Association Of Arsenic Methylation Metabolism With Cardiovascular Disease Risk In Coal-burning Arsenic Exposed Populations: Potential Role Of Vascular Endothelial Injury

Objective:In recent years,an increasing number of scholars have focused on the association between low to moderate arsenic exposure and cardiovascular disease（CVD）.However,there have been no studies on the effect of coal-burning arsenic exposure on CVD risk.In order to understand the metabolism of arsenic in vivo after exposure to coal burning and its effect on the risk of CVD,this study conducted a population-based epidemiological survey.Arsenic methylated metabolites and vascular endothelial injury-related indices were detected.The Atherogenic index of plasma（AIP）and Framingham risk score（FRS）were used to assess CVD risk.The relationship between arsenic exposure and methylated metabolites and vascular endothelial injury and CVD risk was comprehensively analyzed,and the potential role of vascular endothelial injury in arsenic exposure,arsenic methylated metabolites and CVD risk was discussed to provide a reference for subsequent mechanistic studies.Methods:（1）Population epidemiological investigationThe typical case of coal-burning arsenicosis was taken as the investigation point.According to Endemic Arsenic Poisoning Diagnostic Criteria（WS/T 211–2015）standard,244 patients with arsenicosis were recruited as the arsenicosis group,and167 residents were recruited in adjacent non-arsenic-exposed villages as the control group.The data of the respondents was collected through a questionnaire.（2）Indices detectionThe contents of trivalent arsenic(As³⁺),pentavalent arsenic(As⁵⁺),monomethyl arsenic（MMA）and dimethyl arsenic（DMA）were determined by high performance liquid chromatography-Inductively coupled plasma Mass spectrometer（HPLC-ICP-MS）,and calculate the percentage of As³⁺%,As⁵⁺%,MMA%,DMA%.The primary methylation index（PMI）,secondary methylation index（SMI）,inorganic arsenic（i As）and total arsenic（t As）were calculated simultaneously.CVD risk was assessed using AIP and FRS.The levels of endothelial nitric oxide synthase（e NOS）,intercellular adhesion molecule-1（ICAM1）and vascular endothelial growth factor（VEGF）were determined by enzyme-linked immunosorbent assay（ELISA）.The asymmetric dimethylarginine（ADMA）concentration was determined by high performance liquid chromatography-tandem quadrupole mass spectrometry（HPLC-MS/MS）.（3）Statistical methods and analysis1)Bioinformatics analysis:Using GEO database gene chip data GSE110852,using GEO2R to output differential expression results,and using the Cytoscape algorithm to screen hub genes from protein-protein interaction networks mapped on the STRING online platform.R 4.2.1 Pheatmap package was used to draw the heatmaps.2)Statistical analysis method:the independent sample t test was used for comparison between the two groups,Manne-Whitney U test was used for comparison between groups with nonnormal distribution data.Chi-square test was used for rate comparison.A binary logistic regression model was used to assess the difference in CVD risk assessment measures between the two groups.Pearson correlation and linear regression were used to analyze the association between arsenic methylation metabolism and endothelial injury index.Spearman correlation and logistic regression were used to analyze the association between arsenic methylated metabolites and CVD risk.Restricted cubic spline（RCS）analysis was performed using the R 4.2.1rms package and mediation analysis was performed using the R 4.2.1 bruce R package.Bilateral P<0.05 is considered statistically significant.Results:（1）Reduced capacity of arsenic methylation in patients due to arsenic exposure is a risk factor for increased risk of cardiovascular disease1)Arsenic exposure leads to a reduced metabolic capacity for arsenic methylation in patientsIn the present study,DMA,MMA,As³⁺and As⁵⁺were detected in urine.The results showed that DMA,MMA,As³⁺,i As,and t As were significantly higher in the arsenicosis group than in the control group,and As⁵⁺%was significantly lower.Further pairwise comparison showed that the levels of DMA,MMA,As³⁺and t As in mild,intermediate and severe arsenicosis groups were significantly higher than those in control group,while the i As level in mild and severe arsenicosis groups was significantly higher than that in control group,and As⁵⁺%in intermediate arsenicosis group was significantly lower than that in control group.As³⁺%in severe arsenicosis group was significantly higher than that in control group（P<0.05）.Compared with the mild arsenicosis group,DMA%and SMI%of the severe arsenicosis group were significantly decreased and MMA%was significantly increased（P<0.05）.DMA,MMA,As³⁺,i As and t As all showed an increasing trend,while As⁵⁺%showed a decreasing trend(P_trend<0.05).2)Arsenic exposure is a risk factor for increased risk of cardiovascular disease in patientsThe results of logistic regression analysis showed that the risk of CVD was higher in the arsenicosis.Logistic regression analysis and RCS results showed that there was a linear increasing dose-response relationship between MMA,MMA%and CVD risk index FRS,and a linear decreasing dose-response relationship between FRS and DMA%and SMI level.The dose-response relationship between DMA,MMA,i As,t As,As³⁺and cardiovascular risk index AIP was linearly increasing(P_nonlinear>0.05,P_overall<0.05).MMA%was positively correlated with AIP（P<0.05）.（2）Arsenic exposure is a risk factor for increased endothelial injury in patientsIn this study,ADMA,ICAM1,VEGF and e NOS in the plasma of the observation subjects were detected.The results show that:The levels of ADMA,ICAM1 and VEGF in the arsenicosis group were significantly higher than those in the control group,and the Kruskal-Wallis H test further showed that the levels of ADMA and VEGF in the mild,intermediate and severe arsenicosis group were significantly higher than those in the control group.The level of ICAM1 in severe arsenicosis group was significantly higher than that in control group and mild arsenicosis group（P<0.05）.ADMA,VEGF and ICAM1 showed an increasing trend among groups(P_trend<0.05).The endothelial injury caused by arsenic exposure was further analyzed.The results showed that ICAM1 showed an increasing trend with the increase of MMA,DMA,and t As（P<0.05）.MMA and MMA%were positively correlated with ADMA,while SMI and DMA%were negatively correlated with ADMA（P<0.05）.The nonlinear correlation between arsenic methylation metabolism and endothelial injury indexes was further analyzed.The results showed that the dose-response relationship between ADMA and MMA,ADMA and MMA%,VEGF and As⁵⁺increases linearly.The dose-response relationship between ICAM1and DMA,MMA and t As was linearly increasing.The ADMA level still showed a decreasing trend with increasing SMI.Both e NOS and As⁵⁺,e NOS and As⁵⁺%,and ICAM1 and As⁵⁺all decreased first and then increase.ADMA and DMA%,VEGF and t As were firstly increased and then decreased(P_nonlinear<0.05,P_overall<0.05).（3）ADMA and ICAM1 mediate an increased risk of cardiovascular disease due to arsenic exposure and arsenic methylation metabolismlogistic regression and RCS analysis showed that ADMA and ICAM1 and CVD risk index FRS showed an increasing trend,and ADMA,ICAM1,VEGF and CVD risk index AIP had a linear increasing dose-response relationship(P_nonlinear>0.05,P_overall<0.05).The results of mediate analysis showed that ADMA mediated the association between DMA%and FRS,MMA and FRS,SMI and FRS,and MMA%and FRS,with mediation accounting for 24.94%,25.13%,26.96%and 25.47%,respectively.ICAM1 mediates the association between MMA and FRS,with a mediating effect of 18.27%.The proportion of ICAM1 mediated in DMA,t As,and MMA associated with AIP was 18.05%,16.14%and 10.47%,respectively.ADMA played a partial mediating role in the association between MMA and AIP,with a mediating effect ratio of 12.77%.ADMA was a complete mediating effect in the association between MMA%and AIP（P<0.05）.Conclusions:（1）Arsenic exposure was a risk factor for the increased risk of cardiovascular disease in patients,and the reduced capacity of arsenic methylation metabolism caused by arsenic exposure is associated with the increased risk of cardiovascular diseases in the population;（2）Vascular endothelial damage has a tendency to aggravate with the severity of arsenicosis.Increased arsenic exposure and reduced arsenic methylation metabolism may promote vascular endothelial injury.（3）ADMA and ICAM1 mediate the increased risk of cardiovascular disease due to arsenic exposure and reduced capacity of arsenic methylation metabolism.