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Comet assay and analysis of micronucleus formation in patients with rheumatoid arthritis
Mutation Research 721 (2011) 1–5
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Comet assay and analysis of micronucleus formation in patients with rheumatoid arthritis Ali Karaman a,∗ , Do˘gan Nasır Binici b , Meltem Alkan Meliko˘glu c a b c
Department of Medical Genetics, Erzurum Nenehatun Obstetrics and Gynecology Hospital, Erzurum, Turkey Department of Internal Medicine, Erzurum Training and Research Hospital, Erzurum, Turkey Department of Romatology, Erzurum Training and Research Hospital, Erzurum, Turkey
a r t i c l e
i n f o
Article history: Received 27 July 2010 Received in revised form 5 October 2010 Accepted 5 November 2010 Available online 20 January 2011 Keywords: Rheumatoid arthritis Comet assay Micronucleus test Pathogenesis
a b s t r a c t Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of rheumatoid arthritis (RA). Genetic factors that predispose individuals to RA are considered to play an important role in the development of the disease. The aim of this study was to determine, by use of the comet assay and the micronucleus (MN) test, whether DNA damage has an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and DNA damage in RA. This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. We analyzed lymphocytes from patients with RA (12 in active and 31 in inactive periods) and 30 healthy controls for effects in the comet assay and the MN test. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), the erythrocyte sedimentation rate (ESR) and the high-sensitivity C-reactive protein (hs-CRP) rate were determined in all the subjects. The comet-tail length, the MN frequencies and the MDA levels were significantly higher in patients – both in the active and the inactive period – than in the controls. In contrast, the SOD and GSH-Px levels were significantly lower in both patient groups than in the controls. Our results suggest that an increased plasma MDA level and decreased plasma GSH-Px and SOD levels reflect the higher degree of oxidative stress in RA patients, a situation that may impair genetic stability in those patients. Thus, the results suggest that increased DNA damage may play an important role in the pathogenesis of RA. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune syndrome, which produces degradation of articular cartilage and bone erosion. The long-term outcomes of this progressive disease are significant morbidity, loss of functional capacity, and increased mortality [1]. RA affects 1–2% of the general population worldwide [2], and the occurrence in women is three times higher than in men. Although the onset of RA can occur at any age, the incidence increases with age. The aetiology of RA is basically unknown, but several studies have implicated a combination of a genetic background and environmental factors, such as infections and smoking, leading to defects in immunoregulation and a host of inflammatory mechanisms involved in joint-tissue damage, including a role for oxidative stress [3]. The formation and scavenging activity of free radicals in biological systems have been closely linked to a number of pathological
∗ Corresponding author. Tel.: +90 442 317 2295; fax: +90 442 317 2294. E-mail address: [email protected] (A. Karaman). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.11.014
conditions. In healthy individuals, reactive oxygen species (ROS) and associated oxidative stress factors are kept in check by a combination of antioxidant activities [4,5]. There is increasing evidence that ROS and the resulting pro-oxidant/antioxidant imbalance play a major role in RA, as well as in other disease states [6,7]. The comet assay is a fast, simple and sensitive method for the quantification of genetic damage in a small number of cells [8–11]. For these reasons, the comet assay has been used to evaluate DNA damage induced by physical and chemical agents in numerous studies involving environmental monitoring and in medical research [9]. A micronucleus (MN) is an acentric chromosome fragment or a whole chromosome that is left behind during mitotic cell division, and it appears in the cytoplasm of interphase cells as a small additional nucleus [12]. The MN test, widely accepted for in vitro and in vivo genotoxicity investigations, provides a sensitive marker of genomic damage [13]. Living organisms contain specific enzymes catalysing the breakdown of the superoxide radical and hydrogen peroxide, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) [14]. Malondialdehyde (MDA) can directly or indirectly affect many functions integral to cellular and organ
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A. Karaman et al. / Mutation Research 721 (2011) 1–5
Table 1 Demographic details of the patients with rheumatoid arthritis and controls.
ing at least 45 min. The study was approved by the hospital’s Ethical Committee. The study subjects were non-smokers and non-alcoholics, without a history of viral infection or any systemic disease. None of the subjects had received antibiotics, systemic steroids and mineral or vitamin drugs during the past 2 months. All patients were analyzed prior to therapy.
Sex (female/ male)
Age, years (mean ± SD), range
Disease duration, years (mean ± SD), range
Patients (n = 43)
32/11
4.25 ± 2.8; 1–22
2.2. Comet assay
Controls (n = 30)
17/13
39.84 ± 8.32; 25–66 37.21 ± 6.83; 20–61
–
Forty microlitres of blood were taken from the collected samples for the comet assay, which was performed under alkaline conditions, as described by Singh et al. [22], with modifications. Cell viability, determined by the trypan-blue exclusion technique, ranged from 94 to 96% (data not shown). Slides were prepared in duplicate for each subject. Analysis was performed with a 400× objective on an Olympus BX 51 fluorescence microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Slides were randomized and coded to blind the scorer. All slides were scored by one person, to avoid inter-scorer variability. A total of 100 individual cells were screened per subject (50 cells from each slide). Undamaged cells resemble an intact nucleus without a tail and damaged cells have the appearance of a comet. The length of the DNA that had migrated in the comet tail, which is an estimate of DNA damage, was measured with an ocular meter and calculated as comet tail length (m) = (maximum total length) − (head diameter).
homeostasis. The specific determination of MDA is useful for revealing the extent of lipid damage, as a result of oxidative stress, in a variety of lipid systems, such as plasma, organs and cell membranes [15,16]. The increased membrane lipid-peroxidation may evoke immune and inflammatory responses, active gene expression and cell proliferation, or initiate apoptosis. Thus, a close relationship between ROS production, impairment of the antioxidant defense, peroxidative membrane damage and inflammatory or degenerative pathological processes could be postulated [17]. Despite its important physiological role, an unbalanced redox status may present potentially destructive effects on the biology of the cell [18,19]. Currently, the use of biomarkers of oxidative stress can help explore the relation between oxidative damage to macromolecules (DNA, lipids and proteins) and several diseases. Evaluation, both in vivo and ex vivo, includes measurements of DNA oxidation, lipid peroxidation and protein oxidation [20]. The aim of this study was to determine, by use of the comet assay and the MN test, whether genetic lesions and DNA damage have an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and genomic damage in RA. 2. Materials and methods 2.1. Patients and specimen collection This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. Forty-three patients with diagnosed RA and 30 healthy controls were studied. The demographic information of all study subjects is presented in Table 1. We performed the comet assay and the MN test on blood samples from all patients and controls. The patients were divided into two groups, as active (n = 12, 10 females and 2 males) and inactive (n = 31, 23 females and 8 males), based on the severity of their disease identified on the basis of clinical and laboratory findings. Blood samples were obtained by venous puncture and lymphocytes were collected by standard methods. The samples were transported on ice to the laboratory and were processed within 2 h. The blood samples were obtained with informed consent from the healthy volunteers and from the RA patients. The latter group fulfilled the 1987 criteria for RA by the American Rheumatism Association and were in functional Class I, II, or III (Table 2), according to the revised criteria of the American College of Rheumatology [21]. Patients with an active disease duration of at least 6 months, as manifested by at least three joints that were swollen and six joints that were tender at the time of the blood donation, were accepted for the study. In addition, RA patients had an erythrocyte sedimentation rate (ESR) ≥28 mm/h, a high-sensitivity C-reactive protein (hs-CRP) ≥2.4 mg/l, or morning stiffness dur-
Table 2 American college of rheumatology revised criteria for classification of functional status in RAa . Class I Class II Class III Class IV
Completely able to perform usual activities of daily living (self-care, vocational, and avocational) Able to perform usual self-care and vocational activities but limited in avocational activities Able to perform usual self-care activities but limited in vocational and avocational activities Limited in ability to perform usual self-care, vocational, and avocational activities
2.3. Micronucleus (MN) analysis For MN analysis, 2 ml of heparinized blood was drawn from each individual. Lymphocyte cultures were established by adding 0.5 ml of whole blood to 5 ml karyotyping medium (Biological Industries, Beit Haemek, Israel) with 2% phytohaemagglutinin M (Biological Industries) according to standard techniques [23]. The cultures were incubated at 37 ◦ C for 72 h. Cytochalasin B (6 mg/ml; Sigma) was added after 44 h of culture to block cytokinesis, allowing identification of lymphocytes that divide in culture. Cells that have undergone the first mitosis are thus recognized as binucleated cells and are selectively screened for the presence of MN. The cells were then treated hypotonically with 0.075 M KCl for 5 min at room temperature and fixed in methanol:acetic acid (3:1). Cells were dropped onto slides and stained with 5% Giemsa in phosphate buffer (pH 6.8) for 5 min. About 1000 binucleated cells (mean ± SD = 1007.63 ± 7.45, range = 993–1024) from each subject were examined for MN by an experienced observer [24]. 2.4. Measurement of malondialdehyde (MDA) in serum, and antioxidant enzyme activities For this purpose, heparinized whole blood obtained from patients and controls was centrifuged at 2000 × g for 10 min at +4 ◦ C. The sera were separated. The serum samples for measurement of specific parameters were stored at −80 ◦ C until the day of analysis, but no longer than 1 month. MDA was determined by use of the thiobarbituric acid (TBA) method [25]. One milliliter of the serum was transferred to another tube, with the addition of 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% TBA in 0.5 M NaOH. The contents of the tubes were mixed, kept in a boiling water bath for 15 min, and cooled to room temperature. The anti-oxidant butylated hydroxytoluene (BHT) was added to prevent MDA formation during the assay. The addition of BHT to standard MDA did not affect the colour development with TBA. The absorbance of the supernatant was measured at 532 nm. Total TBA-reactive substances were expressed as MDA, using a molar absorptivity for MDA of 1.56 × 105 per mol/cm. The results were expressed as nmol/ml plasma. SOD activity was measured according to the method of Sun et al. [26]. Activity was assessed in the ethanol phase of the lysate after addition of 1 ml of ethanol/chloroform (5/3, v/v) to the same volume of hemolysate, and subsequent centrifugation. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the nitroblue-tetrazolium reduction rate. The absorbance was measured at 560 nm, and the activity of SOD was expressed as U/ml plasma. GSH-Px activity was measured according to the method of Paglia and Valentine [27]. Enzyme activity was determined from the oxidation of reduced nicotinamideadenine-dinucleotide phosphate (NADP) in the presence of H2 O2 as substrate. The decrease in concentration of NADP was monitored and recorded at 340 nm in a mixture containing reduced glutathione and glutathione reductase (pH 7.8, 25 ◦ C). One unit of GSH-Px was calculated with a molar absorptivity of NADP of 6.22 × 103 per mol/cm at 340 nm and the results are expressed as micromoles of NADP oxidized per minute. Enzyme activity was expressed as IU/l plasma. 2.5. CRP and ESR assay The ESR (mm/h) was determined by the classical Westergren method [28]. The serum concentration of hs-CRP was measured by immunonephelometry (BN2TM System, Dade Behring Inc., Newark, DE, USA). 2.6. Statistical analysis
a
Usual self-care activities include dressing, feeding, bathing, grooming, and toileting. Avocational (recreational and/or leisure) and vocational (work, school, homemaking) activities are patient-desired and age- and gender-specific.
The DNA damage (comet-tail length) and MN data were analyzed statistically with the Mann–Whitney U-test. MDA, SOD, GSH-Px, ESR and PMNL levels were
A. Karaman et al. / Mutation Research 721 (2011) 1–5 Table 3 The mean DNA damage (comet-tail length in m) and micronucleus (MN) frequencies across the patients with rheumatoid arthritis and controls.
Patients Active (n = 12) Inactive (n = 31) Controls (n = 30) a b c d
Sex (female/ male)
DNA damage (m) (mean ± SD)
MN/1000 binucleated cells (mean ± SD)
10/2 23/8 17/13
20.6 ± 3.56a 9.69 ± 2.03c 3.12 ± 1.73
3.33 ± 1.25b 3.21 ± 1.14d 1.90 ± 0.67
Significant at p < 0.0001. Significant at p < 0.0001. Significant at p < 0.0001. Significant at p < 0.05.
analyzed statistically by Student’s t-test. To evaluate the correlations between the disease duration, age, sex, MDA level, SOD and GSH-Px activity, ESR, hs-CRP level, DNA damage (comet-tail length) and MN frequency, the coefficients of Pearson’s correlation were calculated. A p-value of less than 0.05 was considered to correspond with statistical significance.
3. Results The extent of DNA damage evaluated by the comet assay in leukocytes of all study subjects as measured by comet-tail length is presented in Table 3. The comet-tail length (mean ± SD) was significantly higher in both the active (20.6 ± 3.56, range 14.6–26.3) and inactive (9.69 ± 2.03, range 5.40–14.14) RA patients than in the controls (3.12 ± 1.73, range 0.6–7.1). There was a statistically significant difference in the comet-tail length between the two groups of patients (Z = 5.036, p < 0.0001). The number of MN/1000 BNCs (per subject) (mean ± SD) was significantly higher in both the active (3.33 ± 1.25, range 1.52–5.92) and inactive (3.21 ± 1.14, range 1.20–5.62) patients than in the controls (1.905 ± 0.67, range 0.77–3.18), but there was no statistically significant difference in the MN frequency between the two patient groups (Z = 0.541, p > 0.05) (Table 3). The values of MDA, SOD, GSH-Px, ESR and hs-CRP are shown in Table 4. The MDA level was significantly higher in both the active and inactive patients than in the controls. There was also a statistically significant difference in the MDA level between the two patient groups. The SOD level was significantly lower in both the active and inactive patients than in the controls. There was also a statistically significant difference in the SOD level between the two patient groups. Similarly, the GSH-Px level was significantly lower in both the active and inactive patients compared with the controls, but there was no statistically significant difference in the GSH-Px level between the two patient groups. Furthermore, the ESR and hsCRP rates were significantly higher in both patient groups than in the controls. These rates were also significantly higher in the active than in the inactive patients. On the other hand, the comet-tail length was positively correlated with the plasma MDA level (r = 0.532, p < 0.05), but not with the plasma SOD and GSH-Px levels in the RA patients (r = 0.129, p > 0.05 and r = 0.103, p > 0.05, respectively). The MN frequency did not correlate with plasma MDA, SOD and GSH-Px levels in the RA patients (r = 0.105, p > 0.05; r = 0.119, p > 0.05 and r = 0.085, p > 0.05, respectively). Furthermore, in the RA patients, the comettail length did not correlate with ESR; hs-CRP rates and patients’ age, sex or disease duration (r = 0.125, p > 0.05; r = 0.152, p > 0.05; r = 0.063, p > 0.05; r = 0.122, p > 0.05 and r = 0.114, p > 0.05, respectively). Similarly, in the RA patients the MN frequency did not correlate with ESR, hs-CRP rates and patients’ age, sex or disease duration (r = 0.126, p > 0.05; r = 0.082, p > 0.05; r = 0.97, p > 0.05; r = 0.115, p > 0.05 and r = 0.083, p > 0.05, respectively).
3
4. Discussion Rheumatoid arthritis (RA) is an inflammatory disease characterized by chronic inflammation of the synovial joints associated with proliferation of synovial cells and infiltration of activated immunoinflammatory cells, including memory T cells, macrophages and plasma cells, leading to progressive destruction of cartilage and bone [19,29]. Most patients present rheumatoid factors, which are autoantibodies directed to the Fc fraction of immunoglobulin G, and antibodies reactive with citrullinated peptides [30,31]. Hitchon and El-Gabalawy propose that the physiological production of ROS by phagocytes in response to antigen may affect T cell–antigen interactions and possibly induce apoptosis of autoreactive arthritogenic T cells, thereby preventing autoimmune responses [19]. Recently, neutrophils from the synovial fluids (SF) of RA were found to be activated and to produce ROS intracellularly in RA, probably as a result of active processing of endocytosed material [32]. Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of inflammation and rheumatoid arthritis. The processes associated with inflammatory responses are complex and often involve reactive oxygen species. There are many mediators that initiate and amplify the inflammatory response such as histamine, serotonin and metabolic products of arachidonic acid (thromboxane, prostaglandins and leukotrienes) [33,34]. Reactive oxygen and nitrogen species (RNS) directly damage DNA and impair DNA-repair mechanisms. This damage can occur in the form of DNA-strand breakage or individual nucleotide base-damage. DNA reaction products, in particular 8-oxo-7-hydrodeoxyguanosine formed by the reaction of hydroxyl radicals (OH• ) with deoxyguanosine, are elevated in leukocytes and sera of patients with RA [35,36]. This oxydized base is particularly mutagenic and cytotoxic. NO, especially in high concentrations, causes deamination of deoxynucleotides, DNA-strand breakage and oxidative damage from peroxynitrite, and DNA modification by metabolically activated N-nitrosamines, all of which can lead to somatic mutations. There is a great deal of evidence on the important role of oxidative stress in RA pathophysiology. Several groups have demonstrated increased oxidative enzyme activity, along with decreased anti-oxidant levels in RA sera and synovial fluids (SF). Studies with SF and tissues in RA have demonstrated oxidative damage of hyaluronic acid [37], lipoperoxidation products [38], oxidation of low-density lipoproteins [39] and carbonyl increment by protein oxidation [40]. Evidence of oxidative damage in cartilage, extracellular collagen and DNA has also been reported. Jikimoto et al. [41] showed a correlation between disease activity and the presence of oxidative stress in patients with RA. It seems that the strongest correlation is between DNA damage and the oxidative stress-index [30]. Apart from the well-established damage to the lipid bilayer caused by free radicals, DNA damage, which is an important target for oxidative injury, has also been investigated. Studies evaluating the DNA lesions by means of the comet assay in RA patients have demonstrated elevated damage levels, which were related to an increased oxidative stress and a reduced total anti-oxidant capability [30]. ROS-induced genotoxic events have also been linked to mutations of the tumour-suppressor gene p53 observed in RAderived fibroblast-like synoviocytes, which could explain, at least in part, the transformed phenotype of these cells and their inadequate apoptosis [42]. ROS and, in particular, O2•− is produced by osteoclasts during bone resorption, and this formation occurs at the osteoclast-bone surface interface [43]. Experimentally, it has been verified that excessive production of ROS may lead to an accelerated damage to joint cartilage and osteoclast activation [43–45]. In addition
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Table 4 Malondialdehyde (MDA) and antioxidant levels, erythrocyte sedimentation rate (ESR) and high sensitivity cerum reactive proteine (hs-CRP) rate in patients with rheumatoid arthritis. Rheumatoid arthritis Active (n = 12) ESR (mm/h), mean ± SD hs-CRP (mg/l), mean ± SD MDA (nmol/ml plasma), mean ± SD Superoxide dismutase (U/l plasma), mean ± SD Glutathione peroxidase (IU/l plasma), mean ± SD a b
41.3 14.3 5.74 412.50 12.36
± ± ± ± ±
Inactive (n = 31)
2.9a 6.2a 0.6a 7.5a 1.42a
19.5 8.4 4.45 498.43 13.21
± ± ± ± ±
3.6a 4.5a 0.2b 3.6b 1.23b
Controls (n = 30) 6.7 2.15 2.05 634.21 18.45
± ± ± ± ±
1.12 1.2 0.7 7.6 1.14
Significant at p < 0.01. Significant at p < 0.05.
to reactive oxygen species, reactive nitrogen species (RNS) have been investigated in RA. This link occurs because of the participation of RNS in the activation of NF-B, as the formation of peroxynitrite interferes in the redox balance of glutathione. Studies indicate that RNS donors caused NF-B activation and increased activation of proteolytic systems [46]. Excessively produced free radicals by PMNL have been suggested to mediate tissue injury in RA. Indeed, excess H2 O2 -induced MDA production with increased macrophage activity has been shown both in vivo and in vitro, suggesting increased neutrophil-derived ROS production in patients with RA [47]. Recently, the genotoxicity of ROS has been well established, and oxidative stress can cause genomic damage [48,49]. Some authors have examined the comet-assay results in RA patients to understand the mechanism. They reported that increased comet outcomes were observed in patients with RA [6,30]. Furthermore, in a new study, Ramos-Remus et al. [50] found high frequencies of MN in patients with RA. Similarly, we found significantly higher comet-assay outcomes and MN frequencies in both the active and inactive patients than in the controls (p < 0.0001, p < 0.0001, p < 0.0001 and p < 0.05, respectively; Table 3). This result is consistent with that of the previous studies mentioned above [6,30,50]. On the other hand, there was also a statistically significant difference in the comet-assay data between the active and inactive patients (p < 0.0001, Table 3). Our results indicate that there was an increased DNA damage in the RA patients, and this situation may be associated with the pathogenesis of RA. In rheumatoid joints, activated macrophages and neutrophils release several kinds of oxidants, which in high concentrations lead to oxidative stress causing damage to lipids, proteins, carbohydrates and DNA. Important targets for oxidants are the unsaturated fatty acids in cell membranes. MDA is a product of lipid peroxidation and thereby functions as a marker of oxidative stress. The level of MDA in plasma or serum has been reported to be higher in RA patients than in control subjects [38,51,52]. Extracellular superoxide dismutase (SOD3) shows protective effects in animal models of ischaemia and inflammation [19]. In mice that are genetically deficient in SOD3, both the severity of collagen-induced arthritis (CIA) and the production of proinflammatory cytokines are increased. SOD3 gene transfer via the subcutaneous route or into the knee decreases the severity of experimental arthritis in rodents [53,54]. Studies in SF and tissue have demonstrated oxidative damage in hyaluronic acid, which has been shown to induce T-cell hyporesponsiveness in RA through effects on protein and proteosomal degradation, with a significant decrease of the intracellular reduced glutathione (GSH) levels, which has also been correlated with the hyporesponsive state of these cells [55]. In this study, we observed decreased levels of plasma SOD and GSH-Px activities in the patients (p < 0.05, p < 0.01, p < 0.05 and p < 0.05, respectively; Table 4), whereas we found increased levels of plasma MDA (p < 0.01 and p < 0.05, respectively, Table 4). In addition, the comet-assay signal was positively correlated with the MDA level in the patients (p < 0.05). The decreased GSH-Px and SOD and the increased MDA levels reflect increased oxidative stress in
the RA patients, and this situation may lead to the oxidative damage in these patients. Thus, our study shows that the response in the comet assay and the frequency of MN is increased in RA, and suggests that this strong response in the comet assay and the MN test stems from the inflammatory condition, which produces active oxygen species. Many studies suggest that oxidative stress plays an important role in the pathogenesis of RA [56,57]. Earlier studies have shown that the level of MDA is related to RA disease activity. In one study Taysi et al. [38] found that serum MDA correlated positively with disease-activity score, and Deaney et al. [58] reported a correlation between ESR and MDA plus another lipid peroxidation product, 4-hydroxynonenal. These results support the hypothesis that an imbalance in the oxidant–antioxidant system is involved in the pathogenesis of RA, characterized by an increase in ROS production and a decrease in antioxidant activity. In conclusion, our results suggest an increased production of ROS in RA, as reflected by higher plasma MDA levels and lower plasma GSH-Px and SOD levels, and this situation may impair genetic stability, as reflected by a stronger response in the comet assay and the MN test in RA patients. Thus, we consider that the elevated outcome of the comet assay (comet-tail length) and the higher MN frequency in RA can be explained by increased oxidative stress. This information is potentially important, as it enables understanding of the mechanism of action of current therapies and in particular the development of new therapeutic strategies. Conflict of interest statement The societies or individuals had no competing interests. Acknowledgement We wish to thank Dr. I˙ . Pirim for excellent technical assistance. References [1] J.J. Goronzy, C.M. Weyand, Rheumatoid arthritis, Immunol. Rev. 204 (2005) 55–73. [2] L.G. Darlington, T.W. Stone, Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders, Br. J. Nutr. 85 (2001) 251–269. [3] Y. Ozkan, S. Yardym-Akaydyn, A. Sepici, E. Keskin, V. Sepici, B. Simsek, Oxidative status in rheumatoid arthritis, Clin. Rheumatol. 26 (2007) 64–68. [4] P.G. Winyard, C.J. Moody, C. Jacob, Oxidative activation of antioxidant defense, Trends Biochem. Sci. 30 (2005) 453–461. [5] W. Dröge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47–95. [6] P.H.J. Remans, M. van Oosterhout, T.J.M. Smeets, M. Sanders, W.M. Frederiks, K.A. Reesquist, P.P. Tak, F.C. Breedveld, J.M. Ivan Laar, ntracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis, Arthritis Rheum. 52 (2005) 2003–2009. [7] D.A. Lawrence, R. Song, P. Weber, Surface thiols in human lymphocytes and their changes after in vitro and in vivo activation, J. Leukoc. Biol. 60 (1996) 611–618. [8] M. Klaude, S. Eriksson, J. Nygren, G. Ahnström, The comet assay: mechanisms and technical considerations, Mutat. Res. 363 (1996) 89–96. [9] D. Anderson, M.J. Plewa, The international comet assay workshop, Mutagenesis 13 (1998) 67–73.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Comet assay and analysis of micronucleus formation in patients with rheumatoid arthritis Ali Karaman a,∗ , Do˘gan Nasır Binici b , Meltem Alkan Meliko˘glu c a b c
Department of Medical Genetics, Erzurum Nenehatun Obstetrics and Gynecology Hospital, Erzurum, Turkey Department of Internal Medicine, Erzurum Training and Research Hospital, Erzurum, Turkey Department of Romatology, Erzurum Training and Research Hospital, Erzurum, Turkey
a r t i c l e
i n f o
Article history: Received 27 July 2010 Received in revised form 5 October 2010 Accepted 5 November 2010 Available online 20 January 2011 Keywords: Rheumatoid arthritis Comet assay Micronucleus test Pathogenesis
a b s t r a c t Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of rheumatoid arthritis (RA). Genetic factors that predispose individuals to RA are considered to play an important role in the development of the disease. The aim of this study was to determine, by use of the comet assay and the micronucleus (MN) test, whether DNA damage has an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and DNA damage in RA. This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. We analyzed lymphocytes from patients with RA (12 in active and 31 in inactive periods) and 30 healthy controls for effects in the comet assay and the MN test. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), the erythrocyte sedimentation rate (ESR) and the high-sensitivity C-reactive protein (hs-CRP) rate were determined in all the subjects. The comet-tail length, the MN frequencies and the MDA levels were significantly higher in patients – both in the active and the inactive period – than in the controls. In contrast, the SOD and GSH-Px levels were significantly lower in both patient groups than in the controls. Our results suggest that an increased plasma MDA level and decreased plasma GSH-Px and SOD levels reflect the higher degree of oxidative stress in RA patients, a situation that may impair genetic stability in those patients. Thus, the results suggest that increased DNA damage may play an important role in the pathogenesis of RA. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune syndrome, which produces degradation of articular cartilage and bone erosion. The long-term outcomes of this progressive disease are significant morbidity, loss of functional capacity, and increased mortality [1]. RA affects 1–2% of the general population worldwide [2], and the occurrence in women is three times higher than in men. Although the onset of RA can occur at any age, the incidence increases with age. The aetiology of RA is basically unknown, but several studies have implicated a combination of a genetic background and environmental factors, such as infections and smoking, leading to defects in immunoregulation and a host of inflammatory mechanisms involved in joint-tissue damage, including a role for oxidative stress [3]. The formation and scavenging activity of free radicals in biological systems have been closely linked to a number of pathological
∗ Corresponding author. Tel.: +90 442 317 2295; fax: +90 442 317 2294. E-mail address: [email protected] (A. Karaman). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.11.014
conditions. In healthy individuals, reactive oxygen species (ROS) and associated oxidative stress factors are kept in check by a combination of antioxidant activities [4,5]. There is increasing evidence that ROS and the resulting pro-oxidant/antioxidant imbalance play a major role in RA, as well as in other disease states [6,7]. The comet assay is a fast, simple and sensitive method for the quantification of genetic damage in a small number of cells [8–11]. For these reasons, the comet assay has been used to evaluate DNA damage induced by physical and chemical agents in numerous studies involving environmental monitoring and in medical research [9]. A micronucleus (MN) is an acentric chromosome fragment or a whole chromosome that is left behind during mitotic cell division, and it appears in the cytoplasm of interphase cells as a small additional nucleus [12]. The MN test, widely accepted for in vitro and in vivo genotoxicity investigations, provides a sensitive marker of genomic damage [13]. Living organisms contain specific enzymes catalysing the breakdown of the superoxide radical and hydrogen peroxide, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) [14]. Malondialdehyde (MDA) can directly or indirectly affect many functions integral to cellular and organ
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A. Karaman et al. / Mutation Research 721 (2011) 1–5
Table 1 Demographic details of the patients with rheumatoid arthritis and controls.
ing at least 45 min. The study was approved by the hospital’s Ethical Committee. The study subjects were non-smokers and non-alcoholics, without a history of viral infection or any systemic disease. None of the subjects had received antibiotics, systemic steroids and mineral or vitamin drugs during the past 2 months. All patients were analyzed prior to therapy.
Sex (female/ male)
Age, years (mean ± SD), range
Disease duration, years (mean ± SD), range
Patients (n = 43)
32/11
4.25 ± 2.8; 1–22
2.2. Comet assay
Controls (n = 30)
17/13
39.84 ± 8.32; 25–66 37.21 ± 6.83; 20–61
–
Forty microlitres of blood were taken from the collected samples for the comet assay, which was performed under alkaline conditions, as described by Singh et al. [22], with modifications. Cell viability, determined by the trypan-blue exclusion technique, ranged from 94 to 96% (data not shown). Slides were prepared in duplicate for each subject. Analysis was performed with a 400× objective on an Olympus BX 51 fluorescence microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Slides were randomized and coded to blind the scorer. All slides were scored by one person, to avoid inter-scorer variability. A total of 100 individual cells were screened per subject (50 cells from each slide). Undamaged cells resemble an intact nucleus without a tail and damaged cells have the appearance of a comet. The length of the DNA that had migrated in the comet tail, which is an estimate of DNA damage, was measured with an ocular meter and calculated as comet tail length (m) = (maximum total length) − (head diameter).
homeostasis. The specific determination of MDA is useful for revealing the extent of lipid damage, as a result of oxidative stress, in a variety of lipid systems, such as plasma, organs and cell membranes [15,16]. The increased membrane lipid-peroxidation may evoke immune and inflammatory responses, active gene expression and cell proliferation, or initiate apoptosis. Thus, a close relationship between ROS production, impairment of the antioxidant defense, peroxidative membrane damage and inflammatory or degenerative pathological processes could be postulated [17]. Despite its important physiological role, an unbalanced redox status may present potentially destructive effects on the biology of the cell [18,19]. Currently, the use of biomarkers of oxidative stress can help explore the relation between oxidative damage to macromolecules (DNA, lipids and proteins) and several diseases. Evaluation, both in vivo and ex vivo, includes measurements of DNA oxidation, lipid peroxidation and protein oxidation [20]. The aim of this study was to determine, by use of the comet assay and the MN test, whether genetic lesions and DNA damage have an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and genomic damage in RA. 2. Materials and methods 2.1. Patients and specimen collection This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. Forty-three patients with diagnosed RA and 30 healthy controls were studied. The demographic information of all study subjects is presented in Table 1. We performed the comet assay and the MN test on blood samples from all patients and controls. The patients were divided into two groups, as active (n = 12, 10 females and 2 males) and inactive (n = 31, 23 females and 8 males), based on the severity of their disease identified on the basis of clinical and laboratory findings. Blood samples were obtained by venous puncture and lymphocytes were collected by standard methods. The samples were transported on ice to the laboratory and were processed within 2 h. The blood samples were obtained with informed consent from the healthy volunteers and from the RA patients. The latter group fulfilled the 1987 criteria for RA by the American Rheumatism Association and were in functional Class I, II, or III (Table 2), according to the revised criteria of the American College of Rheumatology [21]. Patients with an active disease duration of at least 6 months, as manifested by at least three joints that were swollen and six joints that were tender at the time of the blood donation, were accepted for the study. In addition, RA patients had an erythrocyte sedimentation rate (ESR) ≥28 mm/h, a high-sensitivity C-reactive protein (hs-CRP) ≥2.4 mg/l, or morning stiffness dur-
Table 2 American college of rheumatology revised criteria for classification of functional status in RAa . Class I Class II Class III Class IV
Completely able to perform usual activities of daily living (self-care, vocational, and avocational) Able to perform usual self-care and vocational activities but limited in avocational activities Able to perform usual self-care activities but limited in vocational and avocational activities Limited in ability to perform usual self-care, vocational, and avocational activities
2.3. Micronucleus (MN) analysis For MN analysis, 2 ml of heparinized blood was drawn from each individual. Lymphocyte cultures were established by adding 0.5 ml of whole blood to 5 ml karyotyping medium (Biological Industries, Beit Haemek, Israel) with 2% phytohaemagglutinin M (Biological Industries) according to standard techniques [23]. The cultures were incubated at 37 ◦ C for 72 h. Cytochalasin B (6 mg/ml; Sigma) was added after 44 h of culture to block cytokinesis, allowing identification of lymphocytes that divide in culture. Cells that have undergone the first mitosis are thus recognized as binucleated cells and are selectively screened for the presence of MN. The cells were then treated hypotonically with 0.075 M KCl for 5 min at room temperature and fixed in methanol:acetic acid (3:1). Cells were dropped onto slides and stained with 5% Giemsa in phosphate buffer (pH 6.8) for 5 min. About 1000 binucleated cells (mean ± SD = 1007.63 ± 7.45, range = 993–1024) from each subject were examined for MN by an experienced observer [24]. 2.4. Measurement of malondialdehyde (MDA) in serum, and antioxidant enzyme activities For this purpose, heparinized whole blood obtained from patients and controls was centrifuged at 2000 × g for 10 min at +4 ◦ C. The sera were separated. The serum samples for measurement of specific parameters were stored at −80 ◦ C until the day of analysis, but no longer than 1 month. MDA was determined by use of the thiobarbituric acid (TBA) method [25]. One milliliter of the serum was transferred to another tube, with the addition of 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% TBA in 0.5 M NaOH. The contents of the tubes were mixed, kept in a boiling water bath for 15 min, and cooled to room temperature. The anti-oxidant butylated hydroxytoluene (BHT) was added to prevent MDA formation during the assay. The addition of BHT to standard MDA did not affect the colour development with TBA. The absorbance of the supernatant was measured at 532 nm. Total TBA-reactive substances were expressed as MDA, using a molar absorptivity for MDA of 1.56 × 105 per mol/cm. The results were expressed as nmol/ml plasma. SOD activity was measured according to the method of Sun et al. [26]. Activity was assessed in the ethanol phase of the lysate after addition of 1 ml of ethanol/chloroform (5/3, v/v) to the same volume of hemolysate, and subsequent centrifugation. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the nitroblue-tetrazolium reduction rate. The absorbance was measured at 560 nm, and the activity of SOD was expressed as U/ml plasma. GSH-Px activity was measured according to the method of Paglia and Valentine [27]. Enzyme activity was determined from the oxidation of reduced nicotinamideadenine-dinucleotide phosphate (NADP) in the presence of H2 O2 as substrate. The decrease in concentration of NADP was monitored and recorded at 340 nm in a mixture containing reduced glutathione and glutathione reductase (pH 7.8, 25 ◦ C). One unit of GSH-Px was calculated with a molar absorptivity of NADP of 6.22 × 103 per mol/cm at 340 nm and the results are expressed as micromoles of NADP oxidized per minute. Enzyme activity was expressed as IU/l plasma. 2.5. CRP and ESR assay The ESR (mm/h) was determined by the classical Westergren method [28]. The serum concentration of hs-CRP was measured by immunonephelometry (BN2TM System, Dade Behring Inc., Newark, DE, USA). 2.6. Statistical analysis
a
Usual self-care activities include dressing, feeding, bathing, grooming, and toileting. Avocational (recreational and/or leisure) and vocational (work, school, homemaking) activities are patient-desired and age- and gender-specific.
The DNA damage (comet-tail length) and MN data were analyzed statistically with the Mann–Whitney U-test. MDA, SOD, GSH-Px, ESR and PMNL levels were
A. Karaman et al. / Mutation Research 721 (2011) 1–5 Table 3 The mean DNA damage (comet-tail length in m) and micronucleus (MN) frequencies across the patients with rheumatoid arthritis and controls.
Patients Active (n = 12) Inactive (n = 31) Controls (n = 30) a b c d
Sex (female/ male)
DNA damage (m) (mean ± SD)
MN/1000 binucleated cells (mean ± SD)
10/2 23/8 17/13
20.6 ± 3.56a 9.69 ± 2.03c 3.12 ± 1.73
3.33 ± 1.25b 3.21 ± 1.14d 1.90 ± 0.67
Significant at p < 0.0001. Significant at p < 0.0001. Significant at p < 0.0001. Significant at p < 0.05.
analyzed statistically by Student’s t-test. To evaluate the correlations between the disease duration, age, sex, MDA level, SOD and GSH-Px activity, ESR, hs-CRP level, DNA damage (comet-tail length) and MN frequency, the coefficients of Pearson’s correlation were calculated. A p-value of less than 0.05 was considered to correspond with statistical significance.
3. Results The extent of DNA damage evaluated by the comet assay in leukocytes of all study subjects as measured by comet-tail length is presented in Table 3. The comet-tail length (mean ± SD) was significantly higher in both the active (20.6 ± 3.56, range 14.6–26.3) and inactive (9.69 ± 2.03, range 5.40–14.14) RA patients than in the controls (3.12 ± 1.73, range 0.6–7.1). There was a statistically significant difference in the comet-tail length between the two groups of patients (Z = 5.036, p < 0.0001). The number of MN/1000 BNCs (per subject) (mean ± SD) was significantly higher in both the active (3.33 ± 1.25, range 1.52–5.92) and inactive (3.21 ± 1.14, range 1.20–5.62) patients than in the controls (1.905 ± 0.67, range 0.77–3.18), but there was no statistically significant difference in the MN frequency between the two patient groups (Z = 0.541, p > 0.05) (Table 3). The values of MDA, SOD, GSH-Px, ESR and hs-CRP are shown in Table 4. The MDA level was significantly higher in both the active and inactive patients than in the controls. There was also a statistically significant difference in the MDA level between the two patient groups. The SOD level was significantly lower in both the active and inactive patients than in the controls. There was also a statistically significant difference in the SOD level between the two patient groups. Similarly, the GSH-Px level was significantly lower in both the active and inactive patients compared with the controls, but there was no statistically significant difference in the GSH-Px level between the two patient groups. Furthermore, the ESR and hsCRP rates were significantly higher in both patient groups than in the controls. These rates were also significantly higher in the active than in the inactive patients. On the other hand, the comet-tail length was positively correlated with the plasma MDA level (r = 0.532, p < 0.05), but not with the plasma SOD and GSH-Px levels in the RA patients (r = 0.129, p > 0.05 and r = 0.103, p > 0.05, respectively). The MN frequency did not correlate with plasma MDA, SOD and GSH-Px levels in the RA patients (r = 0.105, p > 0.05; r = 0.119, p > 0.05 and r = 0.085, p > 0.05, respectively). Furthermore, in the RA patients, the comettail length did not correlate with ESR; hs-CRP rates and patients’ age, sex or disease duration (r = 0.125, p > 0.05; r = 0.152, p > 0.05; r = 0.063, p > 0.05; r = 0.122, p > 0.05 and r = 0.114, p > 0.05, respectively). Similarly, in the RA patients the MN frequency did not correlate with ESR, hs-CRP rates and patients’ age, sex or disease duration (r = 0.126, p > 0.05; r = 0.082, p > 0.05; r = 0.97, p > 0.05; r = 0.115, p > 0.05 and r = 0.083, p > 0.05, respectively).
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4. Discussion Rheumatoid arthritis (RA) is an inflammatory disease characterized by chronic inflammation of the synovial joints associated with proliferation of synovial cells and infiltration of activated immunoinflammatory cells, including memory T cells, macrophages and plasma cells, leading to progressive destruction of cartilage and bone [19,29]. Most patients present rheumatoid factors, which are autoantibodies directed to the Fc fraction of immunoglobulin G, and antibodies reactive with citrullinated peptides [30,31]. Hitchon and El-Gabalawy propose that the physiological production of ROS by phagocytes in response to antigen may affect T cell–antigen interactions and possibly induce apoptosis of autoreactive arthritogenic T cells, thereby preventing autoimmune responses [19]. Recently, neutrophils from the synovial fluids (SF) of RA were found to be activated and to produce ROS intracellularly in RA, probably as a result of active processing of endocytosed material [32]. Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of inflammation and rheumatoid arthritis. The processes associated with inflammatory responses are complex and often involve reactive oxygen species. There are many mediators that initiate and amplify the inflammatory response such as histamine, serotonin and metabolic products of arachidonic acid (thromboxane, prostaglandins and leukotrienes) [33,34]. Reactive oxygen and nitrogen species (RNS) directly damage DNA and impair DNA-repair mechanisms. This damage can occur in the form of DNA-strand breakage or individual nucleotide base-damage. DNA reaction products, in particular 8-oxo-7-hydrodeoxyguanosine formed by the reaction of hydroxyl radicals (OH• ) with deoxyguanosine, are elevated in leukocytes and sera of patients with RA [35,36]. This oxydized base is particularly mutagenic and cytotoxic. NO, especially in high concentrations, causes deamination of deoxynucleotides, DNA-strand breakage and oxidative damage from peroxynitrite, and DNA modification by metabolically activated N-nitrosamines, all of which can lead to somatic mutations. There is a great deal of evidence on the important role of oxidative stress in RA pathophysiology. Several groups have demonstrated increased oxidative enzyme activity, along with decreased anti-oxidant levels in RA sera and synovial fluids (SF). Studies with SF and tissues in RA have demonstrated oxidative damage of hyaluronic acid [37], lipoperoxidation products [38], oxidation of low-density lipoproteins [39] and carbonyl increment by protein oxidation [40]. Evidence of oxidative damage in cartilage, extracellular collagen and DNA has also been reported. Jikimoto et al. [41] showed a correlation between disease activity and the presence of oxidative stress in patients with RA. It seems that the strongest correlation is between DNA damage and the oxidative stress-index [30]. Apart from the well-established damage to the lipid bilayer caused by free radicals, DNA damage, which is an important target for oxidative injury, has also been investigated. Studies evaluating the DNA lesions by means of the comet assay in RA patients have demonstrated elevated damage levels, which were related to an increased oxidative stress and a reduced total anti-oxidant capability [30]. ROS-induced genotoxic events have also been linked to mutations of the tumour-suppressor gene p53 observed in RAderived fibroblast-like synoviocytes, which could explain, at least in part, the transformed phenotype of these cells and their inadequate apoptosis [42]. ROS and, in particular, O2•− is produced by osteoclasts during bone resorption, and this formation occurs at the osteoclast-bone surface interface [43]. Experimentally, it has been verified that excessive production of ROS may lead to an accelerated damage to joint cartilage and osteoclast activation [43–45]. In addition
4
A. Karaman et al. / Mutation Research 721 (2011) 1–5
Table 4 Malondialdehyde (MDA) and antioxidant levels, erythrocyte sedimentation rate (ESR) and high sensitivity cerum reactive proteine (hs-CRP) rate in patients with rheumatoid arthritis. Rheumatoid arthritis Active (n = 12) ESR (mm/h), mean ± SD hs-CRP (mg/l), mean ± SD MDA (nmol/ml plasma), mean ± SD Superoxide dismutase (U/l plasma), mean ± SD Glutathione peroxidase (IU/l plasma), mean ± SD a b
41.3 14.3 5.74 412.50 12.36
± ± ± ± ±
Inactive (n = 31)
2.9a 6.2a 0.6a 7.5a 1.42a
19.5 8.4 4.45 498.43 13.21
± ± ± ± ±
3.6a 4.5a 0.2b 3.6b 1.23b
Controls (n = 30) 6.7 2.15 2.05 634.21 18.45
± ± ± ± ±
1.12 1.2 0.7 7.6 1.14
Significant at p < 0.01. Significant at p < 0.05.
to reactive oxygen species, reactive nitrogen species (RNS) have been investigated in RA. This link occurs because of the participation of RNS in the activation of NF-B, as the formation of peroxynitrite interferes in the redox balance of glutathione. Studies indicate that RNS donors caused NF-B activation and increased activation of proteolytic systems [46]. Excessively produced free radicals by PMNL have been suggested to mediate tissue injury in RA. Indeed, excess H2 O2 -induced MDA production with increased macrophage activity has been shown both in vivo and in vitro, suggesting increased neutrophil-derived ROS production in patients with RA [47]. Recently, the genotoxicity of ROS has been well established, and oxidative stress can cause genomic damage [48,49]. Some authors have examined the comet-assay results in RA patients to understand the mechanism. They reported that increased comet outcomes were observed in patients with RA [6,30]. Furthermore, in a new study, Ramos-Remus et al. [50] found high frequencies of MN in patients with RA. Similarly, we found significantly higher comet-assay outcomes and MN frequencies in both the active and inactive patients than in the controls (p < 0.0001, p < 0.0001, p < 0.0001 and p < 0.05, respectively; Table 3). This result is consistent with that of the previous studies mentioned above [6,30,50]. On the other hand, there was also a statistically significant difference in the comet-assay data between the active and inactive patients (p < 0.0001, Table 3). Our results indicate that there was an increased DNA damage in the RA patients, and this situation may be associated with the pathogenesis of RA. In rheumatoid joints, activated macrophages and neutrophils release several kinds of oxidants, which in high concentrations lead to oxidative stress causing damage to lipids, proteins, carbohydrates and DNA. Important targets for oxidants are the unsaturated fatty acids in cell membranes. MDA is a product of lipid peroxidation and thereby functions as a marker of oxidative stress. The level of MDA in plasma or serum has been reported to be higher in RA patients than in control subjects [38,51,52]. Extracellular superoxide dismutase (SOD3) shows protective effects in animal models of ischaemia and inflammation [19]. In mice that are genetically deficient in SOD3, both the severity of collagen-induced arthritis (CIA) and the production of proinflammatory cytokines are increased. SOD3 gene transfer via the subcutaneous route or into the knee decreases the severity of experimental arthritis in rodents [53,54]. Studies in SF and tissue have demonstrated oxidative damage in hyaluronic acid, which has been shown to induce T-cell hyporesponsiveness in RA through effects on protein and proteosomal degradation, with a significant decrease of the intracellular reduced glutathione (GSH) levels, which has also been correlated with the hyporesponsive state of these cells [55]. In this study, we observed decreased levels of plasma SOD and GSH-Px activities in the patients (p < 0.05, p < 0.01, p < 0.05 and p < 0.05, respectively; Table 4), whereas we found increased levels of plasma MDA (p < 0.01 and p < 0.05, respectively, Table 4). In addition, the comet-assay signal was positively correlated with the MDA level in the patients (p < 0.05). The decreased GSH-Px and SOD and the increased MDA levels reflect increased oxidative stress in
the RA patients, and this situation may lead to the oxidative damage in these patients. Thus, our study shows that the response in the comet assay and the frequency of MN is increased in RA, and suggests that this strong response in the comet assay and the MN test stems from the inflammatory condition, which produces active oxygen species. Many studies suggest that oxidative stress plays an important role in the pathogenesis of RA [56,57]. Earlier studies have shown that the level of MDA is related to RA disease activity. In one study Taysi et al. [38] found that serum MDA correlated positively with disease-activity score, and Deaney et al. [58] reported a correlation between ESR and MDA plus another lipid peroxidation product, 4-hydroxynonenal. These results support the hypothesis that an imbalance in the oxidant–antioxidant system is involved in the pathogenesis of RA, characterized by an increase in ROS production and a decrease in antioxidant activity. In conclusion, our results suggest an increased production of ROS in RA, as reflected by higher plasma MDA levels and lower plasma GSH-Px and SOD levels, and this situation may impair genetic stability, as reflected by a stronger response in the comet assay and the MN test in RA patients. Thus, we consider that the elevated outcome of the comet assay (comet-tail length) and the higher MN frequency in RA can be explained by increased oxidative stress. This information is potentially important, as it enables understanding of the mechanism of action of current therapies and in particular the development of new therapeutic strategies. Conflict of interest statement The societies or individuals had no competing interests. Acknowledgement We wish to thank Dr. I˙ . Pirim for excellent technical assistance. References [1] J.J. Goronzy, C.M. Weyand, Rheumatoid arthritis, Immunol. Rev. 204 (2005) 55–73. [2] L.G. Darlington, T.W. Stone, Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders, Br. J. Nutr. 85 (2001) 251–269. [3] Y. Ozkan, S. Yardym-Akaydyn, A. Sepici, E. Keskin, V. Sepici, B. Simsek, Oxidative status in rheumatoid arthritis, Clin. Rheumatol. 26 (2007) 64–68. [4] P.G. Winyard, C.J. Moody, C. Jacob, Oxidative activation of antioxidant defense, Trends Biochem. Sci. 30 (2005) 453–461. [5] W. Dröge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47–95. [6] P.H.J. Remans, M. van Oosterhout, T.J.M. Smeets, M. Sanders, W.M. Frederiks, K.A. Reesquist, P.P. Tak, F.C. Breedveld, J.M. Ivan Laar, ntracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis, Arthritis Rheum. 52 (2005) 2003–2009. [7] D.A. Lawrence, R. Song, P. Weber, Surface thiols in human lymphocytes and their changes after in vitro and in vivo activation, J. Leukoc. Biol. 60 (1996) 611–618. [8] M. Klaude, S. Eriksson, J. Nygren, G. Ahnström, The comet assay: mechanisms and technical considerations, Mutat. Res. 363 (1996) 89–96. [9] D. Anderson, M.J. Plewa, The international comet assay workshop, Mutagenesis 13 (1998) 67–73.
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