The Role Of Peripheral Blood BDNF Ⅳ,LINE-1 Methylation And Synapse-associated Protein Levels In The Association Of Occupational PAHs Exposure With Cognition Impairment In Coke Oven Workers

Objective:Polycyclic aromatic hydrocarbons（PAHs）is an organic pollutant that can cause a variety of hazards to the human body,and the previous research of the research group found that coke oven workers exposed to PAHs have cognitive decline,and its severity is closely related to the exposure level of PAHs,but the mechanism of action is not very clear.This study aims to analyze the correlation between PAHs exposure and cognitive function scores in coking plant workers who are occupationally exposed to PAHs,and explore the methylation levels of brain derived neurotrophic factor（BDNF）promoters,long interspersed nuclear element-1（LINE-1）methylation levels Changes in levels of postsynaptic density-95（PSD95）and presynaptic membrane synaptophysin（SYP）,the possibility of using these indicators as biomarkers for early screening of mild cognitive impairment（MCI）candidates,and possible mediating effects in the association between PAHs and MCI were analyzed,and Infer the process and possible mechanism of B[a]P’s action on MCI at the population level.Methods:From April to June 2019,a cluster sampling was conducted on a state-owned enterprise in Shanxi Province,selecting 632 people from a coking plant as the exposure group and 477 people from an active plant as the control group.The two groups of people were paired 1:1 based on age（±1）,smoking status,and educational level.Finally,89pairs of study subjects were determined as the control group and the exposure group.The questionnaire survey collected basic information of the study subjects,including demographic characteristics（gender,age,length of service,ethnicity,education level,etc.）,lifestyle habits（smoking,drinking,eating fried or pickled foods,cooking frequency,cooking and heating fuel use）,occupational history,family medical history,medication history,and personal disease history.The Montreal Cognitive Assessment Scale（Beijing Version）（Mo CA）was used to assess the cognitive function of the study subjects and determine mild cognitive impairment（MCI）.The fasting elbow vein blood of workers was collected in the morning,and the plasma levels of 16 priority controlled PAHs pollutants listed by the United States Environmental Protection Agency were measured using gas chromatography mass spectrometry（GC-MS）.BDNF Ⅳ and LINE-1 were pyrophosphate sequenced using Pyro Mark Q96.The plasma levels of postsynaptic membrane dense substance 95（PSD 95）and presynaptic membrane synaptophysin（SYP）in workers were measured using enzyme linked immunosorbent assay（ELISA）.The sum of 12 concentrations of PAHs with a detection rate of>75%(Sum₁₂PAHs),Low Ring Polycyclic Aromatic Hydrocarbons（LR-PAHs）,and High Ring Polycyclic Aromatic Hydrocarbons（HR-PAHs）were used to evaluate long-term exposure levels of PAHs.The relationship between plasma PAHs and levels of Mo CA scores,BDNF Ⅳ,LINE-1methylation,and synaptic related proteins was analyzed using logistic regression models,further predict the association between PAHs and Mo CA scores in sensitivity analysis using Bayesian kernel machine regression（BKMR）;The sensitivity,specificity,and accuracy of peripheral blood BDNF Ⅳ methylation and synaptic associated proteins（PSD95,SYP）as markers for early screening for MCI effects were calculated by using Origin software to draw a subject performance characteristic curve（ROC curve）;The mediation package in R language was used to analyze simple mediation effects by calculating regression coefficients and 95%confidence intervals,and the LISREL software was used to analyze chain multiple mediation effects.Results:In this study,the average age of the workers in the two groups was（48.06±5.56）years,the average length of service was（26.89±6.66）years,The plasma concentration of Sum₁₂PAHs in the exposure group was significantly higher than that in the control group（P<0.05）。The exposure group’s concentrations of LR-PAHs（4 rings and below）and HR-PAHs（above 4 rings）were significantly higher than those in the control group.The Mo CA scores of workers in the exposure group was significantly lower than that of the control group,mainly reflected in the significant reduction of Visuospatial and Executive function,Delayed recall scores（P<0.05）,and the detection rate of MCI in the exposure group（46.0%）was higher than that in the control group（24.7%）.Spearman correlation analysis did not find the correlation between PAHs and Mo CA scores.After adjusting for covariates（length of service,educational level,frequency of smoking,frequency of eating fried food,frequency of eating pickled food,and frequency of cooking）,a multiple linear regression analysis was performed,and it was found that the correlation between the two was not statistically significant;Further,dummy variables were grouped according to the interquartile spacing of HR-PAHs levels in workers’blood.Unconditional binary logistic regression found that the risk of MCI in the high exposure group to HR-PAHs was 2.54（95%CI=0.71～9.14）times higher than that in the low exposure group,and the risk of MCI in the high exposure group to PAHs homologue benzo（k）fluoranthene B（k）F was 3.99（95%CI=1.00～16.00）times higher than that in the low exposure group.The mean level of methylation at four sites of BDNF Ⅳ in peripheral blood of the exposed group（4.21±1.39）was significantly higher than that of the control group（3.38±1.04）（P<0.05）,and the methylation level at four sites of LINE-1（71.19±3.20）was significantly higher than that of the control group（67.27±2.55）（P<0.05）.The peripheral blood PSD95 and SYP concentrations in the exposed group were significantly higher than those in the control group（P<0.05）.Spearman correlation analysis did not find a correlation between PAHs and BDNF Ⅳ,LINE-1 methylation levels,but HR-PAHs were positively correlated with PSD95 and SYP levels.After adjusting for the influence of covariates,multiple linear regression analysis did not find any associations between PAHs and BDNF Ⅳ,LINE-1 methylation levels,PSD 95,and SYP;Further,dummy variables were grouped according to the interquartile spacing of HR-PAHs levels in workers’blood.Unconditional binary logistic regression analysis found that the risk of BDNF Ⅳ CpG4 hypermethylation in the moderate HR-PAHs exposure group was 3.55（95%CI=0.85～14.93）times higher than that in low exposure;the risk of LINE-1 CpG3hypomethylation with moderate exposure was 2.79（95%CI=0.88～8.87）times higher than that of low exposure;The risk of PSD95 reduction in the moderate exposure group of HR-PAHs was 86%lower than that in the low exposure group（1-0.14=0.86）;The risk of SYP reduction in the middle and high exposure groups of HR-PAHs was 89%（1-0.11=0.89）and 76%（1-0.24=0.76）lower than that in the low exposure group,respectively.Spearman correlation analysis showed that BDNF Ⅳ CpG4 methylation level,LINE-1 CpG3 methylation level,PSD95 concentration,SYP concentration and MCI were positively correlated（P<0.10）;Multiple linear regression analysis did not find a correlation between BDNF Ⅳ CpG4,LINE-1 CpG3 methylation levels,PSD95,and SYP concentrations and MCI;Through further unconditional binary logistic regression,it was found that workers with BDNF Ⅳ CpG4 hypermethylation had a 3.37（95%CI=0.82～13.93）times higher risk of MCI than those with hypomethylation.The risk of MCI in workers with LINE-1 CpG3 hypermethylation was 3.07（95%CI=1.02～9.25）times that of hypomethylation,and the risk of MCI in the group with high concentration of peripheral blood PSD95 was 4.00（95%CI=1.37～11.70）times that of the group with low concentration.The risk of MCI in the high-concentration group of SYP was 5.65（95%CI=1.72～18.61）times that of the low-concentration group;The combined area under curve（AUC）of BDNF Ⅳ CpG4 methylation,PSD 95,and SYP was 0.64（95%CI=0.51～0.77,P=0.04）,which was greater than the respective AUC values of the three indicators.When combined,the diagnostic sensitivity and specificity of the three indicators were 64.3%and 70.4%,respectively,and the corresponding optimal cutoff threshold was 19.62.Adjust the covariates such as length of service,education level,frequency of smoking,frequency of eating fried food,frequency of eating pickled food and frequency of cooking,and use the mediation package in R Studio to analyze the simple mediation effect.The results show that SYP has a significant indirect effect in connecting PAHs and Mo CA scores,but because the total effect is not significant,SYP may play a role of masking the effect between HR-PAHs and Mo CA.The analysis of chain multiple mediation model was carried out by LISREL software,and it was found that HR-PAHs→BDNF Ⅳ CpG4→PSD95→Mo CA and HR-PAHs→BDNF Ⅳ CpG4→SYP→Mo CA were saturated models,and the absolute value of T-Value corresponding to the path coefficient was less than 1.96,It is suggested that BDNF Ⅳ CpG4 and PSD95,BDNF Ⅳ CpG4 and SYP may not have multiple mediating effects in MCI induced by PAHs exposure.Conclusion:1.Peripheral PAHs are independent risk factors for MCI in coking plant workers,among the HR-PAHs homologues,B（k）F has the most obvious effect.2.Peripheral blood PAHs can cause a decrease in LINE-1 CpG3 methylation levels,affecting overall DNA methylation levels;Exposure to PAHs can also cause elevated levels of BDNF Ⅳ CpG4 methylation,PSD 95,and SYP.3.The area under curve（AUC）,sensitivity,and specificity of peripheral blood BDNF Ⅳ CpG4 methylation,PSD95,and SYP are the highest after their addition.The combined detection of BDNF Ⅳ CpG4 methylation,PSD95,and SYP levels may be used as a biomarker for MCI with high diagnostic performance.4.SYP may play a masking effect between HR-PAHs and Mo CA,while BDNF Ⅳ CpG4 and PSD 95,BDNF Ⅳ CpG4 and SYP may not have multiple mediating effects in MCI induced by PAHs exposure.This project is supported by the National Natural Science Foundation of China（project number:81973093）and the Shanxi Province Merit-based Funding Program for Returning Overseas Students（2019-028）.