Study On The Roles Of DNA Adducts And Genomic Polymorphisms Of Associated Repair Genes In The Pathogenesis Of COPD

Chronic obstructive pulmonary disease (COPD) is a major and increasing cause ofmorbidity and mortality worldwide and its prevalence is still increasing. At the present time,there is no specific and effective treatment for COPD, because the pathogenesis is still notclear. Cigarette smoke is a major risk factor for COPD and lung cancer, and almost 90% ofCOPD and lung cancer patients are smokers. The present study found DNA adducts-thetoxic components combined with DNA, which could cause the gene mutation, deletion andmismatching, caused the lung cancer eventually. However, whether DNA adducts isassociated with COPD and which DNA adducts is associated with COPD is still notstudied.Now, there is ample evidence that oxidative stress plays a major role in COPD.Cigarette smoke is a rich source of reactive oxygen species (ROS), which can attack DNAand cause DNA damage. Among the various forms of DNA damage induced by oxygenradicals, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a major form of oxidative DNAdamage and an important marker of cellular oxidative stress. If 8-OHdG is not effectivelyrepaired, it will cause G:C→T:A transversion. Some researches found the 8-OHdG level inlung cancer tissue was higher than non-lung cancer tissue. However, whether 8-OHdG isassociated with COPD is still not studied.There are many DNA repair pathways to maintain the stabilization of gene. Human8-oxoguanine DNA glycosylase (hOGG1), a major component in the DNA repair pathways,which can excise 8-OHdG specifically and avoid G:C→T:A transversion, plays a majorrole in gene stability. The results about the relationship between the expression of hOGG1and 8-OHdG were inconsistent, In addition, the substitution of cysteine for serine at codon326 (Ser326Cys) was associated with significant reduction in the repair capacity. X-rayrepair cross complementing group 1 (XRCC1), is another major DNA repair gene, whichinteracts with hOGG1 directly. So far, more than 60 single nucleotide polymorphisms werefound. The most studied were 26304C→T,27466G→A and 28152G→A, which caused the corresponding amino acid transition: 194 Arg→Trp,280 Arg→His and 399Arg→Gln.Although smoking is the most important risk factor, not all smokers develop COPD, whichsuggests that there is a genetic susceptibility to the development of COPD. Whether thepolymorphisms of hOGG1 and XRCC1 are associated with COPD is not studied.The present study consisted of two parts. One part investigated the relationshipbetween 8-OHdG and COPD: section one investigated the association between 8-OHdGlevel in lymphocyte and the 8-OHdG level in lung tissue; section two investigated therelationship among cigarette smoke,8-OHdG and COPD. The other part investigated therelationship between the polymorphisms of hOGG1 and XRCC1 and COPD.Partâ… Study on 8-OHdG and COPD related with cigarette smokeSection 1 The association between 8-OHdG level in lymphocyte and the 8-OHdG levelin lung tissue【Objective】To investigate the association between 8-OHdG level in lymphocyte and the8-OHdG level in lung tissue.【Methods】8-OHdG was detected by high press liquid chromatography withelectrochemical detection in peripheral blood lymphocyte and lung tissue of 8 COPD casesand 8 controls.【Results】(1) The level of 8-OHdG in lymphocyte was significantly higher in COPD cases(7.25±2.29/10⁵dG) than in controls (3.30±1.94/10⁵dG), (P＜0.05).(2) The level of 8-OHdG in lung tissue was significantly higher in COPD cases(8.15±2.19/10⁵dG) than in controls (3.19±1.51/10⁵dG), (P＜0.05).(3) In COPD group, the level of 8-OHdG in lymphocyte was positively correlated with thelevel of 8-OHdG in lung tissue (r=0.768, P＜0.05); and in control, the level of 8-OHdGin lymphocyte was also positively correlated with the level of 8-OHdG in lung tissue(r=0.787, P＜0.05).【Conclusions】The level of 8-OHdG in lymphocyte and lung tissue of COPD cases washigher than the level of 8-OHdG in lymphocyte and lung tissue of controls. And in COPD cases and controls, the level of 8-OHdG in lymphocyte was significantly associated withthe level of 8-OHdG in lung tissue, which suggested that 8-OHdG in lymphocyte could beused to investigate the relationship between cigarette smoke and COPD.【Keywords】COPD, 8-OHdG, lymphocyte, lung tissueSection 2 The relationship among cigarette smoking,8-OHdG and COPD【Objective】To investigate the relationship among cigarette smoking,8-OHdG inlymphocyte and COPD and the influence of ROS,hOGG1 mRNA and hOGG1 protein on8-OHdG level.【Methods】A total 140 healthy non-smokers, 143 healthy smokers and 129 smokers withCOPD were recruited. 8-OHdG was detected by high press liquid chromatography withelectrochemical detection; DCFH-DA was used to detect ROS level; Real-time PCR wasused to detect the expression of hOGG1 mRNA; Western Blot was used to detect theexpression of hOGG1 protein.【Results】(1) The level of 8-OHdG in lymphocyte was significantly higher in healthy smokers andsmokers with COPD than in healthy nonsmokers (P＜0.05); and the level of 8-OHdG inlymphocyte was significantly higher in smokers with COPD than in healthy smokers.(2) The level of ROS,hOGG1 mRNA and hOGG1 protein in lymphocyte was significantlyhigher in healthy smokers and smokers with COPD than in healthy nonsmokers(P＜0.05); the level of ROS in lymphocyte was significantly higher in smokers withCOPD than in healthy smokers (P＜0.05); the expression of hOGG1 mRNA and hOGG1protein in lymphocyte was significantly lower in smokers with COPD than in healthysmokers (P＜0.05).(3) In healthy smokers and smokers with COPD, the level of 8-OHdG in lymphocyte waspositively correlated with smoking index (P＜0.05); in all three groups, the level of8-OHdG in lymphocyte was positively correlated with ROS level (P＜0.05), andnegatively correlated with the expression of hOGG1 mRNA (P＜0.05) and hOGG1protein (P＜0.05).(4) In healthy nonsmokers and healthy smokers, the level of 8-OHdG in lymphocyte was not significantly correlated with FEV₁% predicted and FEV₁/FVC (P＞0.05); however,in smokers with COPD, the level of 8-OHdG in lymphocyte was significantlynegatively correlated with FEV₁% predicted and FEV₁/FVC (P＜0.05).【Conclusions】The level of 8-OHdG in lymphocyte of smokers with COPD was higherthan the level of 8-OHdG in lymphocyte of healthy nonsmokers and healthy smokers,which suggested that the elevation was associated with COPD caused by cigarette smoke.Partâ…¡: The relationship between the polymorphisms of hOGG1 and XRCC1 and therisk of COPD【Objective】To investigate the relationship between the polymorphisms of hOGG1Ser326Cys,XRCC1 Arg194Trp,Arg280His and Arg399Gln and the risk of COPD.【Methods】A total 201 COPD cases and 309 controls were recruited, hOGG1 genotypeand XRCC1 genotype were determined by PCR-restriction fragment length polymorphismanalysis.【Results】(1) Compared with those with the hOGG1 Ser326Ser genotype, the risk for COPD was notsignificantly different among individuals with Ser326Cys genotype and Cys326Cysgenotype. Compared with those with the XRCC1 Arg399Arg genotype, the risk forCOPD was not significantly different among individuals with Gln399Gln genotype, butthe risk for COPD was significantly elevated among individuals with Arg399Glngenotype (adjusted odds ratios (OR)=1.55, 95% confidence intervals (CI) 1.05-2.29,P＜0.05). After stratifying by smoking status, in current smokers, compared with thosewith the hOGG1 Ser326Ser genotype, the risk for COPD was significantly elevatedamong individuals with Cys326Cys genotype (adjusted OR=5.07, 95% CI 1.84-13.95,P＜0.05); compared with those with the XRCC1 Arg399Arg genotype, the risk forCOPD was significantly elevated among individuals with Arg399Gln genotype(adjusted OR=2.77, 95% CI 1.52-5.07, P＜0.05). After stratifying by smoking exposure,in light smokers, compared with those with the hOGG1 Ser326Ser genotype, the riskfor COPD was significantly elevated among individuals with Cys326Cys genotype (adjusted OR=4.02, 95% CI 1.05-16.80, P＜0.05); compared with those with the XRCC1Arg399Arg genotype, the risk for COPD was significantly elevated among individualswith Gln/Gln genotype (adjusted OR=4.48, 95% CI 1.35-14.90, P＜0.05). In heavysmokers, compared with those with the XRCC1 Arg399Arg genotype, the risk forCOPD was significantly elevated among individuals with Arg399Gln genotype(adjusted OR=2.55, 95% CI 1.42-4.58, P＜0.05).(2) Cmpared with those with the XRCC1 Arg194Arg genotype, the risk for COPD was notsignificantly different among individuals with Trp194Trp genotype. However, afterstratifying by smoking status, in former smokers, compared with those with the XRCC1Arg194Arg genotype, the risk for COPD was significantly reduced among individualswith Trp194Trp genotype (adjusted OR=0.22, 95% CI 0.06-0.85, P＜0.05); afterstratifying by smoking exposure, in light smokers, compared with those with theXRCC1 Arg194Arg genotype, the risk for COPD was significantly reduced amongindividuals with Arg194Trp genotype and Trp194Trp genotype (adjusted OR=0.39,95% CI 0.16-0.94, P＜0.05; 0.24, 95% CI 0.07-0.79, P＜0.05, respectively).(3) There was no significant deviation in three XRCC1 Arg280His genotypic frequenciesbetween the COPD patient group and the control group (P＞0.05). And after stratifyingby smoking status and smoking exposure, there was also no significant deviation inthree XRCC1 Arg280His genotypic frequencies between the COPD patient group andthe control group (P＞0.05).【Conclusions】The risk for COPD is significantly elevated among current/light smokerswith hOGG1 326Cys and XRCC1 399Gln; XRCC1 194Trp genotype is associated with ared uced risk of developing COPD among former and light smokers; XRCC1 280His wasnot significantly associated with the risk of COPD caused by cigarette smoke.