The Role Of DNA Methylation Modification And Single Nucleotide Polymorphism In The Abnormal Expression Of Metabolic Enzymes Induced By Benzo[a]pyrene

Objective:To investigate the possible role of DNA methylation modifications and single nucleotide polymorphisms of the PAHs metabolic enzymes CYP1A1,GSTP1,and GSTM1 genes in the expression of CYP1A1,GSTP1,and GSTM1 genes in peripheral blood among population studies;To investigate the methylation level and gene expression of Cp G islands in CYP1A1,GSTP1 and GSTM1 DNA promoter regions in liver,kidney,lung,cerebral cortex and cerebellum of subchronic poisoned mice induced by Benzo[a]pyrene(0.2,2,20 mmol/L).Reveal the possible role of DN A methylation modification in BaP-induced of PAHs metabolic enzyme genes expressed abnormally Based on the above studies,the possible factors affecting the abnormal gene expression of PAHs metabolizing enzymes caused by BaP were found,which have important scientific significance and potential application value for early detection of high-risk groups of health-related damage to coke oven workers and protection of workers' health.Methods:In the population study,158 occupational healthy coking workers exposed PAHs for Contact Group,and 60 healthy workers who are worked water supply for the control group.Through cross-sectional investigation and research,population gender,age,and Smoking and drinking conditions,types of work,educational level,living habits,personal disease history,family history was investigated by self-designed questionnaires.Urine samples were collected after class,the content of 1-hydroxy-pyrene(1-OH-Py)in urine were determined by high performance liquid chromatography.Fasting cubital venous blood were collected,and the expression of CYP1A1,GSTP1,and GSTM1 genes in the peripheral blood of the study subjects were detected by fluorescence quantitative PCR(FQ-PCR).the methylation levels of CYP1A1,GSTP1 and GSTM1 DNA CpG islands in the promoter regions of in the peripheral blood of the study subjects were detected by Methylation-specific PCR(MSP).The gene polymorphisms of CYP1A1 and GSTP1 were detected by competitive allele-specific PCR(KASP),and the polymorphism of GSTM1 gene was detected by multiplex PCR(Multiply-PCR).Data were analysised by covariance analysis and chi-square test of SPSS 22.0 software.In the animal research: Thirty-two SPF male ICR mice were randomly divided into four groups according to body weight: inchuding solvent control group(peanut oil),low dose exposure group(0.5 mg/kg BaP),medium doseexposure group(2.0 mg/kg BaP)and high dose exposure group(10.0 mg/kg BaP),each group including eight mice.The solvent control group was injected with the same volume of peanut oil as the toxication group and administered by intraperitoneal injection.The exposure time was fixed at 9 am,The frequency of exposure was determined every other day.The exposure period was 2 months.CYP1A1,GSTP1,and GSTM1 DNA promoter regions methylation levels were detected by Methylation-specific PCR(MSP);CYP1A1,GSTP1,and GSTM1 mRNA expression changes detected by Real-time quantitative PCR(qPCR).Multiple groups were compared using single-factor analysis of variance,and comparisons were performed using LSD tests.Results: The population study results are as follows:1)The relationship between BaP exposure and CYP1A1,GSTP1 and GSTM1 methylation modification and gene expression in peripheral blood of coke oven workers(1)Compared with the control group,the 1-OH-Py level in the urine of the coke oven was increased significantly,and the difference was statistically significant(P < 0.05).Compared with the control group,the gene expression levels of CYP1A1 and GSTM1 methylation in the peripheral blood of coke oven workers were decreased significantly,and the difference was statistically significant(P < 0.05).The methylation level and gene expression of GSTP1 were decreased significantly.The difference was statistically significant(P < 0.05).(2)All study subjects were divided into four dose levels according to 1-OH-Py levels in the urine.The results showed that with the increase of 1-OH-Py in urine,the methylation of CYP1A1 and GSTM1 increased,and the expression of CYP1A1 and GSTM1 gene decreased,The difference was statistically significant(P < 0.05);There was no difference in GSTP1 methylation level(P > 0.05),and its gene expression was significantly decreased(P < 0.05).2)Analysis of the relationship between CYP1A1,GSTP1 and GSTM1 methylation modification and gene expression in peripheral blood(1)CYP1A1 methylation level was negatively correlated with gene expression(r=-0.183,P=0.035).The CYP1A1 methylation levels were divided into low,medium,and high levels according to interquartile range(P33,P66).Compared with the CYP1A1 hypomethylated group,the CYP1A1 gene expression levels in the middle and high methylation groups were decreased significantly.The difference was statistically significant(P < 0.01),and the hypermethylation group was lower than the middle methylation group.(2)The level of GSTP1 methylation was positively correlated with gene expression(r=0.175,P=0.043).The level of GSTP1 methylation was divided into low,middle and high levels according to interquartile range(P33,P66),compared with the low GSTP1 methylated group,the expression level of GSTP1 gene in the medium and high methylation group was significantly increased(P < 0.01).The hypermethylation group was lower than the middle methylation group.(3)GSTM1 methylation levels were negatively correlated with gene expression(r=-0.178,P=0.047).The methylation levels were divided into low,medium,and high levels according to interquartile range(P33,P66),and GSTM1 Compared with the hypomethylated group,the GSTM1 gene expression levels in the medium and high methylation groups were decreased significantly(P < 0.001),and the hypermethylation group was lower than the middle methylation group.3)relationship between CYP1A1,GSTP1 and GSTM1 single nucleotide polymorphisms and gene expression in peripheral blood(1)There was no significant difference in genotype between the contact group and the control group(P > 0.05).The allele frequencies of the genotype locus were tested by Chi-square test and were in accordance with the Hardy-weinberg equilibrium law.(2)The person of CYP1A1 rs1048943 mutations heterozygous(GA)and mutant homozygous(GG)were significantly higher in peripheral blood CYP1A1 gene expression than wild-type(AA),the difference was statistically significant(P < 0.05);The person of GSTP1 rs1695 locus mutations heterozygous(GA)and mutant homozygous(GG)were significantly lower in peripheral blood GSTP1 gene expression than wild-type(AA),the difference was statistically significant(P < 0.05);Compared with GSTP1 rs762803 locus wild type(AA),There was no significant difference in mutant heterozygous(CA)and mutant homozygous(CC)in GSTP1 gene expression,there was no statistical significance(P > 0.05);The expression of GSTM1 gene in GSTM1-deficient peripheral blood was lower than that in non-deleted type,and the difference was statistically significant(P < 0.05).4)Analysis of possible interaction between CYP1A1,GSTP1,and GSTM1 methylation modification and single nucleotide polymorphism in peripheral blood for gene expression.(1)CYP1A1 rs1048943 locus polymorphism and gene methylation level had an interaction effect on gene expression,the difference was statistically significant(P < 0.05);mutation and hypomethylation combined decreased CYP1A1 gene expression.(2)GSTP1 rs762803 polymorphism or GSTP1 rs1695 site polymorphism and methylation level had interaction effect were not found on GSTP1 gene expression,the difference was not statistically significant(P > 0.05);GSTM1 gene deletion type either Non-deletion type and methylatio n level have interactions effect were not found on GSTM1 gene expression.5)Experimental results in mice indicate:(1)Compared with the control group,the expression of CYP1A1 gene was in the liver,kidney,lung,cerebral cortex and cerebellum significantly increased in BaP-treated group(0.2,2,20 mmol/L),and the difference was statistically significant(P < 0.05);CYP1A1 methylation levels in the liver,lung and cerebral cortex were decreased significantly,the difference was statistically significant(P < 0.05).(2)Compared with the control group,the expression of GSTP1 gene in the cerebral cortex and cerebellumin increased in BaP-treated group(0.2,2,20 mmol/L),in the kidney and lung decreased,and the difference was statistically significant(P < 0.05).There was no significant change in the liver,and the difference was not statistically significant(P < 0.05);the level of GSTP1 methylation in the cerebral cortex decreased,the difference was statistically significant(P < 0.05).There was no significant change in lung and liver.The difference was not statistically significant(P > 0.05).(3)Compared with the control group,the expression of GSTM1 gene in the liver decreased induced in BaP-treated group(0.2,2,20 mmol/L),and decreased first and then increased in the lungs.The difference was statistically significant(P < 0.05).There was no significant difference in the expression level between the cerebral cortex and cerebellum.The difference was not statistically significant(P > 0.05).The level of GSTM1 methylation in the liver and cerebral cortex increased.There was a statistically significant difference(P < 0.05),which increased first and then decreased in the lungs.The difference was statistically significant(P < 0.05).Conclusions:(1)The increased methylation levels of CYP1A1 and GSTM1 in coke oven workers showed a corresponding decreased in gene expression,decreased GSTP1 methylation and gene expression,and a significant dose-dependent relationship with urinary 1-OH-Py levels.(2)The increased of CYP1A1 and GSTM1 methylation levels was correlated negatively with the corresponding gene expression,whereas the methylation level of GSTP1 was correlated positively with gene expression.(3)The single nucleotide polymorphisms of CYP1A1 rs1048943 and GSTP1 rs1695 and the GSTM1 deletion were associated with gene expression decreased of the corresponding genes.(4)The CYP1A1 rs1048943 locus single nucleotide polymorphism(SNP)and methylation interacted with CYP1A1 gene.Mutation combined with hypermethylation decreased gene expression.(5)With increasing dose of BaP,CYP1A1 the methylation level in mice tissues was decreased significantly,GSTP1 Methylation decreased significantly in mouse cerebral cortex and was closely related to the increase of GSTP1 gene expression in the brain.GSTM1 methylation was increased significantly in mouse liver,and GSTM1 gene expression was decreased.