| Phenanthrene is a dense aromatic hydrocarbon consisting of three benzene rings,with a molecular formula of C14H10.Phenanthrene is widely distributed in the environment,and human exposure to phenanthrene is almost inevitable.After entering the human body,phenanthrene is metabolized and converted into hydroxyphenanthrenes(OHPhs)under the action of hepatic cytochrome P450 enzyme system.OHPhs are mainly eliminated from the urine.Epidemiological studies with large sample sizes usually use the sum of urinary monoOHPh concentrations,known as total hydroxyphenanthrene(ΣOHPh),as a biomarker of internal exposure to phenanthrene.Currently,the effects of phenanthrene exposure on human health have received widespread attention.Although many researchers consider that phenanthrene,similar to other polycyclic aromatic hydrocarbons,may have various negative effects on human health,including blood glucose metabolism,some plantsynthesized phenanthrene and its derivatives also show analgesic,antibacterial,antiinflammatory,antioxidant,hypoglycemic,cancer cytotoxic and other medicinal properties,some of which have also been used as drugs to treat diseases.Therefore,more research is needed on the relationship between exposure to phenanthrene and health.High fasting plasma glucose(FPG)is an important indicator of type 2 diabetes(T2D)and was the fifth leading risk factor for the global burden of disease mortality in 2019.In the same year,high FPG caused approximately 6.23 million deaths,accounting for approximately 11.5%of all deaths.The human physiological status is the result of the interaction between genes and the environment,and humans’ FPG level are no exception.Genetic and epigenetic factors are also important influences on FPG level,and many genes and their methylation have been observed to play an important role in glucose metabolism.A few cross-sectional studies have begun to focus on the relationship between phenanthrene exposure as an environmental factor and FPG,but the research results are inconsistent,and there is currently no longitudinal study exploring the impact of phenanthrene exposure on fasting blood sugar.In summary,the effects of phenanthrene exposure on FPG and potential gene-environment interactions are still unclear.Based on the above reasons,this study took the participants of the Wuhan-Zhuhai(WHZH)community cohort baseline and the second follow-up six years later as the study subjects.Semi-structured questionnaires were used to collect information on the basic demographic characteristics,lifestyle,disease history,and medication history of the study subjects.Blood and urine samples were collected during the physical examination.After measuring the concentrations of all five mono-hydroxyphenanthrene(1hydroxyphenanthrene,2-hydroxyphenanthrene,3-hy droxyphenanthrene,4hydroxyphenanthrene and 9-hydroxyphenanthrene)in the urine and the levels of FPG of all the subjects,the cross-sectional association between urinary ∑OHPh and FPG was explored.Then,since phenanthrene exposure may affect FPG level through deoxyribonucleic acid(DNA)methylation,this study used DNA methylation chips and epigenome-wide association studies(EWAS)to analyze urinary ∑OHPh related and FPG related DNA methylation,and mediation analysis was used and found at the epigenetic level that adenomatous polyposis coli membrane recruitment protein 3(AMER3)gene methylation mediated the association between urinary ∑OHPh concentration and FPG level.Based on the above research,genome-wide genotyping of participants was performed using gene chips to further investigate the effect modification of AMER3 gene polymorphisms on the association between urinary ∑OHPh concentration and FPG level at the genetic level.Finally,a longitudinal study was used to further investigate the longitudinal association between urinary ∑OHPh concentration and FPG level,and the joint effect of genetic risk and urinary ∑OHPh concentration on longitudinal change of FPG level was investigated by constructing the polygenic risk scores(PRS).This study was divided into four parts as follows:Part Ⅰ.Association between urinary total hydroxyphenanthrene concentration and fasting plasma glucose level in community residents Objective:To explore the association between urinary ∑OHPh concentration and FPG level in the community residents.Method:The subjects of this study were all participants in the WHZH community cohort baseline(2011-2012)and the second follow-up(2017-2018)6 years later.After excluding hypoglycemic drug and insulin users,kidney disease patients,and those with missing information on urinary ∑OHPh,FPG,and related covariates,3913 baseline participants and 3170 second follow-up participants were included,totaling 7083 observations.Five urinary OHPhs concentrations were detected by gas chromatography-mass spectrometry and corrected by urinary creatinine(Cr)concentration.The urinary ∑OHPh concentration was the sum of five urinary OHPhs concentrations.Linear mixed models were used to analyze the association between urinary ∑OHPh concentration and FPG level,using individual numbers and batches of urinary ∑OHPh tests as random terms,adjusting for age,sex,body mass index(BMI),smoking status,drinking status,and city.Sensitivity analyses and stratification analyses were used to determine the robustness of the above association.Both urinary ∑OHPh concentration and FPG level were naturally log-transformed due to their right-skewed distribution.Results:The average age of the study subjects was 54.42 years,32.87%were male,16.36%were smokers,the median urinary ∑OHPh concentration was 1.34 μg/mmol Cr,and the median FPG level was 4.90 mmol/L.After adjusting for confounding factors such as age,gender,BMI,smoking status,drinking status,and city,the linear mixed model found that urinary ∑OHPh concentration was significantly negatively associated with FPG level(P<0.05),and each unit increase in natural log-transformed urinary ∑OHPh concentration was associated with a 0.011[95%confidence interval(CI):0.005,0.017]unit decrease in natural log-transformed FPG level,that is,for each 2-fold increase in urinary ∑OHPh concentration,the FPG level decreased by 1.227%(95%CI:0.549%,1.909%).By further adjusting for more covariates or excluding current smokers for sensitivity analysis,a significant negative association(P<0.05)could still be observed between urinary ∑OHPh concentration and FPG level.In addition,stratification analysis was conducted based on factors such as age,gender,body mass index,smoking,drinking,cooking,exercise,income,and city,and no significant effect modification of the above stratified factors was observed on the negative association between urinary ∑OHPh concentration and FPG level(P modification>0.05).Conclusions:Urinary ∑OHPh concentration was significantly negatively associated with FPG level in the community residents of WHZH cohort.Part Ⅱ.Mediation effect of DNA methylation in the association between urinary total hydroxyphenanthrene concentration and fasting plasma glucose level in community residentsObjective:To explore urinary ∑OHPh concentration related DNA methylation and FPG level related DNA methylation,and analyze the mediation effect of DNA methylation in the association between urinary ∑OHPh concentration and FPG level in community residents.Method:In this part,283 participants were randomly selected from the second follow-up of the WHZH cohort,and their whole blood cell epigenome-wide DNA methylation was detected using the Illumina Infinium MethylationEPIC BeadChips(hereafter referred to as EPIC chip).After excluding hypoglycemic drug and insulin users,kidney disease patients,smokers and ex-smokers(because smoking could affect DNA methylation),and those with missing information on urinary ∑OHPh,FPG,and related covariates,the remaining 212 people were included in this part of the study.The EPIC chips data was subjected to sample and probe quality control for subsequent analysis.Urinary ∑OHPh concentration related epigenome-wide association study(EWAS)and FPG level related EWAS were conducted using general linear models with adjusting for age,gender,BMI,passive smoking,drinking status,city,blood cell proportions,and chip detection batch information.Statistically significant was defined as a false discovery rate(FDR)less than 0.05,and gene set enrichment analysis was performed.The screened relevant DNA methylation sites were further included in the mediation analysis to explore the potential mediating role of these sites in the negative association between urinary ∑OHPh concentration and FPG level.Both urinary ∑OHPh concentration and FPG level were naturally log-transformed.Results:The average age of the 212 study subjects in this part of the study was 59.78 years,12.26%were male,the median urinary ∑OHPh concentration was 0.95 μg/mmol Cr,and the median FPG level was 5.08 mmol/L.The urinary ∑OHPh concentration showed a significant negative association with FPG level in this study subjects(P<0.05),and the association results were the same as those in the first part.After sample and probe quality control for the EPIC chips data,a total of 775927 probes and 212 samples were included in the EWAS.Under the condition of FDR<0.05,no DNA methylation sites and DNA methylation regions significantly related to urinary ∑OHPh concentration were found.Under the condition of FDR<0.05,28 DNA methylation sites and 5 DNA methylation regions related to FPG level were found.Among the 28 FPG level related DNA methylation sites,mediation analysis found that cg11350141 located in the AMER3 gene region of chromosome 2 significantly mediated the negative association between urinary ∑OHPh concentration and FPG level,with a mediation proportion of 30.57%.Conclusions:In this part of the study,28 DNA methylation sites related to FPG were identified in the never-smoking community residents of the WHZH cohort,and the methylation of the AMER3 gene partially mediated the negative association between urinary∑OHPh concentration and FPG level.Part Ⅲ.Effect modification of the AMER3 gene polymorphism in the association between urinary total hydroxyphenanthrene concentration and fasting plasma glucose level in community residentsObjective:The second part of the study found that the methylation of the AMER3 gene mediated the association between urinary ∑OHPh concentration and FPG level at the epigenetic level.The aim of this part of the study is to explore the effect modification of the AMER3 gene polymorphism on the association between urinary ∑OHPh concentration and FPG level at the genetic level.Method:The Illumina Infinium OmniZhongHua-8 BeadChips(hereafter referred to as the ZhongHua-8 chip)were used to perform genome-wide genotyping of the participants in the WHZH cohort who had been followed up at least once.Based on the study population of the first part,this part excluded those with missing valid genotyping information,and finally a total of 4,104 observations were included.All single nucleotide polymorphism(SNP)loci detected by the ZhongHua-8 chip in the AMER3 gene region and eligible for quality control were included in this part of the study.The multiplicative interaction term of SNP genotype and urinary ∑OHPh was included in linear mixed models to analyze the effect modification of AMER3 gene on the association between urinary ∑OHPh and FPG,with individual numbers and batches of urinary ∑OHPh tests as random terms in the models,with adjusting for age,gender,BMI,smoking status,drinking status,and city.Both urinary ∑OHPh concentrations and FPG level were natural log-transformed.Results:The average age of the study subjects was 55.62 years,32.55%were male,16.16%were smokers,the median urinary ∑OHPh concentration was 1.38 μg/mmol Cr,and the median FPG level was 4.92 mmol/L.A total of 12 SNP loci in the AMER3 gene region were detected by the ZhongHua-8 chip and quality-controlled for inclusion in the following analyses.Gene(AMER3)-environment(∑OHPh)interaction analyses revealed that two common variants(rs72854995 and rs77687733)and one low-frequency variant(rs189405908)in the AMER3 gene region significantly modified the association between urinary ∑OHPh concentration and FPG level(Pmodification<0.05).Among them,the negative association between urinary ∑OHPh concentration and FPG level was still observed in the major allele homozygous carriers of rs72854995 and rs189405908 and the minor allele carriers of rs77687733(Ptrend<0.05).But the negative association between urinary ∑OHPh concentration and FPG level could not be observed in the minor allele carriers of rs72854995 and rs189405908 and the major allele homozygous carriers of rs77687733(Ptrend>0.05).The above results suggested that the FPG levels of community residents with different AMER3 genotypes may have different responses to phenanthrene exposure,and the negative association between urinary ∑OHPh concentration and FPG level can be observed mainly in some allele carriers of the AMER3 gene.Conclusions:AMER3 gene polymorphism could significantly modify the negative association between urinary ∑OHPh concentration and FPG level in the community residents of the WHZH cohort.Part Ⅳ.Longitudinal association between urinary total hydroxyphenanthrene concentration and fasting plasma glucose level in community residents and the joint effect of genetic factorsObjective:To explore the longitudinal association between urinary ∑OHPh concentration and FPG level in the community residents,and analyze the joint effect of genetic risk and urinary ∑OHPh concentration on the longitudinal change of FPG level.Method:Based on the study population in the third part,this part included participants who completed the baseline and the second follow-up.Based on the two measured median urinary ∑OHPh concentrations,study subjects with both baseline and second follow-up lower than median concentration were divided into a low longitudinal exposure group,those with both baseline and second follow-up higher than median concentration were divided into a high longitudinal exposure group,and the remaining subjects were divided into an exposure unstable group.The 6-year change of FPG level was estimated as the difference between the second follow-up FPG level and the baseline FPG level.General linear models and logistic regression models were used to analyze the longitudinal association of urinary∑OHPh longitudinal exposure groups with the 6-year change of FPG level and 6-year risk of T2D,with adjusting for age,gender,BMI,smoking status,drinking status,and city.Since there is no publicly available data of large sample size genome-wide association studies(GWAS)related to FPG and T2D in the Chinese population,this study chose to use the publicly available data of the GWAS in the Japanese population(BioBank Japan)to construct a polygenic risk score(PRS)to assess the genetic risk of FPG and T2D.Then the PRS was divided into low genetic risk group(lowest quartile group),medium genetic risk group(second and third quartile groups),and high genetic risk group(highest quartile group).Participants were further classified based on urinary ∑OHPh longitudinal exposure groups and genetic risk groups.The high genetic risk-low longitudinal exposure group was used as the reference group to be included in a general linear model to analyze the joint effect between the urinary ∑OHPh longitudinal exposure and the genetic risk on the 6-year change of FPG level with adjusting for age,gender,BMI,smoking status,drinking status,city,and the top 10 principal components of ancestry.Here,the top 10 principal components of ancestry representing the population structure were obtained by principal component analysis using PLINK.Results:A total of 1,327 participants included in this part of the study for analysis had a mean age of 52.59 years at the baseline,31.27%were men and 16.88%were smokers.After adjusting for age,gender,BMI,smoking status,drinking status,and city,compared with the low longitudinal exposure group,the 6-year change of FPG level in the high longitudinal exposure group significantly decreased by 0.248(95%CI:0.072,0.423),and no significant change in the 6-year risk of T2D was observed in the high longitudinal exposure group or unstable exposure group(P>0.05).In addition,FPG-related PRS was positively associated with the 6-year change of FPG level(P<0.05)and T2D-related PRS was positively associated with the 6-year risk of T2D(P<0.05),which indicated the validity of the PRS constructed in this study.After adjusting for relevant confounders,compared with the high genetic risk-low longitudinal exposure group,this study observed that the 6-year change of FPG level in the T2D-related low genetic risk-high longitudinal exposure group significantly decreased by 0.370(95%CI:0.023,0.717)and the 6-year change of FPG level in the FPG-related low genetic risk-high longitudinal exposure group significantly decreased by 0.412(95%CI:0.747,0.078).Conclusions:Urinary ∑OHPh longitudinal exposure was negatively associated with the 6year change of FPG level in the WHZH cohort of community residents,and there was a joint effect of genetic risk and urinary ∑OHPh longitudinal exposure on the 6-year change of FPG level.Summary:This study found a negative association between urinary ∑OHPh concentration and FPG level in community residents,AMER3 gene methylation could partially mediate the above negative association,and AMER3 gene polymorphism could significantly modify the above negative association.A 6-year follow-up longitudinal study also found a longitudinal negative association between urinary ∑OHPh concentration and FPG level.FPG-related genetic risk and T2D-related genetic risk and urinary ∑OHPh concentration were observed joint effect on longitudinal change in FPG level.This study has increased the knowledge of the relationship between phenanthrene exposure and FPG,providing new ideas for human blood glucose control. |
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