Mediation Effects Of DNA Methylation On The Association Between Benzo[a]pyrene Exposure And Lung Cancer Development

Objectives: Benzo[a]pyrene(B[a]P)is a widely distributed pollutant in the environment,and is also the most abundant and typical carcinogenic polycyclic aromatic hydrocarbons(PAHs).It is mainly produced by cigarette smoking,hightemperature cooking fume,petroleum,asphalt and automobile exhaust.Once absorption,B[a]P can be metabolized and activated into the ultimate carcinogen anti-7,8,-dihydrodiol-9,10-epoxide benzo[a]pyrene(BPDE),which can bind to DNA and form BPDE-DNA adducts,and then initiate the development of cancer.BPDE can also bind to the blood albumin to form anti-7,8,-dihydrodiol-9,10-epoxide benzo[a]pyrenealbumin(BPDE-Alb)adducts,which could transfer the BPDE to human tissues and organs through the blood stream.Many epidemiological studies have used BPDE-Alb adducts as the internal exposure marker for assessing B[a]P exposure level and as the biological effective dose marker for measuring the biological dose of B[a]P.In this study,we aimed to investigate the associations between plasma concentrations of BPDE-Alb adducts and DNA methylation patterns in peripheral blood leukocytes by using the genome-wide DNA methylation Bead Chip,and further to explore the mediation effects of DNA methylation on the association of B[a]P exposure with lung cancer risk.This study will help to provide clues and scientific data for exploring the roles of DNA methylation changes caused by environmental pollution on the development of lung cancer.Methods: 109 paired lung cancer cases and controls were included in this study.The basic demographic information,lifestyles,disease histories,and anthropometric indicators of each person were collected by using the face-face questionnaire,and the venous blood of the lung cancer patients and healthy controls,as well as lung cancer tissues and adjacent tissues removed during surgical operation of the lung cancer patients,were collected.Enzyme-linked immunosorbent assay was used to determine the plasma concentrations of BPDE-Alb adducts.Illumina Human Methylation 450 K Bead Chip was used to determine the methylation levels at > 485000 Cp Gs.The multivariate logistic regression model,with adjustment for age,sex,body mass index(BMI),smoking and drinking status,and experimental batch,was used to investigate the association between plasma concentrations of BPDE-Alb adducts with lung cancer risk.For all participants,we used the multiple linear regression models to analyze associations of plasma BPDE-Alb adducts with peripheral blood DNA methylation levels,with adjustment for age,gender,BMI,smoking and drinking status,disease status(lung cancer,control),white blood cell components,and experimental batch.Based on the previous studies,we selected a total of 59 DNA methylation sites(Cp G sites)with P value <1.0×10-4 for subsequent analyses.For these 59 Cp Gs,we further analyzed their associations with smoking status among total population,by using the multiple logistic regression models,with adjustment for age,gender,BMI,drinking,disease status(lung cancer,control),plasma BPDE-Alb adducts,and experimental batch.We compared the differences in DNA methylation levels between the abovementioned 59 Cp Gs in lung cancer and adjacent tissues of 109 lung cancer patients.For the 14 Cp Gs,whose difference between lung cancer tissues and adjacent tissues reached a significant level(P<0.05)and the direction of the difference was consistent with the association direction of plasma BPDE-Alb adducts,we further analyzed their associations with lung cancer risk by using the multivariate logistic regression models,with adjustment for age,gender,BMI,smoking and drinking status,plasma BPDE-Alb adducts,and experimental batch.Then,the mediation effects of the 4 significant Cp Gs on the association of BPDE-Alb adducts with lung cancer risk were estimated by using the causal mediation models,and their diagnostic values on lung cancer were also evaluated by plotting the receiver operating characteristic(ROC)curve.Results: The plasma concentrations of BPDE-Alb adducts among the lung cancer cases were significantly higher than those among the controls,and there was a significant positive association between plasma BPDE-Alb adducts and lung cancer risk(P=0.048).With per SD increase in BPDE-Alb adducts,the lung cancer risk increased 45% [odd ratio(OR)and 95% confidence interval(CI)= 1.45(1.00,2.10)].This association was mainly shown among smokers and subjects with BMI <24.0 kg/m2.With 1 kg/m2 increase in BMI,the lung cancer risk increased 13% [OR(95%CI)=0.87(0.80,0.96)],and smokers have a 3.64-fold risk of lung cancer than the non-smokers [OR(95%CI)= 3.64(1.56,8.49)].We identified 59 Cp Gs whose methylation levels in peripheral blood were significantly associated with plasma BPDE-Alb adducts at P < 1.0×10-4.Among these Cp Gs,the methylation levels of 10 Cp Gs were different among smokers and nonsmokers(P <0.05).We then compared the methylation levels of above 59 Cp Gs between lung tumor and adjacent normal tissues among the 109 lung cancer patients,and found that the differences of 14 Cp Gs cites showed consistence directions with their associations of plasma BPDE-Alb adducts.There are 5 Cp Gs sites,including NUP160 gene cg14931686,PTAFR gene cg20460771,RNF11 gene cg24808674,P4 HTM gene cg07707611,and INPP5 A gene cg16544463,showed higher methylation levels in lung cancer tissues than in adjacent normal lung tissues(fold change>1,P <0.05),and had positive associations with plasma BPDE-Alb adducts(β>0,P < 1.0×10-4);there are 9 Cp Gs sites,including SHANK2 gene cg03371662,SCGB1D1 gene cg26907473,FAM83 A gene cg10424125,cg23225748(introgenic),C3orf22 gene cg24389347,SLC8A1 gene cg10103850,FAT1 gene cg11396712,cg25105578(introgenic),and KIF6 gene cg15478981 showed lower methylation levels in lung cancer tissues than in adjacent normal lung tissues(fold change <1,P <0.05),and had negative associations with plasma BPDE-Alb adducts(β <0,P < 1.0×10-4).Among the above 14 Cp Gs sites,we then estimated the associations of their methylation levels in peripheral blood with lung cancer risk.We found that the blood methylation levels of 5 Cp Gs sites were significantly associated with lung cancer risk: the hyper-methylations of SCGB1D1 gene cg26907473,PTAFR gene cg20460771,and RNF11 gene cg24808674 were significantly associated with increased risk of lung cancer [OR(95%CI)= 1.78(1.26,2.58),10.27(5.58,21.17),and 1.47(1.06,2.07),respectively,and P = 0.002,5.89×10-12,and 0.022,respectively].The hypermethylations of FAM83 A gene cg10424125 and SLC8A1 gene cg10103850 were associated with decreased risk of lung cancer [OR(95%CI)= 0.45(0.31,0.63)and 0.13(0.07,0.22),respectively,P= 1.34×10-5 and 1.89×10-11,respectively].However,we excluded the SCGB1D1 gene cg26907473 in the subsequent analysis,because it showed negative association with plasma BPDE-Alb adducts,but was associated with increased risk of lung cancer.We then estimated the mediation effects of the left 4 Cp Gs,including FAM83 A gene cg10424125,PTAFR gene cg20460771,RNF11 gene cg24808674 and SLC8A1 gene cg10103850,on the associations of BPDE-Alb adducts with lung cancer risk.The mediation analysis showed that the FAM83 A gene cg10424125,PTAFR gene cg20460771,and SLC8A1 gene cg10103850 showed significant mediation effects on the association of plasma BPDE-Alb adducts with lung cancer risk.The hypomethylations at FAM83 A gene cg10424125 and SLC8A1 gene cg10103850 could mediate a separate 42.2% and 44.0%,and hyper-methylation of the PTAFR gene cg20460771 could mediate 35.7% of the association between plasma BPDE-Alb adducts and lung cancer risk(P values were 0.002,0.032,and 0.002,respectively).We also found that the joint mediation proportion of FAM83 A gene cg10424125 and SLC8A1 gene cg10103850 was 64.5%(P=2.0×10-16).The cg20460771 had the highest diagnositic value for lung cancer with the area under the curve(AUC)and 95%CI was 0.889(0.839,0.927),the sensitivity and specificity were 80.73% and 81.76% respectively.The AUC(95%CI)for cg10103850 was 0.864(0.811,0.906),the sensitivity and specificity were 85.32% and 76.15%,respectively and the AUC(95%CI)for cg10424125 was 0.690(0.624,0.751),the sensitivity and specificity were 66.97% and 69.72% respectively.However,cg24808674 had the lowest predictive value for the risk of lung cancer [AUC(95%CI)= 0.578(0.509,0.644),the sensitivity = 65.14%,specificity = 54.13%].Conclusions: In this study,the plasma concentration of BPDE-Alb adducts was associated with the blood methylation levels of 59 Cp Gs at P < 1.0×10-4.We further found that the hyper-methylation levels of PTAFR gene cg20460771 and RNF11 gene cg2480867 were positively associated with the plasma concentrations of BPDE-Alb adducts and risk of lung cancer,and their methylation levels were higher in lung cancer tissues than in adjacent normal tissues.While,the hyper-methylation levels of FAM83 A gene cg10424125 and SLC8A1 gene cg10103850 were negatively associated with the plasma concentrations of BPDE-Alb adducts and risk of lung cancer,and their methylation levels were lower in lung cancer tissues than in adjacent normal tissues.The blood methylation levels of FAM83 A gene cg10424125,PTAFR gene cg20460771 and SLC8A1 gene cg10103850 had significant mediation effects on the association of B[a]P exposure with lung cancer risk.The above results preliminarily suggested the possible role of DNA methylation in the occurrence of lung cancer caused by the exposure of B[a]P,but the underlying biological mechanisms still need further investigations.