Increased DNA damage and oxidative stress in patients with rheumatoid arthritis

Clinical Biochemistry 40 (2007) 167 – 171

Increased DNA damage and oxidative stress in patients with rheumatoid arthritis Ozlem Altindag a,⁎, Mehmet Karakoc b , Abdurrahim Kocyigit c , Hakim Celik c , Neslihan Soran a a

Harran University, Medical Faculty, Research Hospital, Department of Physical Medicine and Rehabilitation, TR-63100 Sanliurfa, Turkey b Ozel Yasam Physical Medicine and Rehabilitation Hospital, Aksaray, Turkey c Harran University Faculty of Medicine, Department of Biochemistry, Sanliurfa, Turkey Received 26 May 2006; received in revised form 3 October 2006; accepted 5 October 2006 Available online 19 October 2006

Abstract Objectives: Oxidative stress has been described as an important mechanism that underlies chronic inflammation in rheumatoid arthritis (RA). The aim of the study was to investigate the peripheral DNA damage, total antioxidant status (TAS), and total oxidative status (TOS) in patients with RA. Design and methods: The study population contained 25 patients with RA and 26 healthy controls. DNA damage was assessed by alkaline comet assay in peripheral lymphocyte, plasma levels of total antioxidant status (TAS) and total oxidative status (TOS) were determined, and OSI was calculated using a novel automated measurement method. Disease activity was evaluated by DAS-28 score. Results: In RA patients, DNA damage was significantly higher than in controls (20.0 ± 9.6 AU, 7.6 ± 4.3 AU; p < 0.001). Plasma TOS and OSI were higher in patients than in healthy controls (9.9 ± 2.6 vs. 7.3 ± 1.1, p < 0.001; 1.04 ± 0.4 vs. 0.7 ± 0.1, p < 0.001, respectively). Plasma TAS level in patients was lower than in healthy controls (0.9 ± 0.7 vs. 1.01 ± 0.7, p < 0.001). DNA damage was correlated with TOS, OSI, and DAS-28 scores (r = 0.682, p < 0.001; r = 0.753, p < 0.001; r = 0.519, p = 0.008, respectively). Conclusions: The findings indicated that lymphocyte DNA damage level increases in patients with RA. Elevated DNA damage may be related with increased oxidative stress and decreased antioxidant capacity. However, the mechanism of this association, and whether it is direct or indirect, remains to be explored. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Rheumatoid arthritis; Oxidative stress; DNA damage

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovial hyperplasia and progressive joint damage [1]. Although the pathophysiological basis of RA is not yet fully understood, reactive oxygen species (ROS) have been implicated in its pathogenesis [2]. ROS are highly reactive transient chemical species, such as nitric oxide, superoxide, and hydroxyl radical anion. Normal cellular metabolism appears to be a primary source for endogenous ROS, release of ROS from these cellular processes and their evasion from antioxidant pathways (e.g. glutathione peroxidase, vitamin E) result in the background levels of ⁎ Corresponding author. Fax: +90 414 3151181. E-mail address: [email protected] (O. Altindag).

modification of cellular molecules, including DNA, which can be detected in normal tissue. However, when the production of damaging ROS exceeds the capacity of the body's antioxidant defenses to detoxify them, a condition known as oxidative stress occurs [3,4]. Deoxyribonucleic acid (DNA) is a particular target for oxidation as damage may lead to important alterations [5]. ROS produced by activated neutrophils during the inflammatory response play an important role in the pathogenesis of inflammatory disease including hepatitis, gastritis, colitis, chronic renal failure, rheumatoid arthritis, and AS [6–10]. Chronic inflammation has long been recognized as being associated with increased risk for human cancer at various sites, such as gastritis for gastric cancer, chronic hepatitis for liver cancer, and inflammatory bowel disease for colorectal cancer [11].

0009-9120/$ - see front matter © 2006 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2006.10.006

168

O. Altindag et al. / Clinical Biochemistry 40 (2007) 167–171

The inflammatory nature of RA, particularly during periods of exacerbation, implies that a state of oxidative stress may exist in this disease [12]. It has been proposed that DNA damage induced by ROS may contribute to increased mutation rates, genome instability, apoptosis and associated tissue regeneration and cell proliferation [13]. Therefore, we compared to oxidative status, total antioxidant capacity and values of DNA damage in peripheral blood lymphocytes in patients with RA and healthy controls.

Lymphocyte separation An amount of 1 mL heparinized blood was carefully layered over 1 ml Lympoprep (Oslo Norway) and centrifuged for 35 min at 500×g and 25 °C. The interface band containing lymphocyte was washed with phosphate buffered saline (PBS) and then collected by 15 min centrifugation at 400×g. The resulting pellets were resuspended in PBS. Membrane integrity was assessed by means of Trypan Blue exclusion method.

Materials and methods Measurement of lymphocyte DNA damage This study was conducted at the Physical Medicine and Rehabilitation Outpatients Clinic of Harran University, Sanliurfa, South-eastern, Turkey. A consecutive sample of outpatients with joint complaints was screened for RA. In a 6-month period, among the 105 cases, referred for the first assessment, 53 did not meet inclusion criteria and 27 subjects refused to participate. The patients satisfied the 1987 revised American College of Rheumatology criteria for classification of RA. Two patients diagnosed with RA before 16 years of age were excluded from the study. Patients with arthritis due to other disease, such as gout, ankylosing spondylitis, Reiter's syndrome, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, Behçet's disease, and adult onset Still disease, neoplastic disease, established deficiency of vitamin B12 or folate and having been received any drugs were also excluded. None of the patients was smoker or consumed alcohol. RA patients were prospectively selected to have an active inflammatory status based on elevated levels of DAS-28 score. Out of 53 patients, 22 were smoker, 4 had infectious arthritis, 25 were undergoing treatment for RA, and 2 were pregnant. Twenty five RA patients (17 females, 8 males) who never took medical treatment were included in the study. Patients with disease of at least 6 months duration were recruited in this study. Informed consent was obtained from each RA patient. Control group was consisted of 26 healthy individuals (16 females, 10 males). The controls were recruited from the family of those in the patients group. Controls had no joint complaints and any rheumatological disease. Age and sex distributions in the group of control subjects were similar to those of RA patients. Informed consent was obtained from each control. Blood samples After an overnight fasting, venous blood was withdrawn into heparinized tubes and citrated tubes. One milliliter of heparinized blood was pipetted into another tube immediately to measure lymphocyte DNA damage. Remaining blood was centrifuged at 3000 rpm for 10 min to separate plasma. The plasma samples were stored at − 80°C until analysis of total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), and erythrocyte sedimentation rate (ESR) was measured from citrated blood.

Endogenous lymphocytes DNA damage was analyzed by alkaline comet assay according to Singh et al. [14] with minor modifications. Ten microliters of fresh lymphocyte cell suspension (around 20,000 cells) was mixed with 80 μl of 0.7% low-melting-point agarose (LMA) (Sigma) in PBS at 37°C. Subsequently, 80 μL of this mixture was layered onto slides that had previously been coated with 1.0% hot (60°C) normal melting point agarose (NMA) and covered with a coverslip at 4°C for at least 5 min to allow the agarose to solidify. After removing the coverslips, the slides were submersed in freshly prepared cold (4°C) lysing solution (2.5 M NaCl, 100 mM EDTA–2Na; 10 mM Tris–HCl, pH 10–10.5; 1% Triton X-100; and 10% DMSO added just before use) for at least 1 h. Slides were then immersed in freshly prepared alkaline electrophoresis buffer (0.3 mol/L NaOH and 1 mmol/L Na2ETDA, pH > 13) at 4°C for unwinding (40 min) and then electrophoresed (25 V/300 mA, 25 min). All of the above steps were conducted under red light or without direct light in order to prevent additional DNA damage. After electrophoresis, the slides were stained with ethidium bromide (2 μ/mL in distilled; 70 μL/slide), covered with a coverslip and analyzed using a fluorescence microscope (Nikon, Japan) vided with epi-flourescence and equipped with rhodamine filter (excitation wavelength, 546 nm; barrier filter, 580 nm). The images of 100 randomly chosen nuclei (50 cells from each of two replicate slides) were analyzed visually from each subject, as described elsewhere [15]. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of either of 0, 1, 2, 3, or 4 (from undamaged class 0 to maximally damaged class 4), so that the total scores of slide could be between 0 and 400 arbitrary units (AU). All procedures were completed by the same biochemistry staff, and DNA damage was detected by a single observer who was not aware of subject's diagnosis. Measurement of total antioxidant status Plasma TAS levels were determined using a novel automated measurement method, developed by Erel [16]. In this method, hydroxyl radical, which is the most potent radical, is produced via Fenton reaction. In the classical Fenton reaction, the hydroxyl radical is produced by mixing

O. Altindag et al. / Clinical Biochemistry 40 (2007) 167–171

of ferrous ion solution and hydrogen peroxide solution. In the most recently developed assay by Erel, same reaction is used. In the assay, ferrous ion solution, which is present in the Reagent 1, is mixed by hydrogen peroxide, which is present in the Reagent 2. The sequential produced radicals such as brown colored dianisidinyl radical cation, produced by the hydroxyl radical, are also potent radicals. In this assay, antioxidative effect of the sample against the potent free radical reactions, which is initiated by the produced hydroxyl radical, is measured. The assay has got excellent precision values, which are lower than 3%. The results are expressed as mmol Trolox equiv/l. Measurement of total oxidant status

Table 2 Comparative analysis of DNA damage and oxidative stress parameters in patients with rheumatoid arthritis and healthy subjects Parameters

Patients (n = 25)

Controls (n = 26)

p

DNA damage (arbitrary unit) TOS (μmol H2O2/L) TAS (mmol Trolox equiv/l) OSI (arbitrary unit) CRP (mg/L) ESR (mm/h)

20.0 ± 9.6 9.9 ± 2.6 0.9 ± 0.7 1.04 ± 0.4 2.1 ± 1.8 49.5 ± 14.3

7.6 ± 4.3 7.3 ± 1.1 1.01 ± 0.7 0.7 ± 0.1 1.7 ± 0.8 11.3 ± 3.2

<0.001 <0.01 <0.001 <0.001 0.3 <0.001

TOS: total oxidative status, TAS: total antioxidant status, OSI: oxidative stress Index, CRP: C-reactive protein, ESR: erythrocyte sedimentation rate. The values represent the mean ± SD. *Significance was defined as p < 0.05.

of 100 mm must be obtained. Using these data, the DAS-28 can be calculated using the following formula:

Plasma TOS levels were determined using a novel automated measurement method, developed by Erel [17]. In this method, oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (ìmol H2O2 equiv/l). Oxidative stress index The percent ratio of the TOS to the TAS gave the oxidative stress index (OSI), an indicator of the degree of oxidative stress [17]. To perform the calculation, the result unit of TAS, mmol Trolox equivalent/l, was converted to μmol equivalent/l, and the OSI value was calculated as the formula; OSI = [(TOS, μmol/l) / (TAS, mmol Trolox equivalent/l) × 100]. Disease activity For assessing disease activity, the disease activity score for 28-joint indices was calculated for each patient [18]. In order to calculate the DAS-28, information about some disease variables is needed. The number of swollen joints and tender joints should be assessed using 28-joint counts (tender28 and swollen28). The erythrocyte sedimentation rate (ESR) should be measured (Westerngren method, mm/hour). In addition, disease activity measured on a visual Analogue Scale (VAS) Table 1 Demographic data of patients with RA

Age (years) Gender (M/F) BMI (kg/m2)

169

Patients (n = 25)

Controls (n = 26)

p

37.9 ± 5.4 17/8 29.9 ± 3.3

36.8 ± 4.9 16/10 27.6 ± 3.8

>0.05 >0.05 >0.05

RA: rheumatoid arthritis, BMI: body mass index. The values represent the mean ± SD.

ðDAS28 ¼ 0:56  tender28 þ 0:28  swollen28 þ 0:70  ESR þ 0:014  VASÞ Statistical analysis Student's t test and Pearson's correlation analyses were performed by using the Statistical Package for Social Sciences (SPSS 11.5, SPSS Inc, Chicago, IL) and p ≤ 0.05 was considered statistically significant. Results Demographic and clinical data of patients with RA and controls are shown in Table 1. Mean ages of patients and controls were 37.9 ± 5.4 and 36.8 ± 4.9. There were no significant differences between two groups with respect to age, gender, and body mass index (BMI). As can be seen in Table 2, value of DNA damage was significantly higher in RA subjects than in healthy controls (20.0 ± 9.6 AU, 7.6 ± 4.3 AU; p < 0.001). Plasma TOS and OSI were higher in patients than in healthy controls (9.9 ± 2.6 vs. 7.3 ± 1.1, p < 0.001; 1.04 ± 0.4 vs. 0.7 ± 0.1, p < 0.001, respectively). Plasma TAS level in patients was lower than in healthy controls (0.9 ± 0.7 vs. 1.01 ± 0.7, p < 0.001). Mean scores of DAS-28 were 4.7 ± 4.1 in patients group. As can be seen in Table 3, the value of DNA damage showed significantly positive correlations with TOS, OSI, and DAS-28 (r = 0.682, p < 0.001, r = 0.753, p < 0.001, r = 0.519, p = 0.008, respectively). Discussion The results of the present study showed that DNA damage in peripheric blood lymphocytes was significantly Table 3 Correlations between DNA damage, TOS, and DAS-28 in patients Parameters

r

p⁎

DNA damage–DAS-28 DNA damage–TOS DNA damage–OSI

0.519 0.682 0.753

0.008 <0.001 <0.001

⁎ Significance was defined as p < 0.05.

170

O. Altindag et al. / Clinical Biochemistry 40 (2007) 167–171

higher in patients with RA than in healthy subjects. In addition, DNA damage was related to severity of the disease in patients. Oxidative damage to DNA has been associated with a number of pathologies including neoplastic, neurodegenerative, cardiovascular, and autoimmune diseases [5]. We found decreased TAS levels, increased TOS levels, and increased DNA damage in patients with RA. DNA damage was correlated with TOS levels. Therefore, it is thought that DNA damage may be related with insufficient antioxidant capacity and excessive ROS generation which contributed to pathogenesis of the disease in RA patients. Oxidative stress, which often arises as a result of an imbalance in the human oxidative/antioxidative status, has been implicated in aging and a number of diseases such as cancer, atherosclerosis, rheumatoid arthritis, osteoarthritis, fibromyalgia, and osteoporosis [19–21]. Reactive oxygen species is produced at the site of synovitis by macrophage and polymorphonuclear cells or by mechanical reperfusion and may contribute to the maintenance of inflammation through the activation of inflammatory molecules, leading to the destruction of articular cartilage in RA [22]. Jikimoto et al. [23] reported an association between disease activity and presence of oxidative stress in RA patients. It has been suggested that high level of DNA damage induced by oxidative stress was observed in human autoimmune diseases including RA [24]. Pro-inflammatory cytokines including tumor necrosis factorα, interleukin-1β, interleukin-6 and others have been shown to play pathologic roles in RA [25]. Enhance production of inflammatory cytokines induces various enzymes such as NADPH oxidase, nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase. These enzymes which produce free radicals may contribute to increased cancer risk in relation to oxidative DNA damage in inflammation [26]. Our findings indicated that (mean value of ESR was 48.5 ± 14.3 and DAS-28 score was 4.6 ± 4.1) the patients included in the study were in the active period of RA. There were important correlations between DNA damage, oxidative stress, and disease activity in patients with RA. Therefore, we suggest that chronic inflammation may cause increased oxidative stress and DNA damage in patients with RA. To our knowledge, this is the first report which demonstrates the association between DNA damage, oxidative stress, and the disease activity in RA patients. Malins and Haimanot [27] first reported an association of oxidized DNA bases with malignity. Subsequently, Shimoda et al. [28] reported that the levels of 8 hydroxy-2′-deoxyguanosine (8OHdG) as a marker of defective DNA repair in liver tissue with chronic inflammatory liver diseases are elevated compared with control liver. The DNA damage has been proposed as a major mechanism for the association of some chronic inflammatory conditions with cancers such as in the case of Helicobacter pylori infection with gastric cancer, and smoking with lung cancer [29]. It was reported that increased sensitivity to oxidative damage and an altered DNA repair capacity

increase the risk of many types of cancer such as liver, colon, lung, and breast cancers [11]. The mechanisms of carcinogenesis associated with inflammation have not been fully elucidated. It has been proposed that prolonged activation of inflammatory cells generates ROS that can damage DNA and contribute to carcinogenesis in chronic inflammatory disease [30]. In conclusion, our observations suggest that there is a link between the increased levels of oxidative stress and DNA damage seen in patients with RA, as compared with those seen in healthy controls. However, the nature of this link, and whether it is direct or indirect, remains to be explored. References [1] Firestein GS, Echeverri F, Yeo M, Zvaifler NJ, Green DR. Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proc Natl Acad Sci U S A 1997;94:10895–900. [2] Biemond P, Swaak AJ, Koster JF. Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid. Arthritis Rheum 1984;27:760–5. [3] Sarban S, Kocyigit A, Yazar M, Isikan UE. Plasma total antioxidant capacity, lipid peroxidation, and erythrocyte antioxidant enzyme activities in patients with rheumatoid arthritis and osteoarthritis. Clin Biochem 2005;38:981–6. [4] Harma M, Harma M, Erel O. Increased oxidative stress in patients with hydatidiform mole. Swiss Med Wkly 2003;133:563–6. [5] Cooke MS, Olinski R, Evans MD. Does measurement of oxidative damage to DNA have clinical significance? Clin Chim Acta 2006;365:30–49. [6] Karakucuk S, Baskol G, Oner AO, Baskol M, Mirza E, Ustdal M. Serum paraoxonase activity is decreased in the active stage of Behcet's disease. Br J Ophthalmol 2004;88:1256–8. [7] Tak PP, Zvaifler NJ, Green DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today 2000;21:78–82. [8] Lunec J, Halloran SP, White AG, Dormandy TL. Free-radical oxidation (peroxidation) products in serum and synovial fluid in rheumatoid arthritis. J Rheumatol 1981;8:233–45. [9] Blake DR, Merry P, Unsworth J, et al. Hypoxic–reperfusion injury in the inflamed human joint. Lancet 1989;11:289–93. [10] Ho KJ, Chen PQ, Chang CY, Lu FJ. The oxidative metabolism of circulating phagocytes in ankylosing spondylitis: determination by whole blood chemiluminescence. Ann Rheum Dis 2000;59:338–41. [11] Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994;305:253–64. [12] Remans PH, van Oosterhout M, Smeets TJ, Sanders M, Frederiks WM, Reedquist KA. Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis. Arthritis Rheum 2005;52:2003–9. [13] Sawa T, Ohshima H. Nitrative DNA damage in inflammation and its possible role in carcinogenesis. Nitric Oxide 2006;14:91–100. [14] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91. [15] Lampids TJ, Kurtoglu M, Maher JC, et al. Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing “hypoxic tumor cells". Cancer Chemother Pharmacol 2006;58:725–34. [16] Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 2004 (Apr.);37(4):277–85. [17] Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38:1103–11. [18] Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-

O. Altindag et al. / Clinical Biochemistry 40 (2007) 167–171

[19]

[20]

[21] [22]

[23]

[24]

eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995;38:44–8. Demirbag R, Yilmaz R, Erel O, Gultekin U, Asci D, Elbasan Z. The relationship between potency of oxidative stress and severity of dilated cardiomyopathy. Can J Cardiol 2005;21:851–5. Ozgocmen S, Ozyurt H, Sogut S, Akyol O. Current concepts in the pathophysiology of fibromyalgia: the potential role of oxidative stress and nitric oxide. Rheumatol Int 2006;26:585–97. Henrotin Y, Kurz B, Aigner T. Oxygen and reactive oxygen species in cartilage degradation: friends or foes? Osteoarthr Cartil 2005;13:643–54. Winrow VR, Winyard PG, Morris CJ, Blake DR. Free radicals in inflammation: second messengers and mediators of tissue destruction. Br Med Bull 1993;49:506–22. Jikimoto T, Nishikubo Y, Koshiba M, et al. Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. Mol Immunol 2002;38:765–72. Bashir S, Harris G, Denman M, Blake DR, Winyard PG. Oxidative DNA

[25] [26]

[27] [28]

[29]

[30]

171

damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann Rheum Dis 1993;52:659–66. Nishimoto N. Interleukin-6 in rheumatoid arthritis. Curr Opin Rheumatol 2006;18:277–81. Kocyigit A, Keles H, Selek S, Guzel S, Celik H, Erel O. Increased DNA damage and oxidative stress in patients with cutaneous leishmaniasis. Mutat Res 2005;585:71–8. Malins DC, Haimanot R. Major alterations in the nucleotide structure of DNA in cancer of the female breast. Cancer Res 1991;51:5430. Shimoda R, Nagashima M, Sakamoto M, et al. Increased formation of oxidative DNA damage, 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis. Cancer Res 1994;54:3171–2. Pignatelli B, Bancel B, Plummer M, Toyokuni S, Patricot LM, Ohshima H. Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa. Am J Gastroenterol 2001;96:1758–66. Ekstrom K, Hjalgrim H, Brandt L, et al. Risk of malignant lymphomas in patients with rheumatoid arthritis and in their first-degree relatives. Arthritis Rheum 2003;48:963–70.