| Objective:The effects of exposure to individual heavy metals such as lead(Pb),mercury(Hg),and cadmium(Cd)on neurocognitive dysfunction have been extensively studied.People are usually exposed to multiple heavy metal mixtures simultaneously.My research team first reported that exposure to low levels of Pb,Hg,and Cd mixture can produce new synergistic neurocytotoxicity different from the two heavy metals and lead to neurobehavioral dysfunction in rats.However,few studies systematically explore biomarkers and mechanisms of neurocognitive impairment induced by mixed exposure to Pb,Hg,and Cd.In recent years,with the rapid development of omics technology,various kinds of omics(genome,transcriptome,proteome,and metabolomics)analysis have been applied to study the toxic effects and mechanisms of exogenous chemicals.Monomics usually only reflects a certain aspect of the effects of exogenous chemicals on organisms,such as changes in protein expression or metabolite content.However,it is impossible to systematically and comprehensively explain the toxic effects and pathways of exogenous chemicals on organisms.It is well known that proteomics enriches differential proteins and "allocates" them to specific pathways;Metabolomics can display the reactions that occur when exogenous chemicals act on organisms.Integrated metabolomics and proteomics analysis can achieve mutual verification and improve the reliability of analysis results.Therefore,this study established a rat model exposed to low levels of Pb,Hg,and Cd mixture,using metabonomic and proteomic techniques to study the effects of low dose Pb,Hg,and Cd combined exposure on serum metabolism and brain protein mass spectrometry,and further conducted a joint metabonomic and proteomic analysis to reveal the functional changes and toxicity mechanisms related to neurotoxicity caused by low dose Pb,Hg,and Cd combined exposure,To provide a basis for comprehensive analysis of the molecular mechanism of heavy metals induced neurotoxicity.Methods:(1)A rat model exposed to low levels of Pb,Hg,and Cd mixture was established to explore the effects of low dose Pb,Hg,and Cd mixed exposure on animal serum metabolic profiles using non-targeted metabonomics techniques,combined with multiple analytical methods such as differential metabolite screening,classification,location prediction,pathway analysis,and subject performance characteristic curve(ROC)analysis.(2)Using TMT-labeled proteomics technology,combined with bioinformatics analysis methods such as differential protein analysis,subcellular localization prediction,GO function analysis,and KEGG pathway analysis,the effects of low dose Pb,Hg,and Cd mixed exposure on protein mass spectrometry in animal brain tissue were investigated.(3)Based on the results of metabolomics and proteomics analysis,a "Pathview" tool is used to perform a integrative analysis of the two and visualize the analysis results,further validating the critical proteins involved in the joint analysis results.Results:(1)A total of 206 differential metabolites were detected in metabolomics.Compared with the control group,the content of 73 metabolites in the heavy metal mixed exposure group increased,while the content of 133 metabolites decreased;The main categories of differential metabolites are benzene ring compounds,organic heterocyclic compounds,phenylpropanes,polyketones,lipids,organic acids,and their derivatives;Differential metabolite localization prediction showed that epidermis,prostate,central nervous system,neural tissue,and hippocampus were the main sites;Differential metabolite pathway analysis showed that mixed exposure to low doses of Pb,Hg,and Cd interfered with pathways such as unsaturated fatty acid biosynthesis,linoleic acid metabolism,phenylalanine metabolism,tryptophan metabolism,and tyrosine metabolism;ROC analysis showed arachidonic acid,adrenal acid,and dihydrogen-γ-The area under curve(AUC)of linolenic acid,oleic acid,retinoic acid,quinolinic acid,and canine uric acid were 1.000,0.861,0.944,0.972,0.917,0.944,and 0.972,respectively.(2)Proteomic analysis revealed a total of 363 differential proteins.Compared with the control group,176 proteins were upregulated and 187 proteins were downregulated in the heavy metal mixed exposure group;The differential proteins are mainly distributed in the cytoplasm,nucleus,extracellular matrix,mitochondria,and plasma membrane.The GO function enrichment results show that the differential proteins especially involve cell components(such as cell membranes,organelles,cell junctions,and synapses),intracellular biological processes(such as regulation of biological processes,metabolic processes,stimulation reactions,and biological adhesion),and molecular functions(such as binding functions,catalytic activity functions,and molecular structural functions);Enrichment analysis of the KEGG pathway showed that the signaling pathways involved in the differential proteins between groups mainly include actin cytoskeleton regulation,iron death,adhesion spots,axon guidance,and cancer protein polysaccharides.(3)The results of the integrative analysis showed that ferroptosis and tryptophan-kynurenine pathways were closely related to both metabolomics and proteomics,and there were significant changes in multiple proteins or metabolites in these two pathways;Further selecting the key proteins in iron death and tryptophan canine urinary acid pathway for WB validation,the results showed that compared with the control group,the LPCAT3 protein expression level in the brain tissue of rats exposed to heavy metals was significantly increased(P<0.05),while the SLC7A11 and KYAT1 protein expression levels were significantly decreased(P<0.05).The WB results were consistent with the results of the joint analysis.Conclusions:Dysregulations of ferroptosis and tryptophan-kynurenine pathways may be related to neurotoxicity caused by low dose Pb,Hg,and Cd combined exposure,which provides new ideas and theoretical support for in-depth research on the neurotoxicity mechanism of heavy metals. |
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