Toxicogical Effects Of Cadmium And Inorganic Arsenic In Chlamys Farreri Based On Multi-omics Approach

Heavy metals are one of the major contaminants disrupting the marine environment for the high rate of accumulation and low metabolism,and the enrichment with the food chain and food web causes toxic effects on marine organisms at different levels.Bivalves are general primary consumers in the marine environment and are sensitive to variations of the marine environment,especially highly enriched in heavy metals,such as cadmium（Cd）and inorganic arsenic（AsIII&AsV）,and have been widely recognized as monitoring species in marine ecology.In this study,Chlamys farreri,an offshore economically important bivalve,was selected as the experimental subject.The eukaryotic non-reference transcriptomics,TMT-labeled proteomics,and UPLC-MS-based untargeted metabolomics approaches were employed to profile the gene-,protein-,and metabolite-level responses and disturbances in different functional metabolic pathways of the ctenophore C.farreri under acute toxicity of high doses of cadmium and inorganic arsenic.Gene expression,apoptosis and antioxidant factor assays were used to complement the validation of the histological results,which collective revealed the mechanism of toxic effects of cadmium and inorganic arsenic in C.farreri.The main conclusions are as follows:Differential expression of 427 genes,258 proteins and 68 metabolites were caused in C.farreri under Cd stress.Transcriptomic and proteomic results showed that the majority of differential genes and proteins were associated with binding（ATP binding,NAD⁺binding,metal ion binding）,catalytic activity（ATPase activity,NAD⁺kinase activity,oxidoreductase activity,amino acid kinase activity）,signal transduction and membrane components;differential metabolites were mainly classified as phospholipids.Multi-omics functional pathway analysis showed that cadmium-induced oxidative stress was generated and resulted in DNA damage and interfered with biochemical pathways such as lipid peroxidation,cell differentiation,and energy metabolism.The NFκB,RIG-I-like receptors,and TNF signaling pathways were activated and interfered with the synthesis of inhibitor of apoptosis-associated protein（XIAP）to induce apoptosis.Covalent metal ions(Mg²⁺,Ca²⁺,Zn²⁺,Fe²⁺)in substance transport were replaced by Cd,altering membrane composition and instead interfering with osmotic pressure regulation and small molecule metabolism,especially for phospholipids.C.farreri induced differential expression of 1630 genes,125 proteins and 60metabolites under AsIII stress;395 genes,153 proteins and 66 metabolites under AsV stress.Transcriptomic results showed similar functional classification of differential genes for both valence states of inorganic arsenic,with the vast majority associated with binding,catalytic activity,transcriptor activity,cellular composition,metabolic processes and biological regulation.Enrichment analysis showed that AsIII differential genes were clustered in their effects on membrane components,while AsV differential genes were less numerous and more functionally dispersed.Proteomics results showed that AsIII differential proteins were enriched in protein fibrils,phenylalanine-t RNA processes,signal transducer activity and energy metabolism-related enzymes,while AsV differential proteins were enriched in disruption of cell division,cysteine-type peptidase activity and histone dephosphorylation processes.The metabolomic results showed that AsIII stress interfered with D-glutamine and D-glutamate metabolism,steroid hormone biosynthesis,and necroptosis;AsV interfered with protein digestion and absorption,phenylalanine metabolism,and amyl-t RNA biosynthesis.Combined with the multi-omics results,the toxic effects of inorganic arsenic were mainly mediated by oxidative stress-dependent signaling pathways in C.farreri.For AsIII,ctenophores were subjected to oxidative stress to generate ROS,activate MAPK,AMPK and PI3K-Akt signaling pathways and induce apoptosis;interfere with intermediate fibers and keratin fibers to affect cytoskeleton;affect c GMP-PKG signaling pathway,NADP metabolism and glucose catabolism process,leading to disruption of energy metabolism.For AsV,it mainly relied on NOD-like receptors,RIG-I-like receptors,NF-kappa B,Wnt signaling pathway,interfered with apoptosis,neuronal activity,growth factor and hormone synthesis.At the protein level disruptions of cell division,extracellular matrix interactions and adherent spots were demonstrated,revealing the toxicity of AsV on cell growth and differentiation.At the protein level disruptions of cell division,extracellular matrix interactions and adherent spots were demonstrated,revealing the toxicity of AsV on cell growth and differentiation.At the metabolic level,amino acid metabolism,lipid metabolism as well as cofactor and vitamin metabolism caused disturbances under AsIII and AsV stress.In addition,alterations in ion binding and membrane composition lead to dysregulation of signal transduction and osmotic pressure.In summary,it was revealed that inorganic arsenic-induced oxidative stress leads to lipid peroxidation,protein damage,apoptosis and neurotoxicity and counteracts the toxicity of inorganic arsenic in the organism by promoting protein synthesis degradation processes in C.farreri.Antioxidant and apoptosis assays validated the histological conclusion that heavy metals induce oxidative stress in ctenophores,produce ROS and lead to apoptosis under Cd and inorganic arsenic（AsIII&AsV）stress.The significant rise in MDA indicates that the action of Cd and inorganic arsenic leads to lipid peroxidation in C.farreri.Twelve genes（COXIII,GPX1,COX1,XIAP,AIF,HSD,GPX2,Cyp3A,TNNT,ABCC1,GTPase,SOD4）expression was verified by Real-Time PCR under Cd,AsIII and AsV stresses,which can be used as biomarkers at the gene level.