gill metabolites and related metabolic pathways of crucian carp were explored using non-targeted metabonomic techniques based on UPLC-QTOF-MS.Taking p<0.05 and VIP>1.0 as the screening criteria for differential metabolites（DMs）,a total of 73 metabolites showed statistically significant differences in all experimental groups.KEGG enrichment analysis showed that the low concentration of DEHP had no obvious effect on gill metabolism but mainly affected sphingolipid metabolism and glyceride metabolism.With the further increase of DEHP concentration,many metabolic pathways such as purine metabolism,glycerol phospholipid metabolism,sphingolipid metabolism,arachidonic acid metabolism,linoleic acid metabolism,andα-linolenic acid changed significantly.On this basis,transcriptomics data showed that 20μg/L DEHP acute exposure had significant effects on cytochrome P450 on heterophyte metabolism,steroid biosynthesis,glycolysis/gluconeogenesis,and other metabolic pathways.In contrast,under 100μg/L DEHP exposure,metabolic pathways including ECM-receptor interaction,complement and coagulation cascade,protein digestion and absorption,and arachidonic acid metabolism were significantly enriched.Under 500μg/L DEHP exposure,the metabolic pathways such as protein digestion and absorption,arachidonic acid metabolism,complement and coagulation cascade,and cytochrome P450 were significantly affected.Several metabolic pathways,including arachidonic acid metabolism,cytochrome P450 metabolism of heterophytes,purine metabolism,and steroid biosynthesis,were significantly enriched in the experimental group.Comprehensive multi-group and biochemical analysis data show that acute exposure to medium and high concentrations of DEHP leads to redox imbalance in gill tissue,and the increase of purine metabolism is the potential source of increased reactive oxygen species.The disorder of lipid metabolism,including arachidonic acid metabolism,induces inflammation.（2）In the DEHP long-term exposure experiment,based on UPLC-QTOF-MS non-targeted metabolomic analysis,20,100,500μg/L DEHP exposure groups and the control group were screened out,including glycerides,glycerophospholipids,steroids,and 60 differential metabolites including its analogs,fatty acids,amino acids,nucleotides,organic acids,etc.KEGG enrichment analysis indicated that at low concentrations of DEHP exposure,the most relevant metabolic pathways included major purine metabolism,glycolysis/gluconeogenesis,glutathione metabolism,and phenylalanine glycerophospholipid metabolism.With the further increase of the exposure concentration,under the condition of 100μg/L DEHP exposure,the metabolic pathways including pyruvate metabolism and galactose metabolism in crucian carp gills were further affected;when the DEHP exposure concentration reached 500μg/L,the metabolic pathways,including the metabolic pathways related to lipid metabolism,including sphingolipid metabolism,glycerophospholipid metabolism were most significantly disturbed.In addition,pyruvate metabolism and phenylalanine metabolism were also enriched and significantly changed.On this basis,transcriptomic analysis was performed on the control and 500μg/L high-concentration DEHP exposure groups.Differentially expressed genes（DEGs）were screened with p-adjust<0.05 and|log₂FC|≥1 as criteria.A total of 3275 DEGs were identified between the two groups.Including cytochrome P450 metabolism of xenobiotics,sterol hormone biosynthesis,and cytokine-cytokine receptor interactions were enriched under high DEHP exposure.Comprehensive analysis showed that under long-term high-concentration DEHP exposure,the cytochrome P450 enzyme system of crucian carp gills was unbalanced,and the abnormal arachidonic acid metabolism might be closely related to it.In addition,long-term exposure to DEHP led to disturbance of endocrine and energy metabolism in gill tissue.Histopathological analysis showed that after exposure to the high concentration of 500μg/L DEHP for 30 days,the gill tissue had obvious structural damage,the arrangement of gill filaments was sparse and disordered,the distance between gill filaments became larger,and pathological changes such as shrinkage and deformation and apex enlargement occurred.Overall,this study provides a possibility to understand the changes among metabolites,genes,and biological phenotypes in fish gill tissues under DEHP stress and provides a new theoretical guide for understanding the toxicity mechanism of DEHP exposure to fish gills.