Effect of Prolonged Low-Dose Oral Aspirin on the Oxidative Status of Peripheral Blood

EFFECT OF PROLONGED LOW-DOSE ORAL ASPIRIN ON the Oxidative Status of Peripheral Blood Mononuclear

Cells of Active Vitiligo

Mohammad Z A Zailaie

There is a growing body of evidence that the etiology of vitiligo may relate primarily to an imbalance in the oxidative status of the epidermal melanocytes and other cells that include peripheral blood mononuclear cells (PBMC). Consequently, this pilot placebo-controlled study was designed to evaluate the effect of long-term (l2 weeks) oral administration of single low-dose (300 mg) of the nonsteroidal anti-inflammatory drug, aspirin on the antioxidant enzymes [catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD)] activities and the intracellular levels of reduced glutathione (GSH) and lipid peroxidation of PBMC in patients with vitiligo in the active phase. Thirty two adult patients with active vitiligo (18 females and 14 males) and 32 normal healthy age-and sex-matched subjects were recruited. The vitiligo patients were divided into two equal groups, one group received a single daily dose of oral aspirin and the other one received placebo for 12 consecutive weeks. The antioxidant enzymes, GSH and the marker of lipid peroxidation, malondialdehyde (MDA) were determined in the supernatant of PBMC homogenates of vitiligo patients (aspirin-treated and placebo groups) before the start of treatment and at the end of treatment period. The determination of these cell markers were carried out with the use of commercially available kits, according to standardized spectrophotometric methods.

Compared with the placebo group, the aspirin-treated group of vitiligo patients showed the following: significant increase in catalase and GPx activities (p<0.00l and p<0.05, respectively) and a significant decrease in SOD activity (p<0.05). Moreover, GSH levels was significantly increased (p<0.00l) with concomitant significant decrease in MDA concentration (p<0.05). Parallel to these changes there was a considerable improvement in the disease activity in all aspirin-treated patients, as well as significant repigmentation in two at the end of study period.

Indian J Dermatol 2005; 50 (1) : 9-16

Key Words: Vitiligo, Antioxidant enzymes, Aspirin, Oxidative stress, PBMC

Introduction

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Vitiligo is an acquired idiopathic depigmenting disorder characterized by the loss of melanocytes from the epidermis, and affecting about 1% of the world’s population, with severe psychological and social impact of the afflicted individuals.1 Vitiligo is currently classified in to two major subtypes, segmental vitiligo (SV) including focal lesions and those of quasi-dermatomal pattern that do not progress towards generalized disease and non-segmental vitiligo (NSV), which includes all generalized usually symmetrical forms including acrofacial vitiligo. An extending vitiligo with

enlarging lesions or development of new lesion is

defined as active vitiligo.2 The etiology of vitiligo

remains obscure and controversial and several testable hypotheses have been proposed to explain the loss of melanocytes. These include: intrinsic genetic susceptibility acting as a predisposing factor in certain subsets of patients,3an autoimmune mechanism,4an autocytotoxic destruction of melanocytes,5altered tetrahydrobiopterin homeostasis6 and a neural hypothesis.7 A convergence theory combining the major pathomechanisms has been proposed.8,9

Nowadays there is a growing body of evidence that oxidative stress via hydrogen peroxide (H2O2) may

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From theVitiligo Unit, KingAbdulazizUniversityMedicalCenter, Jeddah, Saudi Arabia. Address correspondence to: Dr.Md. Zailaie, E-mail:

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play a central role in the process of melanocyte apoptosis in vitiligo.10-12 Recent in vivo and in vitro studies have shown that patients with active vitiligo accumulate high levels of H2O2 in their epidermis.10,13 Several sources of this unusual accumulation of H2O2 in the vitiligo epidermis have been documented.10,14,15 Millimolar levels of H2O2 lead to alteration of the antioxidant pattern, with significant reduction of melanocyte catalase activity and increased susceptibility to an external pro-oxidant agent,11 even if catalase mRNA expression was not modified.10 The activity of vitiligo was found to be associated with a systemic oxidative stress. Systemic catalase, glutathione peroxidase (GPx) and Mn-superoxide dismutase (Mn-SOD) activities were decreased,16-18 whereas the Cu-Zn SOD activities were increased.18 In situ and in vitro evidences showed that epidermal acetylcholinesterase can be deactivated by the millimolar levels of H2O2 .19 It was demonstrated that the removal of H2O2 from vitiligo skin results in the recovery of the entire epidermal 6-tetrahydrobiopterin (6BH4) and catalase activities in association with repigmentation of affected skin.6

Several studies have shown that oxidative stress might enhance the expression of the inducible inflammatory cyclooxygenase-2 (COX-2) mRNA.20,21 Acetylsalicylic acid (ASA, aspirin) is a nonsteroidal anti-inflammatory drug that exerts its therapeutic action by irreversibly inhibiting the activity of the COX-2. Increased COX-2 activities are usually associated with the formation of the inflammatory prostanoids, mediators, cytokines and proteases that collectively can lead to cell apoptosis. Moreover, recent reports have also shown that aspirin at relatively low concentration possesses many antioxidant properties through various mechanisms.22-24 It was also demonstrated that ASA treatment to cultured melanocytes from active vitiligo patients results in a significant increase in the release rate of leukotriene (LT) C4, a strong melanocyte mitogen and a concomitant decrease in the release rate of LTB4.25

In this context, the aim of the present pilot placebo-controlled study was to investigate the effect of prolonged administration of oral low-dose aspirin on the imbalanced antioxidant system of the peripheral blood mononuclear cells (PBMC) in patients with active vitiligo.

Materials and methods

Patients

Thirty two consecutive adult vitiligo patients referred to the Vitiligo Unit at KAUMedicalCenter from different dermatology clinics in the Jeddah province, Saudi Arabia, were recruited for this study. All patients had recent active lesions of non-segmental generalized or acrofacial vitiligo with an average duration of 6.4 months (range 3-11 months). The activity of vitiligo was based on the vitiligo disease activity (VIDA) score represented by a 6-point scale.26Of these 32 patients, 14 were male with the mean age of 28.7 years (range 22-40 years) and 18 were female with mean age 24.0 years (range 18-37 years). The selected patients had VIDA scores of +4 and +3 (active in the past 6 weeks and 3 months, respectively) and skin phototype 4 or 5. Controls were 32 age-and-sex matched healthy volunteers with an average age of 25.9 years (range 18-40 years). Clinical examination and laboratory investigations were carried out on the subjects to exclude any systemic disease. The patients and controls were non-smokers and had no peptic ulcer, bleeding tendency, gastrointestinal bleeding, severe anemia or history of aspirin allergy.

Aspirin treatment of patients

Prior to blood collection and aspirin treatment, written consent was obtained from each subject in compliance with the guidelines set by the ethical committee of the KAUMedicalCenter. The patients were asked to stop all forms of medical treatment for at least two weeks. The patients were divided into two equal groups (7 males and 9 females each). Patients in group one were given a daily single low-dose (300 mg) of oral dispersable aspirin (The Boots Company, Nottingham, UK) taken after breakfast, and patients in the second group were given a single dose of oral placebo. The treatment lasted for 12 weeks.

Blood collection and PBMC isolation

Twelve hourly fasting blood samples (10 ml) were collected into EDTA-containing tubes from the controls and patients prior to aspirin treatment and at the end of treatment period. PBMC were isolated from the blood samples using the Ficoll-Hypaque gradient, according to the conventional method.27 Cells were washed twice with 0.9% NaCI at pH 7.4, counted and their viability was determined by trypan blue exclusion.

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Characterization of the oxidative status

Enzyme assays

The enzyme assays were optimized for time, pH, and protein concentration after homogenization of cell suspension in 50 mM cold Tris-HCl buffer with a glass-Teflon Potter (10 strokes up and down at 4,000 rpm). The cell homogenate was centrifuged at 800xg for 10 min at 40 C, and the clarified supernatant was collected and stored at –800C until analyzed. Later, all of the supernatant samples were thawed and the catalase, GPx, SOD activities were determined spectrophotometrically with the use of Bioxytech catalase-520, GPx-340 and SOD-525 enzyme assay kits, respectively (Oxis Research, Portland, OR, USA) according to standardized assay methods.28-30

Reduced glutathione (GSH)

The GSH content in the supernatant of cell homogenates was measured spectrophotometrically using the Bioxytech GSH-400 glutathione assay kit. The assay is based on a two-step reaction: thioesters are first formed and converted to chromophoric thione detectable at 400 nm by alkaline -elimination.

Intracellular lipid peroxidation

Malondialdehyde (MDA) formed during lipid peroxidation is a chromogenic complex. MDA was assayed in the supernatants of cell homogenates using Bioxytech LPO-586 assay kit, according to method previously described.31

Protein determination

Protein concentration of PBMC supernatant was determined by Bradford technique using Bio-Rad kit (Richmond, CA, USA). Bovine serum albumin was used to generate protein concentration standard plot. Enzyme activities were measured in units per mg protein, whereas GSH and MDA were measured in n mole per mg protein. All standards and samples were assayed in duplicate.

Statistical analysis

Results are expressed as means ± SEM. Data from the aspirin-treated and placebo patients groups and matched normal controls were analyzed using the SPSS 10.0 software. Differences and correlations between groups were compared using the unpaired Mann-Whitney U test and Pearson's correlation coefficient test, respectively. P value <0.05 was considered significant.

Results

Effect of aspirin treatment on the PBMC antioxidant enzymes activities

The value of antioxidant enzymes activities in the group of active vitiligo patients were significantly altered compared to the normal control subjects (Table 1). These results are in agreement with results obtained by other workers.16,18 The catalase and GPx activities of PBMC from the active vitiligo group were

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Table 1 Effect of low-dose (300 mg) oral aspirin treatment on antioxidant enzymes activities of PBMC from vitiligo patients in the active phase

Catalase (cat)
U mg-1 protein / GPx
U mg-1 protein / SOD
U mg-1 protein / SOD /cat
Mean / SEM / Mean / SEM / Mean / SEM
Normal control (n=32)
Active vitiligo, aspirin-treated / 186.07 / 3.84 / 27.09 / 0.93 / 8.93 / 0.26 / 0.05
group (baseline) (n=16)
Active vitiligo, aspirin-treated / 144.03** / 3.74 / 18.36** / 1.34 / 11.14* / 0.72 / 0.08
group (n=16)
Active vitiligo, placebo group / 168.22* / 5.14 / 22.91* / 1.41 / 9.56 / 0.54 / 0.06
(baseline) (n=16) / 139.33 / 4.31 / 19.11 / 1.21 / 11.41 / 0.70 / 0.08
Active vitiligo, placebo group
(n=16) / 128.75 / 3.32 / 18.98 / 0.91 / 12.19 / 0.80 / 0.09

*p<0.05, **p<0.001

Active vitiligo, aspirin-treated group (baseline) is compared to normal control, whereas active vitiligo, aspirin-treated group is compared to placebo group

Table 2 Effect of low-dose (300mg) oral aspirin treatment on the intracellular levels of GSH and MDA of PBMC from vitiligo patients in the active phase

GSH
nMol mg-1 protein / MDA
nMol mg-1 protein
Normal control (n=32) / 55.12 / 1.69 / 0.95 / 0.08
Active vitiligo, aspirin-treated group (baseline) (n=16) / 29.24** / 2.77 / 1.71** / 0.14
Active vitiligo, aspirin-treated group (n=16) / 46.68** / 2.31 / 1.12* / 0.10
Active vitiligo, placebo group (baseline) (n=16) / 28.12 / 1.41 / 1.64 / 0.12
Active vitiligo, placebo group (n=16) / 26.81 / 1.44 / 1.79 / 0.16

*p<0.05, **p<0.001

active vitiligo, aspirin-treated group (baseline) is compared to normal control, whereas active vitiligo, aspirin-treated

group is compared to placebo group

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significantly lower than that of the normal control (22.6% and 32.2% respectively, p<0.001), whereas the SOD activity was significantly increased (22.7%, p<0.05). Consequently, the SOD/cat activity ratio, a parameter of cell susceptibility to oxidative stress, was significantly increased in the active vitiligo group compared to normal control (Table 1). These results indicate an increased production of reactive oxygen species (ROS) that is not adequately balanced by the cellular antioxidant defense system.

Treatment of patients in the active vitiligo group with prolonged low dose (300 mg) oral aspirin significantly improved the PBMC antioxidant enzymes activities as shown in Table 1. Compared to patients of the placebo group, the catalase and GPx activities were significantly increased (30.7% and 20.7%; p<0.001 and p<0.05 respectively), whereas the SOD activity was significantly decreased (21.6%; p<0.05) in patients of the aspirin-treated group.

Effect of aspirin treatment on the concentrations of intracellular GSH and MDA of PBMC

The intracellular concentration of GSH in PBMC of patients with active vitiligo was significantly lower than that of normal control subjects (47.0%; p<0.001). This considerable decrease in GSH level was also reported previously18 and was paralleled by significant increase in the concentration of MDA (80.0%; p<0.001) (Table2).

Aspirin treatment of the patients with active vitiligo significantly caused an increase in intracellular GSH and a decrease in the MDA concentrations,compared to patients in the placebo group (74.1% and 37.4%; p< 0.001 and p<0.05 respectively).

MDA is a biomarker of cellular lipid peroxidation, a normal component of aging in human that contributes to the initiation of many degenerative diseases that may include vitiligo. A significantnegative correlation was found to exist between low levels of intracellular GSH and decreased catalase activity in the PBMC of the active vitiligo patients on one hand and high MDA levels on the other hand (r=-0.48and-0.49, respectively, p<0.001).A significantpositive correlation was also obtained between increased SOD activity and high MDA concentration (r = + 0.44, p<0.001). Whereas the negative correlation between decreased GPx activity and high MDA concentration was relativelyweak and not significant (r = -0.24, p>0.05).

Effect of aspirin treatment on vitiligo activity

The improvement in the oxidative status ofPBMC was associated with considerable clinical improvement of the disease activity, at the end of aspirin treatmentof active vitiligo patients.Thus, the activity of vitiligo

was arrested in all patients. No new vitiligo macula developed during treatment, at the end of treatment period or during one month of follow-up. Two female patients aged 18 and 22 years with recent onset vitiligo (3 and 4 months respectively) showed significant repigmentation (over 30%) of their vitiligo lesions located on the face and forearm.

Discussion

There is compelling evidence suggesting that active vitiligo is a systemic disease involving changes in the oxidative status of PBMC. Hence, PBMC may represent a simple and readily accessible model to study mechanisms relevant to the apoptosis of skinmelanocytes in vitiligo. A recent report has shown thatPBMC from patients with active vitiligo demonstrate

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an imbalance of antioxidants correlated with an increased production of ROS due to impaired mitochondrial permeability that allow the excessive leakage of superoxide anions(O-2).16 This may explain the increased activities of SOD (namely the cytosolic/nuclear isoform, Zn-Cu-SOD), the main defense against O-2 radical-mediated oxidative damage, in the present study and others.16,18Increased activities of SOD lead to an increased production of H2O2 inside the PBMC, that can be detoxified to H2Oby the catalase and GPx activities. However, since the catalase and GPx activities in PBMC of the active vitiligo patients are significantly reduced, then it is expected that H2O2 will accumulate inside the cells at toxic level. Consequently, ROS burst and the cells may enter a state of oxidative stress. It is not really known whether the reduced catalase and GPx activities are due to a primary defect or a result of an increased ROS production. The present study advocates the latter, since both enzymes activities have increased following the aspirin treatment. Reduction in the O-2 release from the impaired mitochondria may lead to reduced formation of H2O2. Here comes the vital role of intracellular GSH. Decreased intracellular GSH concentration may be the primary event that may cause changes in cellular antioxidant enzymes and lipid peroxidation. It was shown that cells that had decreased level of GSH undergo apoptosis when exposed to external oxidative stimuli.32 U SOD/U cat ratio is a parameter of cell susceptibility to oxidative stress.33 However, this parameter of susceptibility becomes critical if intracellular GSH concentration is significantly reduced. In stable vitiligo, in spite of increased SOD activities of PBMC, the susceptibility to oxidative stress was decreased,likely due to the normal level of GSH.18 Intracellular GSH functions in so many ways. It serves as a substrate for GPx in a reaction leading to detoxification of H2O2; hence a reduction in its concentration may cause decreased GPx activities and consequently H2O2 burst. By extrapolation, a consequence of reduced intracellular level of GSH in melanocytes of active vitiligo is an increased ROS production, which makes these cells more sensitive to oxidative burst triggered by external stimuli such as trauma, emotional stress or viral infection.Consequently,this may lead to further depletion of GSH that may upregulate the release of apoptotic cytokines that could ultimately bring about the destruction of melanocytes in vitiligo. This is supported by previous studies that showed cultured melanocytes from active vitiligo patients to have greater sensitivity to oxidative stress21 and increased lipid peroxidation34 than normal melanocytes. It was also shown that certain types of cell clones over-expressing glucose-6-phosphate dehydrogenase results in an increase in the level of GSH, which in turn leads to a decreased ROS production.35Moreover, those clones exhibited strong protection against oxidant-mediated cell killing and inhibition of the nuclear factor-kappa B (NF-B) activation in response to tumor necrosis factor-(TNF-). Therefore, it may be assumed that significant reduction in intracellular GSH concentration may be responsible for the other changes of imbalanced antioxidant enzymes and increased MDA concentrations. In the present study, aspirin is shown to improve significantly the oxidative status of the PBMC and possibly epidermal melanocytes as evidenced by the halting of the activity of vitiligo and induction of stability in all patients. This improvement in the oxidative status following the aspirin treatment may be attributed primarily to the significant increase in the intracellular GSH concentration, which may be explained as follows. The salicylate moiety of aspirin may stimulate the de novo biosynthesis of GSH by yet unknown mechanism. A recent study showed that in rat brain tissue subjected to hypoxia, oral administration of aspirin reduces oxidative stress (increased GSH level and GPx activities and decreased MDA concentrations) and inducible nitric oxide synthase (iNOs) activities.36 However, salicylic acid produced greater reduction in oxidative stress and iNOs activities than aspirin, which may indicate that the salicylate moiety of aspirin may have a stimulatory effect on GSH biosynthesis and subsequently modulating the related antioxidant enzymes activities. In addition, aspirin per se was found to be a strong antioxidant. Aspirin was reported to be an efficient hydroxyl radical scavenger and inhibitor of lipid peroxidation, DNA strand breakage, NF-B and TNF-.37,38 Moreover, aspirin was found to reduce both O-2 and lipid peroxidation in rat brain homogenate.39This may explain the decreased SOD activities of PBMC that may be significantly increased in the presence of high O-2. Consequently, the overall result of aspirin treatment is a reduction in ROS, mediated primarily by the increased level of intracellularGSH that eventually can lead