combined genetic variants 8oxodG (nmol/mmol creatinine) The role of functional variants of phase I and II genes and their contribution to oxidative stress in Serbian patients with COPD Marija Stankovic*, Aleksandra Nikolic*, Djordje Francuski*, Aleksandra Topic**, Marija Mitic-Milikic***, Ljudmila Nagorni-Obradovic***, Natasa Petrovic-Stanojevic****, Marina Andjelic-Jelic*****, Dragica Radojkovic* *Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia; **Faculty of Pharmacy, University of Belgrade, Department of Medical Biochemistry, Serbia; ***Institute for Tuberculosis and Lung Disease, University Clinical Center of Serbia, Serbia; University Clinical Center Zvezdara, **** Department of Pulmonology, *****Department of Endocrinology, Serbia Introduction Methods Chronic obstructive pulmonary disease (COPD) is a complex disorder influenced by multiple genetic and environmental factors, as well their interactions. Increased oxidative stress is a prominent feature of COPD. Oxidants from cigarette smoke and air pollutants are processed by phase I, cytochrome P450 system (CYPs), and phase II, glutathione S-transferases (GSTs) and microsomal epoxide hydrolase (mEH) enzymes. Hence, functional variants of genes of phase I and II might be involved in COPD pathogenesis. Urinary 8-oxo-7,8-dihydro-2’-deoxyguanosine (8oxodG) is sensitive, noninvasive biomarker of systemic oxidative stress, that could be influenced by functional variants of phase I and II genes. The patients and controls (Table 1) genotypes were determined by PCR-RFLP for CYP1A1 *1A/*2A, CYP2E1 *1A/*5B, GSTP1 Ile105Val, mEPHX slow (S) and fast (F), and by multiplex PCR for GSTM1 null and GSTT1 null variants. The distribution of alleles, single and combined genotypes, among groups, was compared using χ2-test, Odds Ratios (ORs) and 95% Confidence Intervals (95%CIs) were calculated using SPSS software. The 8-oxodG was determined using ultra performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Urinary creatinine was measured by the kinetic Jaffé method. Table 1. Characteristics of patients and controls. COPD Controls Subjects (n) 122 100 Age (years) 44.96±1.56 50.9±1.37 Sex, male (%) 70 35 Smokers (%) 50 Smoking history (pack-years) 36.7±2.0 26.3±2.3 FEV1 (% pred) 45.3±2.3 110.1±1.7 FEV1/FVC 62.4±2.0 98.3±0.9 Aim To investigate the role of single and combined functional variants of CYP1A1, CYP2E1, GSTM1, GSTT1, GSTP1 and mEH genes in COPD pathogenesis in Serbian population. To measure the level of 8oxodG, depending on the genotype, in available COPD patients. Results The frequency of GSTM1 homozygote deletion was significantly increased in the patients with COPD (Table 2). According to analysis of combined genotypes the frequency of GSTM1 null and GSTP1 Val/(Val) alleles, as well as CYP1A1 *1A/*2A, GSTM1 null and mEH S/(S) combinations were significantly higher in COPD group (Table 3). Table 2. The allelic and genotypes distributions in patients and controls. gene variant genotype COPD n=122 (%) controls n=100 (%) OR (95%CI) CYP1A1 T3801C *1A/*1A 97 (79.5) 83 (83.0) 1 *1A/*2A 24 (19.7) 17 (17.0) 1.2 (0.6-2.4) *2A/*2A 1 (0.8) 0 (0.0) *2A 0.107 0.085 1.3 (0.7-2.4) CYP2E1 PstI/RsaI 115 (94.3) 96 (96.0) *1A/*5B 7 (5.7) 3 (3.0) 1.9 (0.5-7.7) *5B/*5B 1 (1.0) *5B 0.029 0.025 2.4 (0.8-6.7) GSTM1 deletion + 47 (38.5) 53 (53.0) null 75 (61.5) 47 (47.0) 1.8 (1.1-3.1)* GSTT1 95 (77.9) 76 (76.0) 27 (22.1) 24 (24.0) 0.9 (0.5-1.7) GSTP1 Ile105Val Ile/Ile 48 (39.3) 45 (45.0) Ile/Val 59 (48.4) 46 (46.0) 1.2 (0.7-2.1) Val/Val 15 (12.3) 9 (9.0) 1.6 (0.6-3.9) Val 0.365 0.320 1.2 (0.8-1.8) EPOE3 Tyr113His (S) wt / wt 58 (47.5) 48 (48.0) S / wt 48 (39.4) 40 (40.0) 1.0 (0.6-1.8) S / S 16 (13.1) 12 (12.0) 1.1 (0.5-2.6) S 0.328 1.0 (0.7-1.5) EPOE4 His139Arg (F) 82 (67.2) 66 (66.0) F / wt 38 (31.1) 29 (29.0) 1.1 (0.6-1.9) F / F 2 (1.7) 5 (5.0) 0.3 (0.1-1.7) F 0.172 0.195 0.9 (0.6-1.5) Table 3. The distribution of combined genotypes. combined genes genotype COPD n=122 (%) controls n=100 (%) OR (95%CI) GSTM1, GSTP1 other 75 (61.5) 76 (76.0) 1 null , Val/(Val) 47 (38.5) 24 (24.0) 2.0 (1.1-3.6)* CYP1A1,GSTM1 and mEH 113 (92.6) 99 (99.0) *2A, null and S/(S) 9 (7.4) 1 (1.0) 7.9 (1.0-63.4)** *p=0.03; **p=0.025 In the group of COPD totally nine patients with CYP1A1 *1A/*2A, GSTM1 null and mEH S/(S) genotype and eighteen patients with CYP1A1 *1A/*1A, GSTM1 + and mEH wt/wt genotype were identified, but only one from each of the genotype cohort was available for urinary 8-oxodG measurement. Obtained results showed that COPD patient, carrier of CYP1A1 *1A/*2A, GSTM1 null and mEH S/(S) genotype had greater level of 8oxodG, than patient carrier of wild-type alleles, Table 4. Table 4. Urinary 8oxodG in COPD patients with specific genotype. combined genetic variants 8oxodG (nmol/mmol creatinine) CYP1A1 *1A/*2A, GSTM1 null and EPO S/(S) 1.61 CYP1A1 *1A/*1A, GSTM1 + and EPO wt/wt 0.74 *p=0.04 Conclusion One single genotype, GSTM1 null, and two combined genotypes of GSTM1 and GSTP1, and CYP1A1, GSTM1 and mEH variants, were associated with COPD in Serbian population. The combination CYP1A1 *1A/*2A, GSTM1 null and mEH S/(S), includes alleles connected with hyperactivity of phase I and deficiency/decreased activity of phase II genes which could lead to increased oxidative stress. The 8oxodG level was about two folds higher in the carrier of CYP1A1 *1A/*2A, GSTM1 null and mEH S/(S) genotypes than in the carrier of wild-type alleles, indicating its possible contribution to increased oxidative stress in COPD development. Determining the level of 8oxodG, as a systemic biomarker of oxidative stress, might be useful in COPD prevention, as well antioxidant therapy monitoring.