OR IG INAL AR T ICLE

JEADV (2004) 18, 683 686 DOI: 10.1111/j.1468-3083.2004.01080.x

The role of oxidants and antioxidants in generalized vitiligo at tissue level

Blackwell Publishing, Ltd.

M Yildirim,* V Baysal, HS Inaloz, M Can Departments of Dermatology and Biochemistry, Faculty of Medicine, University of Suleyman Demirel, Isparta, Turkey, and Department of Dermatology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey. *Corresponding author, Suleyman Demirel Universitesi, Tip Fakultesi, Dermatoloji Anabilim Dali, 32100 Isparta, Turkey, tel. +90 246 211 25 03; fax + 90 246 237 17 62; E-mail: yildirim@med.sdu.edu.tr

ABSTRAC T
Background The role of oxidative stress has not been fully understood in the aetiopathogenesis of vitiligo

in different studies. Aim We aimed to investigate the role of the oxidative stress in the pathogenesis of vitiligo. Methods In this study, we examined levels of superoxide dismutase, glutathione peroxidase, malondialdehyde and nitric oxide in tissue of 25 patients with generalized vitiligo and 25 healthy controls. Results Our results revealed that levels of superoxide dismutase, glutathione peroxidase and malondialdehyde in tissue were signicantly increased in patients with generalized vitiligo (P < 0.05). However, there was no statistically signicantly difference between two groups at tissue level of nitric oxide (P > 0.05). Conclusion Our results demonstrate the presence of an imbalance in the oxidantantioxidant system in vitiligo at tissue level and provide further support for a free radical-mediated damage as an initial pathogenic event in melanocyte degeneration in vitiligo.
Key words: antioxidant system, oxidative stress, tissue, vitiligo.
Received 26 January 2004; accepted 4 March 2004

Introduction
Vitiligo is an acquired, relatively common, pigmentary anomaly of the skin manifested by well circumscribed depigmented white patches. It is frequently associated with a positive family history and affects approximately 1 4% of the world population. It usually begins in childhood or young adulthood and both sexes are equally affected. The aetiology of the disease is still unknown. Traditionally, there have been three hypotheses to explain vitiligo: neural, immune and self-destruct hypothesis.13 Free radicals (FRs) are atoms or molecules [superoxide, hydrogen peroxide (H2O2) and nitric oxide (NO)] that occur during several physiological and pathological processes.4,5 FRs can damage cell compounds such as protein, carbohydrate, DNA and, particularly, lipid.4 In recent studies, it has been suggested that FRs may play a part in the pathogenesis of vitiligo.6 10 In this study, we investigated the role of FRs by determining superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde
2004 European Academy of Dermatology and Venereology

(MDA) and NO in the tissue of patients with vitiligo and healthy controls.

Patients and methods

Patients
Twenty-ve patients with stable generalized vitiligo (20 women, ve men) and 25 healthy volunteers (19 women, six men) were gathered as a control group in this study. The control group were chosen from the healthy staff in our hospital with any systemic and dermatological disease. The mean age was 32.4 13.8 years (range 1660) in the patient group and 32.8 10.3 years (range 1752) in the control group. The mean duration of the disease in vitiligo patients was 11.8 years. The vitiligo patients had no concomitant dermatological and/or systemic diseases. The patients did not use any systemic or topical treatment at least for a month. The patient and control groups had no history of smoking, use of vitamin and anti-inammatory drugs and excessive exercise except daily life activities.
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Methods
After obtaining consent forms from all patients and healthy controls, incisional biopsy specimens were taken from vitiliginous skin of patients and controls after local anaesthesia with prilocaine HCl. The biopsies were taken from the middle of the lesional skin in the patient group. The control biopsies were taken from the inner aspect of the forearm in healthy volunteers. Subcutaneous fat tissue was separated and remaining material was immediately transported to the Department of Biochemistry, Faculty of Medicine, University of Suleyman Demirel (Isparta, Turkey). Tissue samples were washed in ice-cold saline and then quickly frozen in liquid nitrogen and then were stored at 80 C for 2 months before assay. Tissue samples were thawed and homogenized ( 1 / 10, w / v) in ice-cold buffer (150 mmol /L potassium phosphate buffer, pH 7.4) (Ultra-Turrax T25, IKA-Labor-technick, Staufen, Germany). Then homogenized samples were sonicated while on ice (Bandelin Sonopuls UW 2070, Berlin, Germany) and centrifuged at 6000 g for 10 min at +4 C. Supernatant was used for determination of protein, thiobarbituric acid reactive substance (TBARS), NO levels and GSH-Px and SOD activities. The protein content of homogenate samples was measured using a commercially available kit (Abbot Aeroset, 1385, Shimoi-Shigami, Ohtawara, Japan) with turbidimetric method. It is possible to determine protein concentration at milligram level by this method. Superoxide dismutase determination SOD measurement method was based on the principle in which xanthine reacts with xanthine oxidase to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)5-phenyltetrazolium chloride (INT) to form a red formazon dye. The SOD activity is then measured by the degree of inhibition of this reaction.11 Glutathione peroxidase determination Determination of GSH-Px activity was based on the method of Paglia and Valentine.12 The principle of the method was as follows: GSH-Px catalyses the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance of NADPH was measured at 340 nm.
Table 1 The mean results and statistical evaluation SOD (U / mg protein) Patients Controls P 102.39 56.29 37.61 17.99 < 0001

Malondialdehyde determination TBARS was determined by the double heating method of Draper and Hadley.13 The principle of the method was based on the spectrophotometric measurement of the colour that occurred during the reaction to thiobarbituric acid with malondialdehyde. Concentration of TBARS was calculated by the absorbance coefcient of the MDA-TBA complex 1.56 105 cm1 M1 and expressed in nmol /mg protein. Nitric oxide determination Serum and tissue concentration of nitrite was determined using Griess reaction after deproteinization. Total nitrite concentration (nitrite + nitrate) was measured by using cadmium reduction method. This method employs granular cadmium metal for chemical reduction of nitrate to nitrite. In acid solution, nitrite is converted to nitrous acid, which diazotizes sulfanilamide. This sulfanilamide-diazonium salt is then reacted with N-(1Naphtyl)-ethylenediamine to produce a chromophore, which is measured at 545 nm.14 An autoanalyser (Abbott Aeroset), was used to determine the activities of SOD and GSH-Px, and a spectrophotometer, Shimadzu UV-1601 (Japan), was used to determine the other parameters.

Statistical analysis
Statistical analysis was performed using the t-test.

Results
The activity of SOD and GSH-Px and the levels of MDA in the tissue of patients were elevated more than the controls. The NO level in tissue of the patients and controls was found to be similar. The activity of SOD (P < 0.001), GSH-Px (P < 0.001) and levels of MDA (P < 0.001) between the patient and control groups were signicantly different, but there was no signicant difference in the NO level (P > 0.05). The mean results as mean SD and statistical values are presented in Table 1.

Discussion
Vitiligo is a chronic, common, hypomelanotic disease characterized by depigmented macules. Despite the fact that the aetiology and pathogenesis of vitiligo is not completely known yet, many factors may be responsible for the disease. Many

GSH-Px (U /mg protein) 3.72 2.09 1.58 0.48 < 0001

MDA (nmol /mg protein) 521.89 324.41 139.92 25.87 < 0001

NO (mol/L) 0.12 0.09 0.11 0.10 > 0.05

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Oxidants and antioxidants in vitiligo 685

possible causes, including stress, accumulation of toxic compounds, infections, autoimmunity, mutations, melatonin receptor dysfunction, altered cellular environment and impaired melanocyte migration or proliferation can lead to the acquired hypopigmentation that characterizes vitiligo.15 Oxidative stress can be induced by increasing generation of reactive oxygen species (ROS) and other radicals. The generation of ROS can be associated with a decrease in antioxidant levels at the skin.6 Recent studies have shown that FRs were increased and the antioxidant systems were insufcient in vitiligo.79 For this reason, we investigated the tissue levels of MDA and NO for the evaluation of oxidative stress and activity of SOD and GSH-Px for the evaluation of antioxidant system in vitiligo. SOD is a group of metalloenzymes that protects cells from the toxic effects of superoxide radicals are produced as endogen. Superoxides converted to H2O2 reaction is accelerated by SOD.16 Varying results have been found in different studies associated with SOD activity in vitiligo patients. Picardo et al.10 have studied SOD activity in erythrocytes, Passi et al.6 in the epidermis, Maresca et al.8 in cultured melanocytes and they have found no difference between vitiligo patients and controls. Chakraborty et al.17 have studied SOD activity in the serum of vitiligo patients and controls and they reported higher SOD activity in vitiligo patients. They have also investigated SOD activity in an animal depigmentation model and determined higher SOD activity. In our study, tissue SOD activity in vitiligo patients have been determined as higher than controls and the difference was statistically signicant (P < 0.001). In a previous study, we found that SOD activity was signicantly higher in sera of vitiligo patients than the controls.18 It has been proposed that as melanin itself has an antioxidant action, its absence or presence could cause an increased or diminished need for SOD. However, another possibility is that overproduction of superoxide anion could provoke the higher SOD activity in the tissue.17 GSH-Px is another antioxidant enzyme that turns H2O2 and other peroxides into water.16 Picardo et al.10 and Passi et al.6 have found no difference between vitiligo patients and controls in their study. Beazley et al.7 have studied blood GSH-Px activity in vitiligo patients and controls. They have determined a lower level GSH-Px activity in vitiligo patients than controls. We have found higher GSH-Px activity in vitiligo patients and the difference was statistically signicant (P < 0.001). MDA is an end-product of a lipid peroxidation reaction and an indicator of oxidative stress.15 The levels of MDA in tissue has not been investigated in vitiligo previously. In our study, the levels of MDA in the skin of vitiligo patients have been found to be higher than the controls and the difference was statistically signicant (P < 0.001). We also obtained a higher level of MDA in sera of vitiligo patients than the control group in a previous study.18 Lipoperoxidation, the primary reaction sites of which involve membrane associated polyunsaturated fatty acids of

phospholipids, can be considered a major manifestation of the oxidative stress.10 High SOD and GSH-Px activity also has been found to correlate with high MDA levels. NO is a free radical synthesized from l-arginine by one of the family of NO synthase enzymes.5 Particularly under inammatory conditions reactive NO species contribute to the cytotoxic action of inammatory cells against normal host cells. The presence of in situ inammatory cellular inltrates in close proximity to normal human melanocytes has been attributed to the disappearance of these melanin producing cells in vitiligo. Ivanova et al.19 suggested that NO generated in vivo during inammation can interfere with the adhesion of melanocytes in the skin and thereby may contribute to depigmentation. They have conrmed this hypothesis by their ow cytometric analysis and immunocytochemical study.19 Rocha and Guillo20 investigated NO production in cultured normal human melanocytes and suggested that NO could lead to autodestruction of melanocytes. There has been no investigation of levels of NO in tissue in vitiligo patients. In our study, we have found that there is no difference between the level of NO in tissue of vitiligo patients and controls (P > 0.05). In conclusion, we have observed a disturbance in the oxidantantioxidant system in vitiligo. However, further studies are necessary to conrm our results.

References
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16 Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants, and human diseases: where are we now? J Lab Clin Med 1992; 119: 598 620. 17 Chakraborty DP, Roy S, Chakraborty AK. Vitiligo, psoralen, and melanogenesis: some observations and understanding. Pigment Cell Res 1996; 9: 107 116. 18 Yildirim M, Baysal V, Inaloz HS et al. The role of oxidants and antioxidants in generalized vitiligo. J Dermatol 2003; 30: 104 108. 19 Ivanova K, Le Poole IC, Gerzer R et al. Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components. J Pathol 1997; 183: 469 476. 20 Rocha IM, Guillo LA. Lipopolysaccharide and cytokines induce nitric oxide synthase and produce in cultured normal human melanocytes. Arch Dermatol Res 2001; 293(5): 245 248.

2004 European Academy of Dermatology and Venereology JEADV (2004) 18, 683 686