- Email: [email protected]
Increased oxidative DNA damage in mononuclear leukocytes in vitiligo
Mutation Research 556 (2004) 101–106
Increased oxidative DNA damage in mononuclear leukocytes in vitiligo Lisa Giovannellia,∗ , Serena Bellandib , Vanessa Pitozzia , Paolo Fabbrib , Piero Dolaraa , Silvia Morettib a
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Florence, Italy b Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Firenze, Florence, Italy Received 1 March 2004; received in revised form 8 July 2004; accepted 14 July 2004
Abstract Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo. © 2004 Elsevier B.V. All rights reserved. Keywords: Vitiligo; Peripheral blood leukocytes; Oxidative stress; Oxidative DNA damage; Comet assay
1. Introduction Vitiligo is an acquired pigmentary disorder of the skin characterized by circumscribed white spots on ∗ Corresponding author. Tel.: +39 055 4271 322; fax: +39 055 4271 280. E-mail address: [email protected] (L. Giovannelli).
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.07.005
the skin that usually progress with enlargment of existing lesions and appearance of new ones during life time. The cause is a substantial loss of functioning melanocytes in the depigmented patches. The aetiology is still unknown and pathogenesis has not been completely clarified so far; various hypotheses have been proposed. They include the genetic hypothesis, pointing out on “intrinsic” inherent melanocyte defect
102
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
[1], the autoimmune hypothesis, based on the presence of melanocyte-specific autoantibodies in some patients [1] and of skin-homing melanocyte-specific cytotoxic T lymphocytes in patients with autoimmune vitiligo [2], and the neural hypothesis, suggesting that an increase in catecholamine release or synthesis is correlated with disease activity [3]. Finally, the autocytotoxic hypothesis suggests that melanocyte impairment could be related initially to an increased oxidative stress [4,5], with consequent induction of H2 O2 accumulation in the epidermis of patients with active disease [6]. Lower levels of catalase were demonstrated in the epidermis of both lesional and nonlesional skin of vitiligo patients [7] and in the active phases of disease an imbalance of antioxidants was found in both the epidermis [8] and peripheral blood mononuclear cells (PBMC), correlated to an increased intracellular reactive oxygen species (ROS) production [9]. These data suggest that the entire epidermis, and even PBMC, may be involved in vitiligo aetiology. ROS can interact with macromolecules such as proteins, membrane lipids and nucleic acids. The lesions induced by ROS on DNA are typically breakage of a single filament or oxidative alteration of purinic and/or pyrimidinic bases [10]. The aim of this study was to evaluate oxidative DNA damage in peripheral blood leukocytes of patients affected by active vitiligo, in order to verify whether an oxidation imbalance was also evident at the nuclear level in these cells. The comet assay was used to assess DNA oxidative damage in PBL of patients affected by active vitiligo and of healthy subjects. DNA strand breaks (SBs) and oxidized bases were measured separately in the polymorphonuclear and mononuclear blood cells.
2. Materials and methods 2.1. Subjects Twenty-one vitiligo patients and 21 healthy control subjects, matched for age, gender and smoking, were studied. There were 16 females and 5 males, and 18 non smokers and 3 smokers, respectively, in both groups, with age ranging from 15 to 70 years (means ± S.E.: 51.06 ± 3.9 in patients and 44.70 ± 3.5 in healthy subjects). The study was conducted according to the
Helsinki Declaration. All patients were affected by active (with new or expanding lesions in the last 3 months) non-segmental vitiligo, and had suspended any specific therapy at least 2 months before testing; three patients were afflicted by thyreoiditis and other three patients presented arterial hypertension (these last three patients were taking antihypertensive drugs). 2.2. Collection of blood samples and cell isolation Peripheral blood samples (total 3 ml) were collected from an antecubital vein into EDTA-containing vacutainer tubes, stored at 10 ◦ C and kept in the dark to prevent further DNA damage. For the analysis of DNA damage in leukocytes, 15 l of fresh whole blood were transferred to an Eppendorf tube, mixed with 85 l of melted agarose (low melting point, LMA, Fisher Scientific, UK) and layered onto a microscopy slide. After allowing for agarose solidification, the slides were run through the comet assay as previously described [11]. 2.3. Comet assay The slides with the agarose-embedded cells were subjected to a lysis step (1 h incubation at 4 ◦ C in 1% N-lauroyl-sarcosine, 2.5 M NaCl, 100 mM Na2 EDTA, 1% TritonX-100, 10% dimethylsulfoxide, pH 10.0). After the lysis step, slides were washed three times in enzyme buffer (40 mM HEPES-KOH, pH 8.0, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, BSA) and then incubated at 37 ◦ C for 60 min with 80 l of the Escherichia coli enzyme formamidopyrimidine DNA glycosylase (FPG, 1:1000, kindly provided by Dr. A.R. Collins, University of Oslo, Norway) for purine oxidation detection. Control slides of the same sample were incubated in enzyme buffer without FPG. Each experimental point was run in duplicate, thus four slides were run for each subject. All experimental slides were placed in an ice-cold electrophoresis chamber containing alkaline electrophoresis solution (300 mM NaOH, 1 mM Na2 EDTA, pH 13.0) for 20 min to allow DNA unwinding. The electrophoresis was subsequently conducted for 20 min at 0.8 V/cm and 300 mA. At the end of the electrophoresis the slides were washed with neutralization buffer (40 mM Tris–HCl, pH 7.4), stained with ethidium bromide overnight and analyzed the following day.
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
Microscopical analysis was carried out by means of a Labophot-2 microscope (Nikon, Tokyo, Japan) provided with epifluorescence and equipped with a rhodamine filter (excitation wavelength 546 nm; barrier 580 nm). The images of 50 randomly chosen nuclei per slide were captured and analyzed using a custom-made imaging software coupled with a CCD camera (model C5985, Hamamatsu, Sunayama-Cho, Japan). Polymorphonuclear and mononuclear leukocytes (mostly represented by lymphocytes) were visually recognized on the basis of their nuclear morphology and analyzed separately as previously described [11]. Each slide was scored twice, once for granulocytes and once for mononuclear leukocytes, each time capturing 50 images. For each image, the program calculated the total fluorescence (i.e., the sum of the gray levels of all pixels) distribution along the longer axis of the nucleus and the fluorescence distribution of the head and of the tail of the comet, respectively. DNA damage was expressed as the percentage of total fluorescence migrated in the tail for each nucleus (%DNA in tail). This value was then averaged over the 50 nuclei measured per slide and the duplicate values were further averaged. Detection of oxidative DNA base damage was carried out by means of the FPG enzyme, which introduces breaks at sites of oxidized purines such as 8-oxo-2 -deoxyguanosine [12]. Thus, the value of DNA damage obtained in slides without enzyme incubation estimated the basal number of DNA strand breaks, whereas specific oxidative damage on purines was assessed for each subject by subtracting the basal number of breaks (buffer-incubated slides) from the number of breaks obtained incubating the slides with FPG.
103
the blood diluted more than 5000 times, the effects due to the antioxidant plasma activity were avoided. After completing the incubation with H2 O2 (two slides for each experimental point), the slides were transferred to the lysis solution and run through the rest of the procedure as described above. The damage induced by H2 O2 was measured as DNA strand breaks, without employing the FPG enzyme. 2.5. Statistical methods The values were expressed as mean ± S.E. (standard error). Each patient was paired to a control subject on the basis of sex, age and smoking status, and differences between groups were evaluated by means of the paired Student’s t-test. Correlations were performed using linear regression analysis, and the significance level was considered as P < 0.05.
3. Results Fig. 1 shows the endogenous levels of DNA damage (mean %DNA in tail ± S.E.) in mononuclear leukocytes of vitiligo patients compared to control subjects. There were no significant differences in the levels of strand breaks between the two groups, being the
2.4. Exposure to H2 O2 In order to evaluate the response of mononuclear leukocytes to a DNA-damaging agent, whole blood slides obtained from five vitiligo patients and five controls were exposed to H2 O2 (from 1 to 100 mM in PBS). As a reference, slides of both patients and controls were incubated in PBS. The incubation with H2 O2 was conducted for 15 min, at 4 ◦ C to inhibit DNA repair. Immediately after the inclusion of the cells in agarose and the solidification of the gel, the slides were immersed in a large volume (100 ml per slide) of PBS containing H2 O2 at the desired concentration. In this way, being
Fig. 1. DNA damage in non-isolated mononuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black columns) and controls (grey columns). DNA damage is expressed as the mean ± S.E. (n = 21) of the percent DNA migrated in the tail of the comet (%DNA in tail). Both the basal level of DNA damage (breaks, columns on the left) and DNA damage detected as FPGsensitive sites (oxidative damage, columns on the right) are shown. *P < 0.05 statistically significant difference between vitiligo and controls (Student’s t-test).
104
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
Fig. 2. DNA damage in non-isolated polymorphonuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black columns) and controls (grey columns). DNA damage is expressed as the mean ± S.E. (n = 5) of the percent DNA migrated in the tail of the comet (%DNA in tail). Both the basal level of DNA damage (breaks, columns on the left) and DNA damage detected as FPG-sensitive sites (oxidative damage, columns on the right) are shown.
corresponding values 4.38 ± 0.32 in patients and 4.81 ± 0.23 in controls. On the contrary, increased levels of FPG-sensitive oxidative DNA damage were found in vitiligo patients (11.78 ± 2.02) compared to control subjects (7.17 ± 1.60), and the difference was statistically significant (+61%, P < 0.05, paired Student’s t-test). When DNA damage was evaluated in polymorphonuclear cells (Fig. 2), no significant differences between vitiligo patients and controls were found either in strand breaks (6.33 ± 0.56 in vitiligo versus 6.07 ± 0.81 in controls) or in oxidative DNA damage (18.17 ± 1.43 in patients versus 17.60 ± 2.19 in healthy subjects). In order to evaluate whether vitiligo and control mononuclear leukocytes behaved differently when exposed to a DNA-damaging agent, the cells from a subset of patients and controls were exposed in vitro to various concentrations of H2 O2 . The results of these experiments are shown in Fig. 3. A dose-dependent increase in DNA damage was found upon H2 O2 exposure (slopes significantly different from zero for both curves, P < 0.05). Linear regression analysis also showed that the slopes of the two curves were not statistically different from each other, indicating that the response to in vitro oxidative stress was similar in vitiligo patients and control subjects.
Fig. 3. In vitro-induced DNA damage in non-isolated mononuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black dots) and controls (white dots), exposed to increasing concentrations of H2 O2 (from 0 to 100 mM). The level of H2 O2 induced DNA breaks is expressed as the mean ± S.E. (n = 5) of the percent DNA migrated in the tail of the comet (%DNA in tail). Equations calculated by means of linear regression analysis: y = 0.08x + 5.74, R2 = 0.91, P = 0.04 for vitiligo; y = 0.07x + 5.21, R2 = 0.95, P = 0.03 for controls.
4. Discussion In active vitiligo an increased oxidative stress of the entire epidermal compartment has been demonstrated [5–8]. Recently, the activity of vitiligo has been associated with a systemic oxidative stress, evaluated by assessing the intracellular generation of ROS and the antioxidant pattern in PBMC [9]. In particular, catalase activity, reduced glutathione and Vitamin E levels were decreased, and this imbalance of antioxidants was associated with hyperproduction of ROS. These aspects of oxidative stress are related to cytoplasmic events, and no report has been produced so far about the nuclear effects of oxidative stress in vitiligo. To our knowledge, the present study is the first report concerning the assessment of oxidative DNA damage in vitiligo patients. We evaluated by means of the alkaline comet assay, the basal levels of DNA strand breaks and oxidized bases in polymorphonuclear and mononuclear leukocytes from fresh whole blood of patients with active vitiligo. This procedure was chosen because it does not involve previous isolation of the leukocyte subtypes, and it has been previously shown that gradient isolation of these cells induces both an increase in strand breaks and an enhanced response to in vitro oxidative stress
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
[11]. Thus, it is feasible that the artefacts involved in the measurement of DNA damage in isolated blood cells subpopulations would make it difficult to detect variations between experimental groups, especially when the differences are not large. We found a significantly higher level of oxidative DNA damage in vitiligo patients versus controls in mononuclear leukocytes, whereas the strand break levels were equivalent in the two groups. These data suggest the presence of a systemic oxidative stress at nuclear level in active vitiligo. However, mononuclear leukocytes from whole blood of vitiligo subjects challenged in vitro with an oxidizing agent did not show an increased induction of DNA damage as compared to those prepared from control subjects. Thus, although their basal level of oxidation is increased, the short-term response of vitiligo mononuclear leukocytes to oxidative stress appears to be normal. These data suggest that the antioxidant defense system might be normal in patients with vitiligo. Thus, a possible cause of increased oxidative DNA damage in mononuclear leukocytes might rather be increased ROS production, as has been reported in vitiligo PBMC [9]. This work also shows that, at variance with mononuclear cells, DNA damage in polymorphonuclear cells from vitiligo patients was not different from that of controls, indicating that in vitiligo the mononuclear represents the most oxidation-sensitive leukocyte population. Alterations in the anti-oxidant status have been previously reported in vitiligo mononuclear leukocytes and not in red blood cells [9]. The reason for this difference in oxidation status among cell types might be either a differential sensitivity to DNA damage, or repair capacity. The induction and repair of DNA lesions by gamma-irradiation were reported to be comparable in human blood granulocytes and lymphocytes [13,14]. However, it has been shown that lymphocytes are less efficient than granulocytes in removing UV damage from immunoglobulin genes [15]. With regard to DNA oxidation damage, no difference was found between polymorphonuclear and mononuclear leukocytes from normal subjects upon in vitro H2 O2 challenge conducted under experimental conditions identical to those of the present work [11]. On the other hand, it has to be noted that the polymorphonuclear leukocyte subtype has been found
105
to be more damaged than the mononuclear cells in other diseases with an oxidative stress component. For example, in a disease related to energy metabolism perturbation, such as diabetes, the polymorphonuclear leukocytes appeared to be more vulnerable to oxidative stress than the mononuclear [16,17]. Hypertensive patients also showed increased oxidative stress in polymorphonuclear and not in mononuclear leukocytes [18]. As a whole, these data do not support an intrinsic increased sensitivity to oxidation of mononuclear as compared to polymorphonuclear leukocytes in the presence of systemic oxidative stress. An alternative hypothesis might be that an oxidation imbalance in the mononuclear compartment is somehow related to the immune dysfunctions that have been repeatedly reported in vitiligo [1,2]. In fact, increased oxidative DNA damage in lymphocytes and not in polymorphonuclear leukocytes from patients with certain autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, has been reported [19]. Measuring DNA damage with the comet assay in human epidermis is not easy for the difficulty of obtaining viable isolated cells from this tissue. However, future assessment of DNA oxidation in the epidermis might shed further light on the role of oxidative stress in vitiligo. In conclusion, this study supports the hypothesis that in active vitiligo a systemic oxidative stress exists, and shows that it can be demonstrated at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear, and not in the polymorphonuclear, compartment of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.
Acknowledgements The Authors wish to thank Dr. Andrew Collins of the Institute for Nutrition Research, University of Oslo, Norway, for kindly providing the enzyme FPG. Dr. V. Pitozzi was supported by an AIRC–FIRC fellowship. This work was also supported by EU grants QLKI1999-00346, QLRT-1999-00505 and QLK1-CT-199900568, and MIUR (Ministero Istruzione Universit`a e Ricerca) 40 and 60%.
106
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
References [1] J.J. Nordlund, J.P. Ortonne, Vitiligo Vulgaris in the Pigmentary System , Oxford University Press, New York, 1998. [2] G.S. Ogg, P.R. Dumbar, P. Romero, J.L. Chen, V. Cerundolo, High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo , J. Exp. Med. 188 (1998) 1203–1208. [3] M.L. Cucchi, P. Frattini, G. Santagostino, G. Orecchia, Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo , Pigment Cell Res. 13 (2000) 28–32. [4] K.U. Schallreuter, J.M. Wood, M.R. Pittelkow, M. Gutlich, K.R. Lemke, W. Rodl, N.N. Swanson, K. Hitzemann, I. Ziegler, Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin , Science 263 (1994) 1444–1446. [5] V. Maresca, M. Roccella, F. Roccella, E. Camera, G. Del Porto, S. Passi, P. Grammatico, M. Picardo, Increased sensitivity to peroxidative agents as a possibile pathogenic factor of melanocyte damage in vitiligo , J. Invest. Dermatol. 109 (1997) 1081–1085. [6] K.U. Schallreuter, J. Moore, J.M. Wood, W.D. Beazley, D.C. Gaze, D.J. Tobin, H.S. Marshall, A. Panske, E. Panzig, N.A. Hibberts, In vivo and in vitro evidence for hydrogen peroxide (H2 O2 ) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase , J. Invest. Dermatol. Symp. Proc. 4 (1999) 91–96. [7] K.U. Schallreuter, J.M. Wood, J. Berger, Low catalase levels in the epidermis of patients with vitiligo , J. Invest. Dermatol. 97 (1991) 1081–1085. [8] S. Passi, M. Grandinetti, F. Maggio, A. Stancato, C. De Luca, Epidermal oxidative stress in vitiligo , Pigment Cell Res. 11 (1998) 81–85. [9] M.L. Dell’Anna, V. Maresca, S. Briganti, E. Camera, M. Falchi, M. Picardo, Mitochondrial impairment in peripheral blood mononuclear cells during the active phase of vitiligo , J. Invest. Dermatol. 117 (2001) 908–913.
[10] B. Halliwell, O.I. Aruoma, DNA damage by oxygen-derived species , FEBS Lett. 281 (1991) 9–19. [11] L. Giovannelli, V. Pitozzi, S. Riolo, P. Dolara, Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay , Mutat. Res. 538 (2003) 71–80. [12] M. Dizdaroglu, Chemical determination of free-radical induced damage to DNA , Free Radic. Biol. Med. 10 (1991) 225– 242. [13] Vijayalaxmi, G.H. Strauss, R.R. Tice, An analysis of gammaray-induced DNA damage in human blood leukocytes, lymphocytes and granulocytes , Mutat. Res. 292 (1993) 123–128. [14] M.H. Lankinen, L.M. Vilpo, J.A. Vilpo, UV- and gammairradiation-induced DNA single-strand breaks and their repair in human blood granulocytes and lymphocytes , Mutat. Res. 352 (1996) 31–38. [15] M.S. Bianchi, N.O. Bianchi, A. de la Chapelle, Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes , Mutat. Res. 232 (1990) 89–97. [16] V. Pitozzi, L. Giovannelli, G. Bardini, C.M. Rotella, P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes , Mutat. Res. 529 (2003) 129–133. [17] M.P.A. Hannon-Fletcher, M.J. O’Kane, K.W. Moles, C. Weatherup, C.R. Barnett, Y.A. Barnett, Levels of peripheral blood cell DNA damage in insulin-dependent diabetes mellitus human subjects , Mutat. Res. 460 (2000) 53–60. [18] K. Yasunari, K. Maeda, M. Nakamura, J. Yoshikawa, Oxidative stress in leukocytes is a possible link between blood pressure, blood glucose, and C-reacting protein , Hypertension 39 (2002) 777–780. [19] S. Bashir, G. Harris, M.A. Denman, D.R. Blake, P.G. Winyard, Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases , Ann. Rheum. Dis. 52 (1993) 659–666.
Increased oxidative DNA damage in mononuclear leukocytes in vitiligo Lisa Giovannellia,∗ , Serena Bellandib , Vanessa Pitozzia , Paolo Fabbrib , Piero Dolaraa , Silvia Morettib a
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Florence, Italy b Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Firenze, Florence, Italy Received 1 March 2004; received in revised form 8 July 2004; accepted 14 July 2004
Abstract Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo. © 2004 Elsevier B.V. All rights reserved. Keywords: Vitiligo; Peripheral blood leukocytes; Oxidative stress; Oxidative DNA damage; Comet assay
1. Introduction Vitiligo is an acquired pigmentary disorder of the skin characterized by circumscribed white spots on ∗ Corresponding author. Tel.: +39 055 4271 322; fax: +39 055 4271 280. E-mail address: [email protected] (L. Giovannelli).
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.07.005
the skin that usually progress with enlargment of existing lesions and appearance of new ones during life time. The cause is a substantial loss of functioning melanocytes in the depigmented patches. The aetiology is still unknown and pathogenesis has not been completely clarified so far; various hypotheses have been proposed. They include the genetic hypothesis, pointing out on “intrinsic” inherent melanocyte defect
102
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
[1], the autoimmune hypothesis, based on the presence of melanocyte-specific autoantibodies in some patients [1] and of skin-homing melanocyte-specific cytotoxic T lymphocytes in patients with autoimmune vitiligo [2], and the neural hypothesis, suggesting that an increase in catecholamine release or synthesis is correlated with disease activity [3]. Finally, the autocytotoxic hypothesis suggests that melanocyte impairment could be related initially to an increased oxidative stress [4,5], with consequent induction of H2 O2 accumulation in the epidermis of patients with active disease [6]. Lower levels of catalase were demonstrated in the epidermis of both lesional and nonlesional skin of vitiligo patients [7] and in the active phases of disease an imbalance of antioxidants was found in both the epidermis [8] and peripheral blood mononuclear cells (PBMC), correlated to an increased intracellular reactive oxygen species (ROS) production [9]. These data suggest that the entire epidermis, and even PBMC, may be involved in vitiligo aetiology. ROS can interact with macromolecules such as proteins, membrane lipids and nucleic acids. The lesions induced by ROS on DNA are typically breakage of a single filament or oxidative alteration of purinic and/or pyrimidinic bases [10]. The aim of this study was to evaluate oxidative DNA damage in peripheral blood leukocytes of patients affected by active vitiligo, in order to verify whether an oxidation imbalance was also evident at the nuclear level in these cells. The comet assay was used to assess DNA oxidative damage in PBL of patients affected by active vitiligo and of healthy subjects. DNA strand breaks (SBs) and oxidized bases were measured separately in the polymorphonuclear and mononuclear blood cells.
2. Materials and methods 2.1. Subjects Twenty-one vitiligo patients and 21 healthy control subjects, matched for age, gender and smoking, were studied. There were 16 females and 5 males, and 18 non smokers and 3 smokers, respectively, in both groups, with age ranging from 15 to 70 years (means ± S.E.: 51.06 ± 3.9 in patients and 44.70 ± 3.5 in healthy subjects). The study was conducted according to the
Helsinki Declaration. All patients were affected by active (with new or expanding lesions in the last 3 months) non-segmental vitiligo, and had suspended any specific therapy at least 2 months before testing; three patients were afflicted by thyreoiditis and other three patients presented arterial hypertension (these last three patients were taking antihypertensive drugs). 2.2. Collection of blood samples and cell isolation Peripheral blood samples (total 3 ml) were collected from an antecubital vein into EDTA-containing vacutainer tubes, stored at 10 ◦ C and kept in the dark to prevent further DNA damage. For the analysis of DNA damage in leukocytes, 15 l of fresh whole blood were transferred to an Eppendorf tube, mixed with 85 l of melted agarose (low melting point, LMA, Fisher Scientific, UK) and layered onto a microscopy slide. After allowing for agarose solidification, the slides were run through the comet assay as previously described [11]. 2.3. Comet assay The slides with the agarose-embedded cells were subjected to a lysis step (1 h incubation at 4 ◦ C in 1% N-lauroyl-sarcosine, 2.5 M NaCl, 100 mM Na2 EDTA, 1% TritonX-100, 10% dimethylsulfoxide, pH 10.0). After the lysis step, slides were washed three times in enzyme buffer (40 mM HEPES-KOH, pH 8.0, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, BSA) and then incubated at 37 ◦ C for 60 min with 80 l of the Escherichia coli enzyme formamidopyrimidine DNA glycosylase (FPG, 1:1000, kindly provided by Dr. A.R. Collins, University of Oslo, Norway) for purine oxidation detection. Control slides of the same sample were incubated in enzyme buffer without FPG. Each experimental point was run in duplicate, thus four slides were run for each subject. All experimental slides were placed in an ice-cold electrophoresis chamber containing alkaline electrophoresis solution (300 mM NaOH, 1 mM Na2 EDTA, pH 13.0) for 20 min to allow DNA unwinding. The electrophoresis was subsequently conducted for 20 min at 0.8 V/cm and 300 mA. At the end of the electrophoresis the slides were washed with neutralization buffer (40 mM Tris–HCl, pH 7.4), stained with ethidium bromide overnight and analyzed the following day.
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
Microscopical analysis was carried out by means of a Labophot-2 microscope (Nikon, Tokyo, Japan) provided with epifluorescence and equipped with a rhodamine filter (excitation wavelength 546 nm; barrier 580 nm). The images of 50 randomly chosen nuclei per slide were captured and analyzed using a custom-made imaging software coupled with a CCD camera (model C5985, Hamamatsu, Sunayama-Cho, Japan). Polymorphonuclear and mononuclear leukocytes (mostly represented by lymphocytes) were visually recognized on the basis of their nuclear morphology and analyzed separately as previously described [11]. Each slide was scored twice, once for granulocytes and once for mononuclear leukocytes, each time capturing 50 images. For each image, the program calculated the total fluorescence (i.e., the sum of the gray levels of all pixels) distribution along the longer axis of the nucleus and the fluorescence distribution of the head and of the tail of the comet, respectively. DNA damage was expressed as the percentage of total fluorescence migrated in the tail for each nucleus (%DNA in tail). This value was then averaged over the 50 nuclei measured per slide and the duplicate values were further averaged. Detection of oxidative DNA base damage was carried out by means of the FPG enzyme, which introduces breaks at sites of oxidized purines such as 8-oxo-2 -deoxyguanosine [12]. Thus, the value of DNA damage obtained in slides without enzyme incubation estimated the basal number of DNA strand breaks, whereas specific oxidative damage on purines was assessed for each subject by subtracting the basal number of breaks (buffer-incubated slides) from the number of breaks obtained incubating the slides with FPG.
103
the blood diluted more than 5000 times, the effects due to the antioxidant plasma activity were avoided. After completing the incubation with H2 O2 (two slides for each experimental point), the slides were transferred to the lysis solution and run through the rest of the procedure as described above. The damage induced by H2 O2 was measured as DNA strand breaks, without employing the FPG enzyme. 2.5. Statistical methods The values were expressed as mean ± S.E. (standard error). Each patient was paired to a control subject on the basis of sex, age and smoking status, and differences between groups were evaluated by means of the paired Student’s t-test. Correlations were performed using linear regression analysis, and the significance level was considered as P < 0.05.
3. Results Fig. 1 shows the endogenous levels of DNA damage (mean %DNA in tail ± S.E.) in mononuclear leukocytes of vitiligo patients compared to control subjects. There were no significant differences in the levels of strand breaks between the two groups, being the
2.4. Exposure to H2 O2 In order to evaluate the response of mononuclear leukocytes to a DNA-damaging agent, whole blood slides obtained from five vitiligo patients and five controls were exposed to H2 O2 (from 1 to 100 mM in PBS). As a reference, slides of both patients and controls were incubated in PBS. The incubation with H2 O2 was conducted for 15 min, at 4 ◦ C to inhibit DNA repair. Immediately after the inclusion of the cells in agarose and the solidification of the gel, the slides were immersed in a large volume (100 ml per slide) of PBS containing H2 O2 at the desired concentration. In this way, being
Fig. 1. DNA damage in non-isolated mononuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black columns) and controls (grey columns). DNA damage is expressed as the mean ± S.E. (n = 21) of the percent DNA migrated in the tail of the comet (%DNA in tail). Both the basal level of DNA damage (breaks, columns on the left) and DNA damage detected as FPGsensitive sites (oxidative damage, columns on the right) are shown. *P < 0.05 statistically significant difference between vitiligo and controls (Student’s t-test).
104
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
Fig. 2. DNA damage in non-isolated polymorphonuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black columns) and controls (grey columns). DNA damage is expressed as the mean ± S.E. (n = 5) of the percent DNA migrated in the tail of the comet (%DNA in tail). Both the basal level of DNA damage (breaks, columns on the left) and DNA damage detected as FPG-sensitive sites (oxidative damage, columns on the right) are shown.
corresponding values 4.38 ± 0.32 in patients and 4.81 ± 0.23 in controls. On the contrary, increased levels of FPG-sensitive oxidative DNA damage were found in vitiligo patients (11.78 ± 2.02) compared to control subjects (7.17 ± 1.60), and the difference was statistically significant (+61%, P < 0.05, paired Student’s t-test). When DNA damage was evaluated in polymorphonuclear cells (Fig. 2), no significant differences between vitiligo patients and controls were found either in strand breaks (6.33 ± 0.56 in vitiligo versus 6.07 ± 0.81 in controls) or in oxidative DNA damage (18.17 ± 1.43 in patients versus 17.60 ± 2.19 in healthy subjects). In order to evaluate whether vitiligo and control mononuclear leukocytes behaved differently when exposed to a DNA-damaging agent, the cells from a subset of patients and controls were exposed in vitro to various concentrations of H2 O2 . The results of these experiments are shown in Fig. 3. A dose-dependent increase in DNA damage was found upon H2 O2 exposure (slopes significantly different from zero for both curves, P < 0.05). Linear regression analysis also showed that the slopes of the two curves were not statistically different from each other, indicating that the response to in vitro oxidative stress was similar in vitiligo patients and control subjects.
Fig. 3. In vitro-induced DNA damage in non-isolated mononuclear leukocytes, analyzed in whole-blood slides prepared from vitiligo patients (black dots) and controls (white dots), exposed to increasing concentrations of H2 O2 (from 0 to 100 mM). The level of H2 O2 induced DNA breaks is expressed as the mean ± S.E. (n = 5) of the percent DNA migrated in the tail of the comet (%DNA in tail). Equations calculated by means of linear regression analysis: y = 0.08x + 5.74, R2 = 0.91, P = 0.04 for vitiligo; y = 0.07x + 5.21, R2 = 0.95, P = 0.03 for controls.
4. Discussion In active vitiligo an increased oxidative stress of the entire epidermal compartment has been demonstrated [5–8]. Recently, the activity of vitiligo has been associated with a systemic oxidative stress, evaluated by assessing the intracellular generation of ROS and the antioxidant pattern in PBMC [9]. In particular, catalase activity, reduced glutathione and Vitamin E levels were decreased, and this imbalance of antioxidants was associated with hyperproduction of ROS. These aspects of oxidative stress are related to cytoplasmic events, and no report has been produced so far about the nuclear effects of oxidative stress in vitiligo. To our knowledge, the present study is the first report concerning the assessment of oxidative DNA damage in vitiligo patients. We evaluated by means of the alkaline comet assay, the basal levels of DNA strand breaks and oxidized bases in polymorphonuclear and mononuclear leukocytes from fresh whole blood of patients with active vitiligo. This procedure was chosen because it does not involve previous isolation of the leukocyte subtypes, and it has been previously shown that gradient isolation of these cells induces both an increase in strand breaks and an enhanced response to in vitro oxidative stress
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
[11]. Thus, it is feasible that the artefacts involved in the measurement of DNA damage in isolated blood cells subpopulations would make it difficult to detect variations between experimental groups, especially when the differences are not large. We found a significantly higher level of oxidative DNA damage in vitiligo patients versus controls in mononuclear leukocytes, whereas the strand break levels were equivalent in the two groups. These data suggest the presence of a systemic oxidative stress at nuclear level in active vitiligo. However, mononuclear leukocytes from whole blood of vitiligo subjects challenged in vitro with an oxidizing agent did not show an increased induction of DNA damage as compared to those prepared from control subjects. Thus, although their basal level of oxidation is increased, the short-term response of vitiligo mononuclear leukocytes to oxidative stress appears to be normal. These data suggest that the antioxidant defense system might be normal in patients with vitiligo. Thus, a possible cause of increased oxidative DNA damage in mononuclear leukocytes might rather be increased ROS production, as has been reported in vitiligo PBMC [9]. This work also shows that, at variance with mononuclear cells, DNA damage in polymorphonuclear cells from vitiligo patients was not different from that of controls, indicating that in vitiligo the mononuclear represents the most oxidation-sensitive leukocyte population. Alterations in the anti-oxidant status have been previously reported in vitiligo mononuclear leukocytes and not in red blood cells [9]. The reason for this difference in oxidation status among cell types might be either a differential sensitivity to DNA damage, or repair capacity. The induction and repair of DNA lesions by gamma-irradiation were reported to be comparable in human blood granulocytes and lymphocytes [13,14]. However, it has been shown that lymphocytes are less efficient than granulocytes in removing UV damage from immunoglobulin genes [15]. With regard to DNA oxidation damage, no difference was found between polymorphonuclear and mononuclear leukocytes from normal subjects upon in vitro H2 O2 challenge conducted under experimental conditions identical to those of the present work [11]. On the other hand, it has to be noted that the polymorphonuclear leukocyte subtype has been found
105
to be more damaged than the mononuclear cells in other diseases with an oxidative stress component. For example, in a disease related to energy metabolism perturbation, such as diabetes, the polymorphonuclear leukocytes appeared to be more vulnerable to oxidative stress than the mononuclear [16,17]. Hypertensive patients also showed increased oxidative stress in polymorphonuclear and not in mononuclear leukocytes [18]. As a whole, these data do not support an intrinsic increased sensitivity to oxidation of mononuclear as compared to polymorphonuclear leukocytes in the presence of systemic oxidative stress. An alternative hypothesis might be that an oxidation imbalance in the mononuclear compartment is somehow related to the immune dysfunctions that have been repeatedly reported in vitiligo [1,2]. In fact, increased oxidative DNA damage in lymphocytes and not in polymorphonuclear leukocytes from patients with certain autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, has been reported [19]. Measuring DNA damage with the comet assay in human epidermis is not easy for the difficulty of obtaining viable isolated cells from this tissue. However, future assessment of DNA oxidation in the epidermis might shed further light on the role of oxidative stress in vitiligo. In conclusion, this study supports the hypothesis that in active vitiligo a systemic oxidative stress exists, and shows that it can be demonstrated at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear, and not in the polymorphonuclear, compartment of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.
Acknowledgements The Authors wish to thank Dr. Andrew Collins of the Institute for Nutrition Research, University of Oslo, Norway, for kindly providing the enzyme FPG. Dr. V. Pitozzi was supported by an AIRC–FIRC fellowship. This work was also supported by EU grants QLKI1999-00346, QLRT-1999-00505 and QLK1-CT-199900568, and MIUR (Ministero Istruzione Universit`a e Ricerca) 40 and 60%.
106
L. Giovannelli et al. / Mutation Research 556 (2004) 101–106
References [1] J.J. Nordlund, J.P. Ortonne, Vitiligo Vulgaris in the Pigmentary System , Oxford University Press, New York, 1998. [2] G.S. Ogg, P.R. Dumbar, P. Romero, J.L. Chen, V. Cerundolo, High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo , J. Exp. Med. 188 (1998) 1203–1208. [3] M.L. Cucchi, P. Frattini, G. Santagostino, G. Orecchia, Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo , Pigment Cell Res. 13 (2000) 28–32. [4] K.U. Schallreuter, J.M. Wood, M.R. Pittelkow, M. Gutlich, K.R. Lemke, W. Rodl, N.N. Swanson, K. Hitzemann, I. Ziegler, Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin , Science 263 (1994) 1444–1446. [5] V. Maresca, M. Roccella, F. Roccella, E. Camera, G. Del Porto, S. Passi, P. Grammatico, M. Picardo, Increased sensitivity to peroxidative agents as a possibile pathogenic factor of melanocyte damage in vitiligo , J. Invest. Dermatol. 109 (1997) 1081–1085. [6] K.U. Schallreuter, J. Moore, J.M. Wood, W.D. Beazley, D.C. Gaze, D.J. Tobin, H.S. Marshall, A. Panske, E. Panzig, N.A. Hibberts, In vivo and in vitro evidence for hydrogen peroxide (H2 O2 ) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase , J. Invest. Dermatol. Symp. Proc. 4 (1999) 91–96. [7] K.U. Schallreuter, J.M. Wood, J. Berger, Low catalase levels in the epidermis of patients with vitiligo , J. Invest. Dermatol. 97 (1991) 1081–1085. [8] S. Passi, M. Grandinetti, F. Maggio, A. Stancato, C. De Luca, Epidermal oxidative stress in vitiligo , Pigment Cell Res. 11 (1998) 81–85. [9] M.L. Dell’Anna, V. Maresca, S. Briganti, E. Camera, M. Falchi, M. Picardo, Mitochondrial impairment in peripheral blood mononuclear cells during the active phase of vitiligo , J. Invest. Dermatol. 117 (2001) 908–913.
[10] B. Halliwell, O.I. Aruoma, DNA damage by oxygen-derived species , FEBS Lett. 281 (1991) 9–19. [11] L. Giovannelli, V. Pitozzi, S. Riolo, P. Dolara, Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay , Mutat. Res. 538 (2003) 71–80. [12] M. Dizdaroglu, Chemical determination of free-radical induced damage to DNA , Free Radic. Biol. Med. 10 (1991) 225– 242. [13] Vijayalaxmi, G.H. Strauss, R.R. Tice, An analysis of gammaray-induced DNA damage in human blood leukocytes, lymphocytes and granulocytes , Mutat. Res. 292 (1993) 123–128. [14] M.H. Lankinen, L.M. Vilpo, J.A. Vilpo, UV- and gammairradiation-induced DNA single-strand breaks and their repair in human blood granulocytes and lymphocytes , Mutat. Res. 352 (1996) 31–38. [15] M.S. Bianchi, N.O. Bianchi, A. de la Chapelle, Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes , Mutat. Res. 232 (1990) 89–97. [16] V. Pitozzi, L. Giovannelli, G. Bardini, C.M. Rotella, P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes , Mutat. Res. 529 (2003) 129–133. [17] M.P.A. Hannon-Fletcher, M.J. O’Kane, K.W. Moles, C. Weatherup, C.R. Barnett, Y.A. Barnett, Levels of peripheral blood cell DNA damage in insulin-dependent diabetes mellitus human subjects , Mutat. Res. 460 (2000) 53–60. [18] K. Yasunari, K. Maeda, M. Nakamura, J. Yoshikawa, Oxidative stress in leukocytes is a possible link between blood pressure, blood glucose, and C-reacting protein , Hypertension 39 (2002) 777–780. [19] S. Bashir, G. Harris, M.A. Denman, D.R. Blake, P.G. Winyard, Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases , Ann. Rheum. Dis. 52 (1993) 659–666.