| Lung cancer is a malignant tumor with the highest mortality in China and around the worldwide,and has caused a heavy burden on social and economic development.Insidious onset and rapid progression often make lung cancer patients unable to receive effective treatments.Therefore,it is particularly important to carry out early prevention of lung cancer.Benzo[a]pyrene(B[a]P),as the most carcinogenic polycyclic aromatic hydrocarbon(PAH),can induce DNA damage and genomic changes.B[a]P can be metabolized to benzo[a]pyrene-diol-epoxide(BPDE),catalyzed by the cytochrome P4501 A enzymes.BPDE can bind with plasma albumin,form benzo[a]pyrene diol epoxide-albumin(BPDEAlb)adduct,and then transported throughout the body.Thus,the internal exposure dose of B[a]P can be assessed by measuring plasma BPDE-Alb adduct.Long-term exposure to B[a]P significantly increases the risk of lung cancer.As an essential trace element,zinc(Zn)is involved in the regulation of DNA damage and repair,cell proliferation,differentiation and apoptosis,activation or inhibition of proteases.Zn also plays important roles in antioxidant and immune regulation.Our previous study found that increased plasma Zn was significantly associated with reduced risk of lung cancer,but the effect of Zn on the lung tumorigenesis induced by B[a]P is rarely reported.DNA methylation,the most frequently studied epigenetic modification,plays a crucial role in lung tumorigenesis,mainly via regulating the expression of oncogenes or tumor suppressor genes.Previous studies have shown that exposure to B[a]P can cause alterations of methylation levels at global or specific sites,and DNA methylation may be a potential intermediate process involved in the mechanism of B[a]P carcinogenesis.As a requirement in the activity of various epigenetic enzymes,changes in Zn levels can lead to alterations in DNA methylation or its association with environmental exposures.However,there is limited evidence on whether Zn may influence the association of B[a]P-related epigenetic changes with lung cancer risk.Based on the findings above,we aimed to explore the role of Zn on the association of B[a]P with lung cancer development by using epidemiological study.First,in a lung cancer case-cohort study derived from Dongfeng-Tongji cohort,plasma levels of Zn and BPDEAlb adduct were detected.The modification effect of Zn on BPDE-Alb adduct-lung cancer association was also evaluated.Second,genome-wide DNA methylation beadchips were used to detect genome-wide DNA methylation levels,and its association with plasma BPDE-Alb adduct was analysed.The modification effect of plasma Zn on the association of BPDE-Alb adduct-related DNA methylation with lung cancer risk was estimated.Correlations between DNA methylation levels and corresponding gene expression levels were analyzed by using genome-wide methylation beadchips and m RNA sequencing for paired lung cancer tissues and adjacent normal tissues.Furthermore,differential gene expression in lung cancer and adjacent normal tissues was compared by using The Cancer Genome Atlas(TCGA)database and m RNA sequencing.The present study is mainly divided into the following two parts.Part Ⅰ.The role of zinc on the association between benzo[a]pyrene and risk of lung cancer: a case-cohort studyObjectives: To explore the modification effect of Zn on the association of BPDE-Alb adduct and lung cancer risk in a lung cancer case-cohort study.Methods: This study was based on 38 295 participants in the Dongfeng-Tongji cohort in 2013.After excluding those with electrocardiograms abnormalities,coronary heart disease,stroke,cancers and those missing medical insurance numbers and blood samples,1399 participants were randomly selected as a sub-cohort from the remaining 24 415 retirees, stratified by age and sex,with a sampling rate of 5.8%.The lung cancer case–cohort study(n=1742)included the 1399 sub-cohort subjects and additional 359 incident lung cancer cases(16 cases were in the sub-cohort)in 24 415 participants during the time between enrollment and December 31,2018.The basic information was obtained by questionnaires,enzyme-linked immunosorbent assay(ELISA)and inductively coupled plasmamassspectrometry(ICP-MS)were used to detect the baseline plasma levels of BPDE-Alb adduct and Zn,respectively.Weighted Cox proportional hazards regression models and restricted cubic spline(RCS)curves were used to analyse associations of BPDE-Alb adduct and Zn with incident lung cancer,with adjustment for age,sex,body mass index(BMI),smoking and drinking status,education levels,regular physical exercise and experimental batch.The interaction and combined effects of BPDE-Alb adduct,Zn and basic characteristics including age,gender,smoking and drinking status on incident lung cancer were further evaluated.Finally,all participants were divided into low/medium/high groups according to the 33.3% and 66.7% quantile of Zn in the sub-cohort,the associations between BPDE-Alb adduct and lung cancer risk at different levels of plasma Zn were analyzed,and the modification effect of Zn on BPDE-Alb adduct-lung cancer association was also explored.Results: Each one standard deviation(SD)increase in natural logarithm(ln)-transformed baseline BPDE-Alb adduct was associated with 55% increased [HR(95% CI)= 1.55(1.32,1.82)] risk of lung cancer;each one SD increase in ln-transformed Zn was associated with 21% decreased [HR(95% CI)= 0.79(0.68,0.92)] risk of lung cancer.RCS analysis showed that both BPDE-Alb adduct and Zn were linearly associated with lung cancer risk(P < 0.05).Alcohol consumption significantly modified the association between BPDE-Alb adduct and lung cancer risk(Pinteraction=0.013).Age>65 years-old,smoking and high levels of BPDEAlb adduct had combined effects on increasing risk of lung cancer [HR(95% CI)=3.52(2.33,5.31)and 2.70(1.66,4.37),respectively,Ptrend<0.001];whereas age≤65 years-old,non-smoking and high levels of Zn had combined effects on reduced risk of lung cancer [HR(95% CI)=0.28(0.18,0.42)and 0.34(0.21,0.53),respectively,Ptrend<0.001].All subjects were divided into three groups according to the 33.3% and 66.7% quantile of plasma Zn in the sub-cohort(T1,T2 and T3 corresponding to low,medium and high levels of Zn groups,respectively).Plasma BPDE-Alb adduct was significantly positively associated with lung cancer risk in T1 and T2 groups [HR(95% CI)=2.36(1.65,3.38)and 1.61(1.20,2.15)],but no significant association was found in T3 group [HR(95% CI)=1.29(0.92,1.82),P=0.140].Plasma Zn could significantly modify the harmful effect of BPDEAlb adduct on lung cancer risk(Pinteraction=0.021).Conclusions: Increased plasma BPDE-Alb adduct was associated with increased risk of lung cancer,while increased plasma Zn was associated with decreased risk of lung cancer.High levels of Zn can alleviate the harmful effect of BPDE-Alb adduct on lung cancer risk,which provides preliminary evidence of the protective effect of Zn on lung tumorigenesis induced by B[a]P.Part Ⅱ.The role of zinc on the association between benzo[a]pyrene and risk of lung cancer: an epigenetic studyObjectives: To explore the association of plasma BPDE-Alb adduct-related DNA methylation levels with incident lung cancer,and to evaluate the modification effect of Zn on the above associations.Methods: The Illumina Infinium Human Methylation EPIC Bead Chip(EPIC Bead Chip)was used to measure peripheral blood of DNA methylation levels for all subjects in the lung cancer case-cohort study.In the sub-cohort subjects,the association of plasma BPDE-Alb adduct with genomic DNA methylation was analyzed by using multiple linear regression model,with adjusted for age,sex,BMI,smoking status,drinking status,estimated cell type proportions,and all surrogate variables calculated by Smart SVA.Cp Gs with P<1.0×10-5 were selected as BPDE-Alb adduct-related Cp Gs.The functional annotations,gene ontology(GO)pathway,and DNA co-methylation patterns were further analyzed.In the lung cancer case-cohort subjects,weighted Cox proportional hazards regression models were used to analyse associations of BPDE-Alb adduct-related methylation levels with lung cancer risk.For the Cp Gs associated with lung cancer risk,the association of their methylation levels with plasma Zn was explored,and associations of their methylation levels with BPDE-Alb adduct,and with lung cancer risk in low/medium/high Zn groups(stratified by the 33.3% and 66.7% quantile of Zn in the sub-cohort participants)were further evaluated.The modification effects of Zn on Cp Gs methylation-lung cancer association were also explored.On the other hand,EPIC Bead Chip and m RNA sequencing were used to measure DNA methylation levels and m RNA expression levels for paired tumor and adjacent normal tissues for 32 lung cancer patients,respectively.Gene expression data from 1037 lung cancer tissues and 108 adjacent normal tissues were also downloaded from TCGA online public database.Finally,the correlations of DNA methylation with gene expression,and differences of gene expression in lung cancer tissues and adjacent normal tissues were further analyzed.Results: In epigenome-wide association study,29 Cp Gs were associated with plasma BPDE-Alb adduct at P<1.0×10-5,among which cg07839457 in NLRC5,cg05475649 in B2 M,and cg19748455 in LOC100996291 had FDR<0.05.Annotated genes of BPDE-Alb adduct-related Cp Gs showed enrichment in cell apoptosis,immune response,maintenance of protease activity and tumorigenesis.Among 29 Cp Gs,the methylation levels of cg19748455 in LOC100996291,cg02089348 in TMEM129 and TACC3,and cg17021907 in C1QL2 were significantly associated with decreased risk of lung cancer,whereas methylations levels of cg01156012 in IRS2,cg05407682 in AKAP1,cg12798040 in XRCC3,cg24518943 in FAM135 A were significantly associated with increased risk of lung cancer(P<1.72×10-3,0.05/29 Cp Gs).Plasma Zn was negatively correlated with methylation levels of cg05407682 [β(SE)=-0.136(0.071)].Plasma BPDE-Alb adduct were significantly negatively associated with the methylation levels of cg19748455 and cg02089348 [β(SE)=-1.312(0.257)and-0.514(0.181),respectively],and were significantly positively associated with methylation levels of cg05407682,cg12798040,and cg24518943 [β(SE)=1.163(0.261),0.719(0.232),and 0.437(0.196),respectively] in the low Zn group.However,the above associations were not significant in the high Zn group(all P>0.05).The positive associations of cg01156012 and cg05407682 on lung cancer risk were mainly shown in T1 group [HR(95%CI)=1.88(1.40,2.52)and 1.58(1.23,2.03)] and T2 groups [HR(95%CI)= 1.67(1.18,2.36)and 1.38(1.04,1.83)],whereas the above associations were not significant or decreased in T3 group [HR(95%CI)=1.41(1.00,2.00)and 0.92(0.68,1.26)].Zn could significantly modify the relationship between methylation of cg05407682 and lung cancer risk(Pinteraction= 0.012).Hypermethylation of cg05407682 was associated with high expression of AKAP1(r = 0.341,P = 0.043),and the expression level of AKAP1 was significantly higher in lung cancer tissues than in adjacent normal tissues(P < 0.001).Conclusions: In this study,significant associations of DNA methylation and plasma BPDEAlb adduct were observed at 29 Cp Gs,whose annotated genes were mainly involved in apoptosis,immune response,and tumor initiation.Hypermethylation of cg19748455 in LOC100996291,cg02089348 in TMEM129 and TACC3,and cg17021907 in C1QL2 were associated with decreased lung cancer risk,whereas hypermethylation of cg01156012 in IRS2,cg05407682 in AKAP1,cg12798040 in XRCC3,and cg24518943 in FAM135 A were associated with increased lung cancer risk.High levels of plasma Zn was associated with hypomethylation of cg05407682.With the increase of Zn,the correlation effect of cg05407682 in AKAP1 with lung cancer risk decreased,and Zn could modify association of cg05407682 methylation with lung cancer.Further histological analysis showed that hypermethylation of cg05407682 was associated with high expression of AKAP1,and the expression level of AKAP1 was significantly higher in lung cancer tissues than in adjacent normal tissues.The above results provide epigenetic evidence for elucidating the protective effect of Zn on lung tumorigenesis induced by B[a]P exposure.In conclusion,this study found that high levels of Zn can reduce the risk effect of BPDE-Alb adduct on lung cancer,and Zn significantly modified the correlation between the methylation level of BPDE-Alb adduct-related cg05407682 in AKAP1 and lung cancer risk,indicating that Zn may protect against carcinogenesis of B[a]P by influencing B[a]Prelated epigenetic changes. |
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