Association Between Exposure To Metal Mixtures And Cardiac Enzymes In Ferromanganese Workers And Mechanism Of Autophagy In Manganese-induced Cardiotoxicity

Objective To investigate the associations between the concentrations of metals in blood cell and cardiac maker levels in serum based on a cross-sectional study among occupational population.And to explore the role of autophagy in manganese-induced cardiac toxicity by manganese-treated rats and H9c2 cells.Methods1.Based on manganese-exposed workers healthy cohort（MEWHC）,a cross-sectional study was designed to select 554 manganese exposed workers who were followed up and completed serum myocardial enzymes testing in2021.Cardiac enzymes include creatine kinase（CK）,creatine kinase isoenzyme（CK-MB）,aspartate aminotransferase（AST）,lactic dehydrogenase（LD）andα-hydroxybutyrate dehydrogenase（α-HBD）.And inductively Coupled Plasma Mass Spectrometry（ICP-MS）was used to determine the concentrations of 22metals in blood cells of occupational manganese exposure workers.（1）Generalized linear regression model was used to analyze the influencing factors of metal concentration and myocardial enzyme level,as well as the correlation between a single metal and myocardial enzyme level.（2）The LASSO regression was used to screen the metals related to the myocardial enzyme level,and the composite polymetallic exposure model was constructed in combination with generalized linear regression to analyze the relationship between the composite polymetallic exposure and the myocardial enzyme level.（3）Restricted cubic spline（RCS）was used to analyze the dose-response relationship between the concentration of metal and myocardial enzyme level.（4）The correlation between the concentration of metal and myocardial enzyme level was calculated by stratified analysis.（5）Bayesian Kernel Machine Regression（BKMR）model was used to evaluate the combined effect of polymetallic compound exposure on myocardial enzymes.2.A total of 40 SPF male SD rats were ranked into 4 groups（n=10 per group）,and treated with 0,5,10,20 mg/kg Mn by intraperitoneal injection for 6months,respectively.The content of Mn in the heart of rat was determined by ICP-MS,and the expression levels of apoptosis related proteins,autophagy related proteins and Phosphatidylinositol 3 kinase/Protein kinase B（PI3K/Akt）signaling pathway related proteins were detected by Western blot.3.H9c2 cells were exposed to 0,100,200,and 400μM Mn for 24 h,respectively.The content of Mn in the H9c2 cells was detected by ICP-MS,and the expression levels of PI3K/Akt signaling pathway related proteins were detected by Western blot.H9c2 cells were pretreated with rapamycin（Rapa）,3-Methyladenine（3-MA）,and chloroquine（CQ）,respectively,and then exposed to Mn（400μM）for 24 h.The expression levels of apoptosis related proteins and autophagy related proteins were detected by Western blot.The cell survival rates were detected by MTT assay.After interference of ATG7 si RNA,H9c2 cells were exposed to Mn（400μM）for 24 h,the expression levels of apoptosis related proteins and autophagy related proteins were detected by Western blot.H9c2 cells were pretreated with LY294002 and N-acetyl cysteine（NAC）,respectively,and then exposed to Mn（400μM）for 24 h.The expression levels of apoptosis related proteins,autophagy related proteins and PI3K/Akt signaling pathway related proteins were detected by Western blot.Results1.Single metal model analysis of correlation between the concentration of metal in blood cells and the level of myocardial enzyme in serum among workers who were occupational exposed to manganese.（1）The influencing factors of metal concentration mainly include sex,age,length of service,smoking and drinking.The concentrations of magnesium,iron,arsenic,rubidium,molybdenum,cadmium and lead in blood cells were higher in males（P<0.05）,while the concentrations of manganese and cobalt were higher in females（P<0.001）.The concentrations of zinc,selenium,molybdenum and lead in blood cells were different by age（P<0.05）.The concentrations of magnesium and selenium were higher in BMI≥24 kg/m² group（P<0.05）.The concentrations of manganese and lead in blood cells were different by length of service（P<0.05）.And the concentrations of manganese,rubidium and cadmium in blood cells were higher in drinker（P<0.05）.The concentrations of CK,CK-MB and AST were higher in males（P<0.001）.（3）The results of the single-metal model（the continuous variable model）showed that:In overall participants,the concentrations of manganese,arsenic and rubidium were positively correlated with CK level（P<0.05）.The concentration of manganese was positively correlated with CK-MB level（P=0.029）.The concentrations of calcium,strontium and molybdenum were negatively correlated with LD level（P<0.05）,and calcium and molybdenum were negatively correlated withα-HBD level（P<0.05）.In males,the concentrations of manganese,rubidium and lead were positively correlated with CK level（P<0.05）,calcium and molybdenum were negatively correlated with LD level（P<0.05）.The concentration of arsenic was positively correlated withα-HBD level,while calcium and molybdenum were negatively correlated withα-HBD level（P<0.05）.In females,the concentration of calcium was negatively correlated with CK-MB level（P<0.05）.The concentrations of manganese,iron and antimony were positively correlated with LD（P<0.05）,while strontium was negatively correlated with LD（P<0.05）.The concentrations of magnesium and iron were positively correlated withα-HBD,while calcium and strontium were negatively correlated withα-HBD（P<0.05）.The concentrations of vanadium and chromium were positively correlated with AST（P<0.05）.2.Multiple-metals model analysis of correlation between the concentration of metal in blood cells and the level of myocardial enzyme in serum among workers who were occupational exposed to manganese.（1）LASSO regression:in overall participants,manganese,arsenic and rubidium were screen as CK related metals;magnesium,calcium,cobalt,nickel,zinc,molybdenum and antimony were screen as LD related;and calcium,manganese,iron,cobalt,nickel,zinc,arsenic and molybdenum were selected asα-HBD related.In males,manganese was related to CK level;calcium,zinc,arsenic and molybdenum were related to LD level;calcium,manganese,zinc,arsenic and molybdenum were related toα-HBD level;and tin was related to AST level.In females,magnesium,manganese,iron,cobalt,nickel,strontium and antimony were related to LD level.（2）Generalized linear regression:in overall participants,the concentration of arsenic and rubidium were positively correlated with CK level（P<0.05）,antimony was positively correlated with LD level,while calcium,zinc and molybdenum were negatively correlated with LD level（P<0.05）.Nickel was positively correlated withα-HBD level,while calcium,zinc and molybdenum were negatively correlated withα-HBD level（P<0.05）.In males,the concentration of calcium and zinc were positively correlated with LD level,while molybdenum was negatively correlated with LD level（P<0.05）,and zinc and molybdenum were negatively correlated withα-HBD level（P<0.05）.In females,the concentration of iron,nickel and antimony were positively correlated with LD level,while strontium was negatively correlated with LD level（P<0.05）.（3）Dose-response relationship:In overall participants,manganese was linearly and positively associated with CK level(P_overall=0.019,P_{non-linearity}=0.307),calcium was linearly negative associated with LD level(P_overall=0.008,P_{non-linearity}=0.488),molybdenum was linearly negative associated withα-HBD level(P_overall=0.010,P_{non-linearity}=0.125),and we observed a U shape dose-respose relationship between molybdenum and LD level(P_overall<0.001,P_{non-linearity}=0.488).In males,we also observed a U shape dose-respose relationship between molybdenum and LD level(P_overall<0.001,P_{non-linearity}=0.036)and molybdenum was linearly negative associated withα-HBD level(P_overall=0.028,P_{non-linearity}=0.217).Arsenic was linearly positive associated withα-HBD level(P_overall=0.019,P_{non-linearity}=0.307).In females,manganese,nickel and antimony were linearly positive associated with LD level,respectively(P_overall=0.044,P_{non-linearity}=0.210;P_overall=0.026,P_{non-linearity}=0.494;P_overall=0.043,P_{non-linearity}=0.249,respectively).（4）Interaction:in overall participants,there was an interaction between age and the correlation between calcium concentration in blood cell and LD level(P_interation=0.041).Besides,there was an interaction between smoking and the correlation between molybdenum concentration in blood cell and LD level(P_interation=0.035).（5）BKMR model:in overall participants,manganese in blood cells was positively correlated with CK level,and dominated the overall positive effect of three metal mixtures（rubidium,arsenic and manganese）on CK level.Calcium and zinc were negatively correlated with LD levels,and dominated the overall negative effect of seven metal mixtures（rubidium,molybdenum,zinc,nickel,cobalt,calcium and magnesium）on LD level.In males,zinc and molybdenum were negatively correlated with LD,and dominated the overall negative effect of four metal mixtures（molybdenum,arsenic,zinc and calcium）on CK level.In females,manganese and antimony were positively correlated with LD,and dominated the overall positive effect of seven metal mixtures（rubidium,strontium,nickel,cobalt,iron,manganese and magnesium）on LD level.3.Results of heart in chronic manganese exposure rats.（1）With the increasing exposure dose of Mn,the Mn content in rats’heart increased,and the Mn content in all Mn exposure groups were significantly higher than control group（P<0.05）.（2）Compared with control group,the protein Cleaved-Caspase 3 levels and Cleaved-Caspase 3/Caspase 3 ratios in all Mn treated rats’heart were significantly higher（P<0.05）,and the protein Bcl-2-associated X protein（Bax）level in 20 mg/kg group was significantly higher（P<0.05）,furthermore,the protein B-cell lymphoma 2（Bcl-2）levels and Bcl-2/Bax ratios in 10 and 20mg/kg groups were significantly decreased（P<0.05）.（3）Compared with control group,the protein p-m TOR levels in all Mn treated rats’heart were significantly decreased（P<0.001）,and the protein m TOR and p62 levels in 10 and 20 mg/kg groups were significantly decreased（P<0.05）,besides,the protein LC3Ⅱlevels in 10 and 20 mg/kg groups were significantly increased（P<0.01）.In addition,LC3Ⅱ/LC3Ⅰratio in 20 mg/kg group was significantly increased（P<0.05）.（4）Compared with control group,the levels of protein p-Akt in 10 and 20mg/kg Mn-treated rats were significantly higher（P<0.01）.4.Results of Mn-treated H9c2 cells（1）With the increasing exposure dose of Mn,the Mn content in H9c2 cells increased,and the Mn content in all Mn exposure groups were significantly higher than control group（P<0.05）（2）Compared with Mn group in H9c2 cells,the protein p-m TOR levels in Rapa+Mn and CQ+Mn groups were significantly decreased（P<0.05）,the protein LC3Ⅱlevels in Rapa+Mn and CQ+Mn groups were significantly increased（P<0.05）,and the LC3Ⅱ/LC3Ⅰratio in CQ+Mn group was significantly increased（P<0.05）.Meanwhile,the protein Bcl-2 level in Rapa+Mn was significantly increased（P<0.05）,but the protein Bcl-2 levels in3MA and CQ+Mn groups were significantly decreased（P<0.05）,furthermore,the cell survival rate in CQ+Mn group was significantly decreased（P<0.01）.（3）Compared with si-NC group in H9c2 cells,the protein Atg7 levels in other groups were significantly decreased（P<0.001）.And compared with Mn group,the protein Atg7 level in si-ATG7+Mn was further decreased（P<0.001）.There were no significant differences in apoptosis related protein levels between si-NC group and si-NC+Mn group,but compared with si-NC group,the protein Bcl-2 level in si-ATG7+Mn group were significantly decreased（P<0.01）,moreover,the Bcl-2/Bax ratios in si-ATG7 and si-ATG7+Mn groups were significantly decreased（P<0.05）.（4）Compared with the control group,the levels of protein Akt in 200 and400μM groups were significantly decreased（P<0.05）,but the levels of protein p-Akt were significantly increased（P<0.05）.（5）In H9c2 cells,the protein level of p-Akt in LY294002 group was significantly lower than control group（P<0.01）.The protein level of Akt in LY294002+Mn group was significantly higher than Mn group（P<0.01）,while the protein level of p-Akt was significantly lower（P<0.001）.Compared with Mn group,the protein m TOR level in LY294002+Mn group was significantly increased（P<0.01）,but the protein Bcl-2 level was significantly decreased（P<0.05）.（6）In H9c2 cells,the level of protein Akt in NAC+Mn group was significantly lower than Mn group（P<0.01）.Compared with the control group,the protein m TOR level in NAC+Mn group was significantly lower（P<0.05）,but there was no significant difference between Mn group and control group（P>0.05）.Compared with control group,the protein Bcl-2 level and Bcl-2/Bax ratio in Mn group were significantly decreased（P<0.05）,but there was no significant difference between NAC+Mn group and control group（P>0.05）.Conclusions1.In overall participants,manganese,arsenic and rubidium were screen as CK related metals,and manganese dominated the overall positive effect of these three metal mixtures on CK level.Rubidium,molybdenum,zinc,nickel,cobalt,calcium and magnesium were screen as LD related metals,and calcium and zinc dominated the overall negative effect of these seven metal mixtures on LD level.Besides,manganese was linearly and positively associated with CK level,calcium was linearly negative associated with LD level,molybdenum was linearly negative associated withα-HBD level,and we observed a U shape dose-respose relationship between molybdenum and LD level.In males,molybdenum,arsenic,zinc and calcium were screen as CK related metals,and zinc and molybdenum were dominated the overall negative effect of these four metal mixtures on CK level.And we also observed a U shape dose-response relationship between molybdenum and LD level,and molybdenum was linearly negative associated withα-HBD level,arsenic was linearly positive associated withα-HBD level.In females,rubidium,strontium,nickel,cobalt,iron,manganese and magnesium were screen as LD related metals,and manganese and antimony dominated the overall positive effect of seven metal mixtures（rubidium,strontium,nickel,cobalt,iron,manganese and magnesium）on LD level.And manganese,nickel and antimony were linearly positive associated with LD level.In overall participants,there was an interaction between age and the correlation between calcium concentration in blood cell and LD level.Besides,there was an interaction between smoking and the correlation between molybdenum concentration in blood cell and LD level.2.Chronic manganese exposure can activate autophagy and autophagy plays a protective role in manganese-induced cardiotoxicity.