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

STUDIES

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 Vitiligo is an acquired idiopathic depigmenting enlarging lesions or development of new lesion is disorder characterized by the loss of melanocytes defined as active vitiligo.2 The etiology of vitiligo from the epidermis, and affecting about 1% of the remains obscure and controversial and several world’s population, with severe psychological and testable hypotheses have been proposed to explain social impact of the afflicted individuals.1 Vitiligo is the loss of melanocytes. These include: intrinsic currently classified in to two major subtypes, genetic susceptibility acting as a predisposing factor segmental vitiligo (SV) including focal lesions and in certain subsets of patients,3 an autoimmune those of quasi-dermatomal pattern that do not mechanism,4 an autocytotoxic destruction of progress towards generalized disease and non- melanocytes,5 altered tetrahydrobiopterin segmental vitiligo (NSV), which includes all homeostasis6 and a neural hypothesis.7 A generalized usually symmetrical forms including convergence theory combining the major acrofacial vitiligo. An extending vitiligo with pathomechanisms has been proposed.8,9

Nowadays there is a growing body of evidence that

oxidative stress via hydrogen peroxide (H2O2) may

From theVitiligo Unit, King Abdulaziz University Medical Center, Jeddah, Saudi Arabia. Address correspondence to: Dr. Md. Zailaie, E-mail: [email protected]

Indian J Dermatol 2005; 50 (1) 1 Mohammad Z A Zailaie

play a central role in the process of melanocyte Materials and methods apoptosis in vitiligo.10-12 Recent in vivo and in vitro Patients studies have shown that patients with active vitiligo 10,13 accumulate high levels of H2O2 in their epidermis. Thirty two consecutive adult vitiligo patients referred to the Vitiligo Unit at KAU Medical Center from Several sources of this unusual accumulation of H2O2 in the vitiligo epidermis have been documented.10,14,15 different dermatology clinics in the Jeddah province, Saudi Arabia, were recruited for this study. All patients Millimolar levels of H2O2 lead to alteration of the antioxidant pattern, with significant reduction of had recent active lesions of non-segmental generalized melanocyte catalase activity and increased or acrofacial vitiligo with an average duration of 6.4 susceptibility to an external pro-oxidant agent,11 even if months (range 3-11 months). The activity of vitiligo catalase mRNA expression was not modified.10 The was based on the vitiligo disease activity (VIDA) score 26 activity of vitiligo was found to be associated with a represented by a 6-point scale. Of these 32 patients, systemic oxidative stress. Systemic catalase, 14 were male with the mean age of 28.7 years (range glutathione peroxidase (GPx) and Mn-superoxide 22-40 years) and 18 were female with mean age 24.0 dismutase (Mn-SOD) activities were decreased,16-18 years (range 18-37 years). The selected patients had whereas the Cu-Zn SOD activities were increased.18 In VIDA scores of +4 and +3 (active in the past 6 weeks situ and in vitro evidences showed that epidermal and 3 months, respectively) and skin photo type 4 or 5. acetylcholinesterase can be deactivated by the Controls were 32 age-and-sex matched healthy millimolar levels of H O .19 It was demonstrated that volunteers with an average age of 25.9 years (range 18- 2 2 40 years). Clinical examination and laboratory the removal of H O from vitiligo skin results in the 2 2 investigations were carried out on the subjects to recovery of the entire epidermal 6-tetrahydrobiopterin exclude any systemic disease. The patients and controls (6BH ) and catalase activities in association with 4 were non-smokers and had no peptic ulcer, bleeding 6 repigmentation of affected skin. tendency, gastrointestinal bleeding, severe anemia or Several studies have shown that oxidative stress might history of aspirin allergy. enhance the expression of the inducible inflammatory Aspirin treatment of patients cyclooxygenase-2 (COX-2) mRNA.20,21 Acetylsalicylic acid (ASA, aspirin) is a nonsteroidal anti-inflammatory Prior to blood collection and aspirin treatment, written drug that exerts its therapeutic action by irreversibly consent was obtained from each subject in compliance inhibiting the activity of the COX-2. Increased COX-2 with the guidelines set by the ethical committee of the activities are usually associated with the formation of KAU Medical Center. The patients were asked to stop the inflammatory prostanoids, mediators, cytokines and all forms of medical treatment for at least two weeks. proteases that collectively can lead to cell apoptosis. The patients were divided into two equal groups (7 Moreover, recent reports have also shown that aspirin males and 9 females each). Patients in group one were at relatively low concentration possesses many given a daily single low-dose (300 mg) of oral antioxidant properties through various mechanisms.22-24 dispersable aspirin (The Boots Company, Nottingham, It was also demonstrated that ASA treatment to UK) taken after breakfast, and patients in the second cultured melanocytes from active vitiligo patients group were given a single dose of oral placebo. The results in a significant increase in the release rate of treatment lasted for 12 weeks. leukotriene (LT) C4, a strong melanocyte mitogen and Blood collection and PBMC isolation a concomitant decrease in the release rate of LTB .25 4 Twelve hourly fasting blood samples (10 ml) were In this context, the aim of the present pilot placebo- collected into EDTA-containing tubes from the controlled study was to investigate the effect of controls and patients prior to aspirin treatment and at prolonged administration of oral low-dose aspirin on the end of treatment period. PBMC were isolated from the imbalanced antioxidant system of the peripheral the blood samples using the Ficoll-Hypaque gradient, 27 blood mononuclear cells (PBMC) in patients with according to the conventional method. Cells were active vitiligo. washed twice with 0.9% NaCI at pH 7.4, counted and their viability was determined by trypan blue exclusion.

2 Effect of prolonged low-dose oral aspirin…

Characterization of the oxidative status Protein determination Enzyme assays Protein concentration of PBMC supernatant was determined by Bradford technique using Bio-Rad kit The enzyme assays were optimized for time, pH, and (Richmond, CA, USA). Bovine serum albumin was protein concentration after homogenization of cell used to generate protein concentration standard plot. suspension in 50 mM cold Tris-HCl buffer with a Enzyme activities were measured in units per mg glass-Teflon Potter (10 strokes up and down at 4,000 protein, whereas GSH and MDA were measured in n rpm). The cell homogenate was centrifuged at 800xg mole per mg protein. All standards and samples were for 10 min at 40 C, and the clarified supernatant was assayed in duplicate. collected and stored at –800C until analyzed. Later, all of the supernatant samples were thawed and the Statistical analysis catalase, GPx, SOD activities were determined spectrophotometrically with the use of Bioxytech Results are expressed as means ± SEM. Data from the catalase-520, GPx-340 and SOD-525 enzyme assay aspirin-treated and placebo patients groups and kits, respectively (Oxis Research, Portland, OR, USA) matched normal controls were analyzed using the according to standardized assay methods.28-30 SPSS 10.0 software. Differences and correlations between groups were compared using the unpaired Reduced glutathione (GSH) Mann-Whitney U test and Pearson's correlation The GSH content in the supernatant of cell coefficient test, respectively. P value <0.05 was homogenates was measured spectrophotometrically considered significant. using the Bioxytech GSH-400 glutathione assay kit. The assay is based on a two-step reaction: thioesters Results are first formed and converted to chromophoric thione detectable at 400 nm by alkaline -elimination. Effect of aspirin treatment on the PBMC antioxidant Intracellular lipid peroxidation enzymes activities

Malondialdehyde (MDA) formed during lipid The value of antioxidant enzymes activities in the peroxidation is a chromogenic complex. MDA was group of active vitiligo patients were significantly assayed in the supernatants of cell homogenates using altered compared to the normal control subjects (Table Bioxytech LPO-586 assay kit, according to method 31 1). These results are in agreement with results obtained previously described. by other workers.16,18 The catalase and GPx activities of PBMC from the active vitiligo group were

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

3 Mohammad Z A Zailaie

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 MDA nMol mg-1 protein 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

significantly lower than that of the normal control MDA is a biomarker of cellular lipid peroxidation, a (22.6% and 32.2% respectively, p<0.001), whereas the normal component of aging in human that contributes SOD activity was significantly increased (22.7%, to the initiation of many degenerative diseases that may p<0.05). Consequently, the SOD/cat activity ratio, a include vitiligo. A significant negative correlation was parameter of cell susceptibility to oxidative stress, was found to exist between low levels of intracellular GSH significantly increased in the active vitiligo group and decreased catalase activity in the PBMC of the compared to normal control (Table 1). These results active vitiligo patients on one hand and high MDA indicate an increased production of reactive oxygen levels on the other hand (r=-0.48and-0.49, respectively, species (ROS) that is not adequately balanced by the p<0.001). A significant positive correlation was also cellular antioxidant defense system. obtained between increased SOD activity and high MDA concentration (r = + 0.44, p<0.001). Whereas the Treatment of patients in the active vitiligo group with negative correlation between decreased GPx activity prolonged low dose (300 mg) oral aspirin significantly and high MDA concentration was relatively weak and improved the PBMC antioxidant enzymes activities as not significant (r = -0.24, p>0.05). shown in Table 1. Compared to patients of the placebo group, the catalase and GPx activities were Effect of aspirin treatment on vitiligo activity significantly increased (30.7% and 20.7%; p<0.001 and The improvement in the oxidative status of PBMC was p<0.05 respectively), whereas the SOD activity was associated with considerable clinical improvement of significantly decreased (21.6%; p<0.05) in patients of the disease activity, at the end of aspirin treatment of the aspirin-treated group. active vitiligo patients. Thus, the activity of vitiligo

was arrested in all patients. No new vitiligo macula Effect of aspirin treatment on the concentrations of developed during treatment, at the end of treatment intracellular GSH and MDA of PBMC period or during one month of follow-up. Two female patients aged 18 and 22 years with recent onset vitiligo The intracellular concentration of GSH in PBMC of (3 and 4 months respectively) showed significant patients with active vitiligo was significantly lower repigmentation (over 30%) of their vitiligo lesions than that of normal control subjects (47.0%; p<0.001). located on the face and forearm. This considerable decrease in GSH level was also reported previously18 and was paralleled by significant Discussion increase in the concentration of MDA (80.0%; p<0.001) (Table 2). There is compelling evidence suggesting that active vitiligo is a systemic disease involving changes in the Aspirin treatment of the patients with active vitiligo oxidative status of PBMC. Hence, PBMC may significantly caused an increase in intracellular GSH represent a simple and readily accessible model to and a decrease in the MDA concentrations, compared study mechanisms relevant to the apoptosis of skin to patients in the placebo group (74.1% and 37.4%; p< melanocytes in vitiligo. A recent report has shown that 0.001 and p<0.05 respectively). PBMC from patients with active vitiligo demonstrate

an imbalance of antioxidants correlated with an the destruction of melanocytes in vitiligo. This is increased production of ROS due to impaired supported by previous studies that showed cultured mitochondrial permeability that allow the excessive melanocytes from active vitiligo patients to have - 16 21 leakage of superoxide anions(O 2). This may explain greater sensitivity to oxidative stress and increased the increased activities of SOD (namely the lipid peroxidation34 than normal melanocytes. It was cytosolic/nuclear isoform, Zn-Cu-SOD), the main also shown that certain types of cell clones over- - defense against O 2 radical-mediated oxidative damage, expressing glucose-6-phosphate dehydrogenase results in the present study and others.16,18 Increased activities in an increase in the level of GSH, which in turn leads to a decreased ROS production.35 Moreover, those of SOD lead to an increased production of H2O2 inside clones exhibited strong protection against oxidant- the PBMC, that can be detoxified to H2O by the catalase and GPx activities. However, since the mediated cell killing and inhibition of the nuclear catalase and GPx activities in PBMC of the active factor-kappa B (NF-B) activation in response to vitiligo patients are significantly reduced, then it is tumor necrosis factor- (TNF-). Therefore, it may be expected that H2O2 will accumulate inside the cells at assumed that significant reduction in intracellular GSH toxic level. Consequently, ROS burst and the cells may concentration may be responsible for the other changes enter a state of oxidative stress. It is not really known of imbalanced antioxidant enzymes and increased whether the reduced catalase and GPx activities are due MDA concentrations. In the present study, aspirin is to a primary defect or a result of an increased ROS shown to improve significantly the oxidative status of production. The present study advocates the latter, the PBMC and possibly epidermal melanocytes as since both enzymes activities have increased following evidenced by the halting of the activity of vitiligo and - induction of stability in all patients. This improvement the aspirin treatment. Reduction in the O 2 release from the impaired mitochondria may lead to reduced in the oxidative status following the aspirin treatment may be attributed primarily to the significant increase formation of H2O2. Here comes the vital role of intracellular GSH. Decreased intracellular GSH in the intracellular GSH concentration, which may be concentration may be the primary event that may cause explained as follows. The salicylate moiety of aspirin changes in cellular antioxidant enzymes and lipid may stimulate the de novo biosynthesis of GSH by yet peroxidation. It was shown that cells that had unknown mechanism. A recent study showed that in rat decreased level of GSH undergo apoptosis when brain tissue subjected to hypoxia, oral administration of exposed to external oxidative stimuli.32 U SOD/U cat aspirin reduces oxidative stress (increased GSH level and GPx activities and decreased MDA concentrations) ratio is a parameter of cell susceptibility to oxidative 36 stress.33 However, this parameter of susceptibility and inducible nitric oxide synthase (iNOs) activities. becomes critical if intracellular GSH concentration is However, salicylic acid produced greater reduction in significantly reduced. In stable vitiligo, in spite of oxidative stress and iNOs activities than aspirin, which increased SOD activities of PBMC, the susceptibility may indicate that the salicylate moiety of aspirin may to oxidative stress was decreased, likely due to the have a stimulatory effect on GSH biosynthesis and normal level of GSH.18 Intracellular GSH functions in subsequently modulating the related antioxidant so many ways. It serves as a substrate for GPx in a enzymes activities. In addition, aspirin per se was reaction leading to detoxification of H O ; hence a found to be a strong antioxidant. Aspirin was reported 2 2 to be an efficient hydroxyl radical scavenger and reduction in its concentration may cause decreased inhibitor of lipid peroxidation, DNA strand breakage, GPx activities and consequently H O burst. By 2 2 NF- B and TNF-.37,38 Moreover, aspirin was found to extrapolation, a consequence of reduced intracellular  reduce both O- and lipid peroxidation in rat brain level of GSH in melanocytes of active vitiligo is an 2 39 increased ROS production, which makes these cells homogenate. This may explain the decreased SOD more sensitive to oxidative burst triggered by external activities of PBMC that may be significantly increased - stimuli such as trauma, emotional stress or viral in the presence of high O 2. Consequently, the overall infection. Consequently, this may lead to further result of aspirin treatment is a reduction in ROS, depletion of GSH that may upregulate the release of mediated primarily by the increased level of apoptotic cytokines that could ultimately bring about intracellular GSH that eventually can lead

to an improvement in the oxidative status of PBMC and melanocytes. There is strong evidence suggesting 5. Hann SK, Chun WH. Autocytotoxic hypothesis for the that active vitiligo may be regarded as an inflammatory destruction of melanocytes as the cause of vitiligo. In: skin disease.40,41 Several reports have shown that Hann SK, Nordlund JJ. Vitiligo. Oxford: Blackwell increased ROS production during aging might enhance Science, 2000: 137- 41. 20,21 the expression of the inflammatory COX-2 mRNA. 6. Hasse S, Gibbons NCJ, Rokos H, Marles LK, Therefore, decreasing the ROS production by oral Schallreuter KU. Perturbed 6-tetrahydrobiopterin aspirin treatment may reduce the inflammatory recycling via decreased dihydrobiopterin reductase in prostaglandins, mediators, cytokines and proteases that vitiligo: more evidence for H2O2 stress. J Invest may be responsible for the downregulation of the Dermatol 2004; 122: 307- 13. proliferative capacities of PBMC and melanocytes in the active phase of vitiligo leading to stability of the 7. Orecchia G. Neural pathogenesis. In: Hann SK, disease and increased probability of repigmentation. Nordlund JJ. Vitiligo. Oxford: Blackwell Science, 2000: Furthermore, GPx was found to reduce semi-stable 142-50. hydroxyperoxides to less reactive alcohols and 8. Le Poole IC, Das PK, van den Wijngaard RM et al. removes H O involved in inflammation.42 Therefore, 2 2 Review of the etiopathomechanism of vitiligo: a increased GPx activities following aspirin treatment, convergence theory. Exp Dermatol 1993;2:145-53. further confer protection against ROS production by inflammatory cells. Consequently, this may lead to 9. Ortonne JP, Bose SK. Vitiligo: where do we stand? modulation of the enhanced immune response that may Pigment Cell Res 1993; 6: 62-72. accelerate melanocyte apoptosis in active vitiligo. 10. Schallreuter KU, Moore J, Wood JM, et al. In vivo and In conclusion, oral low-dose aspirin may be used to in vitro evidence for hydrogen peroxide (H2O2) improve the oxidative status of PBMC and accumulation in the epidermis of patients with vitiligo melanocytes that may greatly influence the activity of and its successful removal by a UVB-activated pseudocatalase. J Invest Dermatol Symp Proc 1999; 4: vitiligo and induce stability that is essential before the 91-6. start of any medical or surgical therapeutic modality. 11. Maresca V. Roccella M, Roccella F, et al. Increased Acknowledgement sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo. J The accomplishment of this work was made possible Invest Dermatol 1997; 109: 310-13. with the full cooperation and compliance of patients. Thanks to Dr. J. Atiea, consultant endocrinologist, 12. Jimbow K, Chen H, Park JS, Thomas PD. Increased Good Hope hospital, Birmingham, UK, for his useful sensitivity of melanocytes to oxidative stress and suggestions and comments on the treatment protocol. abnormal expression of tyrosinase-related protein in vitiligo. Br J Dermatol 2001; 144: 55-65.

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