Proteomics And Metabolomics Analysis On The Toxic Mechanism Of Manganese

With the quick development of modern industry,metal pollution in the environment has been exploding in the past decades.Manganese（Mn）is a necessary trace element for humans and animals,but Mn is also an environmental pollutant.Mn can be transported in various ways in the ecosystem and can be accumulated in animal bodies through different intake pathways,resulting in animal die and affecting the safety of animal food.Mn can be absorbed by human body through the food chain.Excessive intaking Mn can accumulate in the body,which could result in neurodegenerative disease like Parkinson’s disease.The disease is characterized as losing dopaminergic neurons and appearing abnormal proteins in the cells.But the pathogenesis is not entirely clear.Due to many neurotoxic effects of Mn,and there is a wide range of sensitive species to Mn,so the neurotoxicity of Mn and chronic low concentration Mn exposure has become a public health issues of wide concern.In our study,the key proteins and sensitive indicators in global proteins and small molecular metabolites in plasma,which global responses to manganism and the global metabolic pathways will be analyzed using proteomics and metabolomics technologies.The main works and results were as follows:1.Establishing manganism rat model.The concentration of Mn in drinking water was200 mg/L,the exposure was lasting for 5 weeks.The results showed that the model groups appeared behavioral change;the movement area and distance were obviously decreased compared to the control groups in open field test using animal behavior video analysis system.And the level ofΑβ_1-40 increased significantly in plasma and brain of Mn-treated groups,which was the typical pathological characteristic of neurodegenerative diseases.2.Each organ has different distribution of Mn,and manganese exposure induced liver damage and intestinal inflammatory response.No statistically significant difference was observed in the body weight of rats between Mn-treated and control groups in the processes of Mn exposure produce neurotoxin.But the liver index decreased significantly and the cholesterol level in plasma was significantly elevated in Mn-treated group.Histology examinations of the hepatic tissue of the two groups depicted the damaged areas.More extensive necrosis and more dissolved nuclei were clearly observed in the Mn-treated group in comparison with the control group.Our research is the first demonstration that rats are vulnerable to Mn,as shown by the liver damage due to the excess Mn.Duodenal villi infiltration with eosinophils and RBC,cells showed degeneration and necrosis in Mn-treated group;these results indicated that inflammatory response was produced in gut.Each organ has the different distribution of trace elements of Mn,Cu,Fe and Zn,and the content from high to low is:Fe>Zn>Cu>Mn.Among them,the highest content of Mn is liver.The liver damage may be related to enrichment of Mn in liver tissue.3.Manganese exposure changed protein expression profile of intestinal mucosa from rat models.We performed a qualitative and quantitative analysis of 3012 proteins for modifiers of cellular Mn content in intestinal mucosa cells by isobaric tags for relative and absolute quantitation（iTRAQ）.Following stringent validation assays and bioinformatics,a total of 175intestinal mucosa proteins were found that may regulate Mn levels under biologically relevant Mn exposures.Among the different proteins,the expressions of 108 different proteins in rat small intestinal mucosa were down-regulated,whereas 67 were up-regulated.These proteins are related to pancreatic secretion,protein digestion and absorption,fat digestion and absorption,biosynthesis of amino acids,glycerolipid metabolism,Alzheimer’s and Parkinson’s disease,mineral absorption associated protein,etc,based on Gene Ontology（GO）pathway annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis.16 proteins have various relationships in protein–protein interactions（PPI）network and were recognized as key nodes:Prss1,Cela2a,Ctrl,Cela3b,etc.4.Plasma metabolomics reveals the metabolic perturbations by manganese exposure in rat models.We use an untargeted metabolomics approach by performing ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry（UHPLC-Q-TOF/MS）on control and Mn-treated rat plasma,to identify metabolic disruptions under high Mn exposure conditions.Using multivariate and univariate analysis,Mn significantly altered the concentrations of 36 metabolites（12 metabolites showed a remarkable increase in number and 24 metabolites reduced significantly in concentration）in the plasma of the Mn-treated group.Major alterations were observed for purine metabolism,amino acid metabolism and fatty acid metabolism.The metabolites of taurodeoxycholic acid,5-Hydroxyindole acetate,tryptamine,urocanic acid,3-methoxy-4-hydroxyphenyl glycol sulfate,D-proline,bata-hydroxypyruvic acid,stearamide,etc,may be potential biomarkers for manganese exposure.5.Manganese exposure activates the T lymphocytes in peripheral blood,induced inflammatory response,and reduced antioxidant capacity.The proportion of CD3⁺and CD4⁺T lymphocytes in the peripheral blood of Mn treatment group was significantly higher than that in the control group from the results of flow cytometry.ELISA showed that the inflammatory cytokines such as NO,IFN-γ,NF-κB increased expression in plasma;and the level of IL-10 which is an anti-inflammatory cytokines were significantly decreased in the control group.Western blot showed a large number of GFAP expressions in the brain of Mn treatment group,indicating that the manganese exposure can activate T lymphocytes and inflammatory response of the whole body,and then activate astrocytes.In addition,manganese exposure could significantly inhibit antioxidant enzymes:glutathione peroxidase（GSH-PX）,superoxide dismutase（SOD）and catalase（CAT）,which act trace elements as the active center;so that the body’s total antioxidant capacity（T-AOC）was reduced.In conclusion,the mechanism of manganese induced organization and neurons damage include:liver damage,intestinal inflammatory response,changed protein expression profile of intestinal mucosa,perturb plasma metabolic profiles,activate T lymphocytes in peripheral blood,induce inflammation reaction,and lower antioxidant capacity.These results greatly enrich the toxic mechanism of manganese,and provide theoretical basis and technical support for manganism in early diagnosis,treatment,and dynamic monitoring.