The Influence Of G6PD On DNA Damage And Methylation Induced By Benzene

Benzene is a common environmental pollutant and widely used in industrial production.The IRAC identified benzene as a human carcinogen in 1982..The toxic mechanism of benzene is mainly related to oxidative damage,gene mutation and chromosomal aberration caused by oxidative metabolites.Glucose-6-Phosphate dehydrogenase(G6PD)deficiency is a red blood cell hemolytic disease caused by a mutation in the G6 PD gene,resulting in a decrease in its own enzyme activity.G6 PD is the initial enzyme of the pentose phosphate pathway,which can protect cells from reactive oxygen species(ROS)damage by catalyzing the synthesis of NADPH and maintaining the reductive nature of glutathione.Our previously study found that G6 PD deficiency could increase the oxidative damage and cytotoxicity induced by benzoquinone exposure in K562 cels.This study will further investigate the effect of G6 PD deficiency on genetic damage and methylation induced by benzene.To study the regulation of G6 PD on benzene-induced genetic damage in mice and its effect,the G6PD-deficiency animal model was used to detect the changes of oxidative stress,genetic damage level,DNA methylation and methyltransferase production in mice after benzene treatment.Furthermore,the epidemiological study of benzene exposed population was used to verify the effects of G6 PD deficiency on benzene-induced genetic damage and methylation changes.1.Study on genetic damage of bone marrow cells induced by benzene in mice induced by G6 PD deficiencyFirstly,the G6PD-deficiency animal model was constructed,and the mouse genotype was identified by sequencing.G6 PD low-expression C3H/He mice and G6 PD normal C3H/He mice were exposed to subcutaneous injection of benzene at a concentration of 150 mg/(kg.d).After the end of the treatment,the visceral coefficient,NADPH/NADP,GSH/GSSG and MDA content were analyzed,and the DNA damage,telomere length,telomerase activity and DNA repair enzyme gene OGG1 and XRCC1 gene expression were detected to study the regulatio n of G6 PD.Effects of benzene on genetic damage in mouse bone marrow cels.The sequencing results showed that G6PD-deficient mice contained CTCAGG-deficie nt sequences,and C3H-deficient mice contained normal CTCTGG sequences,and the G6 PDdeficient animal model was successfully constructed.After exposure,the renal-body ratio of G6PD-deficient mice was lower than that of normal mice(P<0.05),and the ratio of spleen to thymus was higher than that of normal mice(P<0.05).After treatment with benzene,the GSH content and GSH/GSSG ratio of G6PD-deficient mice were lower than those of normal mice(P<0.05),and the MDA content was higher than that of normal mice(P<0.05).The results of comet assay showed that the tail DNA% and Oliver tail moment of G6PD-deficient mice exposed to benzene were significantly higher than those of normal mice(P<0.05).The telomere test results showed that the telomere length and telomerase activity of G6PD-deficient mice were lower than those of normal mice(P<0.05).The results of RT-PCR showed that the relative expression of OGG1 and XRCC1 mRNA in G6PD-deficient mice was lower than that in normal mice(P<0.05).The above results indicate that G6PD-deficient mice may not be sufficient to maintain the reduced form of NADPH when benzene is exposed,resulting in sufficient GSH,so the oxidative damage of G6PD-deficient mice is aggravated and the MDA content is increased.Excessive accumulation of oxidation products leads to DNA damage,shortens telomeres,inhibits DNA repair genes and telomerase production,and further deepens genetic damage in G6PD-deficie nt mice.2.G6 PD Defects on DNA Methylation of Bone Marrow Cells Induced by BenzeneFirst,a G6PD-deficient animal model was constructed,and the mouse genotype was identified by sequencing.G6 PD low-expression C3H/He mice and G6 PD normal C3H/He mice were exposed to subcutaneous injection of benzene at a concentration of 150 mg/(kg.d).At the end of the treatment,the DNA methylation level of the mice was detected by colorimetry.The relative expression of DNMT1,DNMT3 a and DNMT3 b mRNA was detected by RT-PCR.The effect of G6 PD on the methylation changes induced by benzene in mice was studied.The experimental results showed that the relative level of DNA methylation in G6 PDdeficient mice was lower than that in normal mice after benzene exposure(P<0.05).The relative expression levels of DNMT1,DNMT3 a and DNMT3 b in G6PD-deficient mice were lower than those in normal mice(P<0.05).In summary,after exposure to benzene,G6 PD defects cause higher levels of oxidative stress,inhibit the formation of DNMTs,and fail to maintain the original methylation level.3.Effect of G6 PD expression level on DNA repair enzyme expression and telomere length in benzene exposed populationIn this study,the occupational epidemiological survey method was used to select the benzene exposed workshop workers in Yangzhou City as the research object,conduct physical examination and questionnaire survey,and detect the telomere length,G6 PD,hOGG1 and XRCC1 gene relative expression by RT-PCR.The activity of G6 PD was detected by colorimetric assay to investigate the effect of G6 PD expression on DNA repair enzyme expression and telomere length in benzene exposed population.The results showed that the relative expression levels of hOGG1 and XRCC1 genes were different in the relative expression level of G6 PD gene.Linear regression analysis showed that G6 PD mRNA expression level was one of the influencing factors of DNA repair enzymes hOGG1 and XRCC1 expression.It is speculated that after exposure to benzene,the G6 PD deficiency leads to a decrease in the antioxidant system,resulting in insufficient production of DNA repair enzymes hOGG1 and XRCC1,and increased genetic damage.Furthermore,the effect of G6 PD expression level on DNA methylation in benzene exposed population was investigated.The DNA methylation level of benzene exposed population was detected by colorimetry,and the expression level of DNMTs was detected by RT-PCR.The relative expression levels of DNMT1,DNMT3 a and DNMT3 b genes were different in the distribution of G6 PD expression levels(P<0.05).There was no difference in the distribution of DNA methylation levels in the expression level of G6PD(P>0.05).It is speculated that after workers exposed to benzene,oxidative stress occurs in the body,and people with G6 PD deficiency cannot metabolize excess ROS,and finally inhibit the formation of DNMTs.The expression level of G6 PD has an effect on the regulation of benzene-induced DNA methylatio n,but it is still necessary to expand the sample size to study.