ORIGINAL ARTICLE
Membrane Lipid Alterations as a Possible Basis for Melanocyte Degeneration in Vitiligo Maria Lucia Dell’Anna1, Monica Ottaviani1, Veronica Albanesi1, Andrea Paro Vidolin2, Giovanni Leone2, Carmela Ferraro3, Andrea Cossarizza4, Luisa Rossi5 and Mauro Picardo1 The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. In order to evaluate this possible correlation we focused on the lipid component of cell membranes. We observed in vitiligo melanocytes, through FACS methods, an increased median fluorescence intensity of rhodamine 123 and C11-BODIPY581/591 indicating a spontaneous higher production of reactive oxygen species (ROS) and membrane lipoperoxidation, associated with an altered pattern of cardiolipin (CL) distribution, defined on the basis of the fluorescence pattern after staining with 10-nonyl acridine orange. We confirmed membrane peroxidation by confocal and contrast-phase microscopes and demonstrated impaired activity of the mitochondrial electron transport chain (ETC) complex I. Finally, we observed increased apoptotic events following exposure to the pro-oxidant cumene hydroperoxide by Annexin V/propidium iodide fluorescence. We hypothesize that in vitiligo melanocytes lipid instability, with a defect in the synthesis or recycling of CL, induces ETC impairment and ROS production. In basal conditions melanocytes maintain the redox balance whereas following chemical or physical stress ROS-mediated membrane peroxidation is increased with a possible further CL oxidation, leading to cell death or detachment. Journal of Investigative Dermatology (2007) 127, 1226–1233. doi:10.1038/sj.jid.5700700; published online 18 January 2007
INTRODUCTION Vitiligo is an acquired depigmenting disease whose etiopathogenesis has still to be completely defined. Among the possible mechanisms leading to the disappearance of functional melanocytes, the immune-mediated and the toxic ones are the most probable. Clinical and experimental evidence suggests the occurrence of an oxidative stress leading to melanocyte degeneration. However this process, as well as the executive disappearance mechanism, lacks a clear explanation (Le Poole et al., 1993; Taieb, 2000; Gauthier et al., 2003). In the epidermis from subjects with active vitiligo, an increased production of H2O2 has been reported and is associated with a lower expression and lower activity of the antioxidant enzyme catalase (Schallreuter et al., 1991, 1999). This could represent the biological basis 1
Laboratory of Cutaneous Physiopathology, IRCCS, Rome, Italy; 2Unity of Phototherapy, IRCCS, Rome, Italy; 3Department of Clinical Dermatology of San Gallicano Dermatological Institute, IRCCS, Rome, Italy; 4Department of Biological Sciences, Modena e Reggio Emilia University, Modena, Italy and 5 Department of Biology, Tor Vergata Unversity, Rome, Italy Correspondence: Dr Mauro Picardo, Laboratory of Cutaneous Physiopathology, San Gallicano Dermatological Institute, IRCCS, Via San Gallicano 25/a, Rome 00153, Italy. E-mail:
[email protected];
[email protected] Abbreviations: CL, cardiolipin; CxI, complex I; ETC, electron transport chain; MFI, median fluorescence intensity; NAO, 10-nonyl acridine orange; ROS, reactive oxygen species; CuH, cumene hydroperoxide Received 27 July 2006; revised 29 September 2006; accepted 18 October 2006; published online 18 January 2007
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for the reported susceptibility to chemical (cumene hydroperoxide (CuH); phenols) or physical (UV) stressors of the melanocytes in vitro (Maresca et al., 1997; Jimbow et al., 2001). In addition, several authors have described an alteration of the redox balance even in non-epidermal compartments, suggesting a possible systemic involvement in the disease (Akyol et al., 2002; Agrawal et al., 2004; Koca et al., 2004; Schallreuter et al., 2006). Previously, in peripheral blood mononuclear cells of patients with active disease, we demonstrated an increased reactive oxygen species (ROS) generation and a compromised antioxidant capacity indicating mitochondria as a possible site of ROS generation. These cells were particularly susceptible to the inhibition of the electron transport chain (ETC) complex I (CxI), with an increased cell death and decreased mitochondrial membrane potential when exposed to rotenone (Dell’Anna et al., 2001, 2003). ROS production was associated with an alteration of the mitochondrial transmembrane potential, and was reduced by cyclosporin A, an inhibitor of mitochondrial permeability transition pores. The mitochondrial involvement is also supported by the increased expression of the mitochondrial malate dehydrogenase, possibly due to a compensatory mechanism ensuring the proper intramitochondrial nicotinamide adenine dinucleotide (reduced form) level and ATP production at least in basal conditions (Dell’Anna et al., 2001, 2003). The characterization of the biochemical pathways that account for the imbalance of the redox system and for the & 2007 The Society for Investigative Dermatology
ML Dell’Anna et al. Membrane Damage and Vitiligo
RESULTS Membrane peroxidation in primary vitiligo melanocytes and mitochondrial ROS production
Cultured epidermal primary vitiligo melanocytes at passage 0 present a significant membrane peroxidation, evaluated by confocal microscopy and FACS using C11-BODIPY581/591 (median fluorescence intensity (MFI) 43278 in vitiligo and 17973 in normal melanocytes, P ¼ 0.00004) (Figure 1a). The confocal granular pattern of the fluorescence suggests a specific involvement of the mitochondrial membranes (Figure 1c). Moreover, a higher ROS production was detected by FACS analysis as indicated by MFI of the probe rorhodamine 123 (350715 in vitiligo and 19078 in normal melanocytes Po0.01) (Figure 1b), further supporting mitochondrial involvement in vitiligo pathogenesis (Sastre et al., 1996; Wrona et al., 2005). Lipid components of the membranes in primary vitiligo melanocytes
We evaluated the content and the transmembrane distribution of CL in order to assess a possible initial derangement of the mitochondrial membranes. CL is present almost exclusively in membranes harboring ETC complexes. It is required for the correct interaction and assembly of proteins beyond ETC and provides their stability. However, it did not coisolate with proteins not engaged in oxidative phosphorylation (Haines and Dencher, 2002; Gohil et al., 2004). Upon binding to CL (ratio 2:1), but not to oxidized CL, 10-nonyl acridine orange (NAO) shifts the fluorescence emission from
a
b 500 450 400 350 300 250 200 150 100 50
60 50
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mitochondrial alteration could provide a better understanding of vitiligo pathomechanisms and possibly improve therapeutic approaches. Before this, vitiligo melanocytes were reported to be more susceptible to the toxic effects of chemical oxidants or UVB, both able to induce ROS generation and alterations to mitochondrial activities (Maresca et al., 1997; Jimbow et al., 2001; Kroll et al., 2005). The expression and function of the mitochondrial ETC complexes is strictly dependent on the lipid component of the inner mitochondrial membrane and particularly on cardiolipin (CL) (Gohil et al., 2004). CL is a dimeric structure consisting of four fatty acyl chains, and is the most representative phospholipid of the inner membrane responsible for the correct housing and activity of ETC proteins (Haines and Dencher, 2002; Gohil et al., 2004). An impaired assembly or recycling of CL is associated with mitochondrial defects (Xu et al., 2005) reverted in vitro by supplementation with the phospholipid or its components (Valianpour et al., 2003). Here, we studied epidermal primary melanocytes from non-lesional vitiligo skin and we observed altered ROS production, membrane lipoperoxidation, modification of the transmembrane CL distribution, and decreased CxI activity, as well as increased susceptibility to the chemical oxidant CuH. The structural and functional pattern demonstrated here may represent the initial biochemical impairment occurring in vitiligo and account for the loss of melanocyte viability after oxidant challenge.
40 30 20 10
VTG
N
0
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BODIPY581591
c
20 m N
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Figure 1. Membrane peroxidation associates with a high production of ROS in vitiligo melanocytes. Epidermal melanocytes were grown in medium 254 supplemented with human melanocyte growth factors (all from Cascade Biologics). Immediately after the trypsinization, the cells were stained at 371C for 30 minutes with (a) C11-BODIPY581/591 0.1 mM, evaluating the degree of membrane lipoperoxidation, or with (b) rorhodamine 123, in order to detect the intracellular ROS generation. The analysis was performed by means of FACSCalibur and CellQuest was used for the acquisition and analysis of the file acquired in listmode. The MFI of FL1 (520 nm) was recorded in linear scale for both dhrorhodamine 123 and C11-BODIPY581/591. (c) The membrane lipoperoxidation was also evaluated by means of confocal microscope (original magnification 63), after staining with C11-BODIPY581/591 2 mM in Chamber Slides, where the occurrence of membrane peroxidation was indicated by the shift of the emission from the red to the green. Rorhodamine 123 data were reported as mean7SD (n ¼ 5). *Po0.01.
green towards red (from 520 to 640 nm), detectable by FACS in FL3 (Fernandez et al., 2002). The inflection point of the NAO curve, corresponding to the saturation of the outer side of the inner membrane in vitiligo melanocytes, takes place at a lower NAO concentration compared to that observed in normal melanocytes (7.5 vs 10 mM). This indicates a reduced percentage of CL (18 and 56% in vitiligo and normal samples, respectively; Po0.01) (Figure 2). CxI activity is decreased in melanocytes from vitiligo subjects
Mitochondrial energy production is physiologically associated with the production of ROS, but has no effect on cell viability and function. However, defective ETC activity can lead to the generation of reactive species that overcome the cellular detoxifying mechanisms. The impairment of the CxI activity can be a suitable basis for the altered redox status reported in different cell types from vitiligo subjects. We previously demonstrated that peripheral blood mononuclear cells from vitiligo subjects are particularly susceptible to the specific CxI inhibitor rotenone (Dell’Anna et al., 2003). Alteration of the CL may affect CxI activity that in turn can lead to the generation of ROS. In order to test whether impaired CxI activity can provide the basis for the observed high ROS production and membrane lipoperoxidation in melanocytes, we performed a semiquantitative analysis of the www.jidonline.org 1227
ML Dell’Anna et al. Membrane Damage and Vitiligo
100
50 45
Arbitrary units
80 NAO saturation
40
VTG N
60
N VTG
35
*
30 25 20 15
40
10 20
*
5 0 Basal
Rotenone
0
Figure 2. CL could be directly involved. Primary epidermal melanocytes were fixed with 1% paraformaldehyde for 20 minutes at 221C, washed and stained with NAO (0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 mM) for 15 minutes, and immediately analyzed after extensive washing. In all 10,000 total events were acquired by means of FACSCalibur and FL3 fluorescence intensity was acquired and analyzed in a linear scale. The inflection point of NAO saturation was calculated by Grafit program. The data reported are the mean7SD of the five analyzed samples. *Po0.01.
Nk N rot
CxI activity in epidermal melanocytes from non-lesional areas of vitiligo patients and from the skin of healthy subjects. We demonstrate that even melanocytes are characterized by a significant decreased activity of CxI, histochemically indicated by a lower level of black spots produced following 3(4,5-dimethylthiazol-2yl)2,5 diphenyltetrazolium bromide reduction, and further decreased by treatment with the specific CxI inhibitor rotenone (0.6 mg/ml) (Figure 3). The degree of bright white (a.u.) inversely correlates with the activity of the CxI. Basal values of 2072 were found in normal melanocytes and 3074 in vitiligo melanocytes (Po0.001). Treatment with rotenone leads to bright white of 2573 and 4074 in normal and vitiligo melanocytes, respectively (Po0.001 vs basal in vitiligo samples), indicating that vitiligo cells were more susceptible to the effect of the CxI inhibitor. Vitiligo melanocytes are more susceptible to apoptosis by CuH
As vitiligo worsens in stressful conditions, we evaluated the effects of chemical oxidative stress by exposing primary epidermal melanocytes to CuH. Vitiligo melanocytes were characterized by an increased susceptibility to the prooxidant agent, as indicated, on the basis of the Differential Light Scatters, by the enrichment of the apoptotic region and by the increase of apoptotic events inside the viable region. The analysis of the apoptotic process inside the viable region, carried out after the staining with Annexin V and propidium iodide, demonstrated the increased early (melanocytes Annexin V þ /propidium iodide 1174 vs 772% at 20 mM CuH, Po0.05) and late (melanocytes Annexin V þ /propidium iodide þ 3876 vs 1273.2% at 20 mM, Po0.001) apoptotic events (Figure 4; Table 1). In separate experiments the induction of apoptotic pathway by CuH in vitiligo melanocytes was confirmed by caspase 3 activation as evaluated by 1228 Journal of Investigative Dermatology (2007), Volume 127
vtg k vtg rot
20 m
Figure 3. CxI activity is affected in vitiligo melanocytes. Immunohistochemistry allows a semiquantitative evaluation of the CxI activity. The average7SD of five vitiligo and normal subjects are represented in the histogram (*Po0.001). The ability of the cells to transform 3(4,5dimethylthiazol-2yl)2,5 diphenyltetrazolium bromide in the black formazan correlates with the activity of the mitochondrial CxI. We calculated, through a dedicated macro, the intensity (a.u. with a black–white scale provided by the software) of the white for each area of the slide observed by means of contrast-phase microscopy.
FACS analysis using specific monoclonal antibody (data not shown). Our results indicate a correlation between a primitive membrane defect and increased susceptibility to stress. DISCUSSION It has been previously demonstrated that melanocytes from vitiligo subjects show an increased susceptibility to stressful stimuli, such as CuH, phenols, catechols, and UV (Maresca et al., 1997; Yang and Boissy, 1999; Jimbow et al., 2001; Boissy and Manga, 2004; Kroll et al., 2005). This susceptibility is associated with a high level of epidermal ROS production (Schallreuter et al., 1998, 1999, 2001; Rokos et al., 2002). Moreover, increased ROS production affecting the intracellular redox balance and the susceptibility to chemical oxidants also characterizes non-epidermal cells (Dell’Anna et al., 2001). Herein we have added new insight
ML Dell’Anna et al. Membrane Damage and Vitiligo
1.27
104
25.3
FL2-H: PI
FL2-H: PI
104
8.21
103
103 102 101
0
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35.5 1
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Figure 4. Vitiligo melanocytes are susceptible to chemical pro-oxidants. Cells were exposed to 7 and 20 mM CuH and stained with Annexin V and propidium iodide. The dot plots of the FSC-SSC subset melanocytes (debris exclusion) were reported (up: normal, down: vitiligo; left: control, central: 7 mM, right: 20 mM). The histograms correspond to the average 7SD of the percentage of late apoptotic cells in five samples. *Po0.05; **Po0.001.
Table 1. The apoptotic events in normal and vitiligo melanocytes treated with CuH k Early apoptosis Late apoptosis
N
271
VTG
270.5
N VTG
7 lM 2.572
20 lM 772*
372
1174**
372
872
1273.2*
872
1273
3876***,*
CuH, cumene hydroperoxide; N, normal samples. *Po0.05 versus basal and versus VTG; **Po0.01 versus basal; ***Po0.0001 versus basal; *Po0.001 versus N.
into the possible etiopathogenetic mechanisms leading to melanocyte loss in vitiligo lesions. Previous observations reported vacuolization of both melanocytes and keratinocytes in vitiligo epidermis as well as in vitiligo melanocyte cultures as a consequence of H2O2-derived oxidative stress (Schallreuter et al., 1999; Tobin et al., 2000). Now we provide evidence that the alteration of the lipid components of the membrane could be a possible determining factor in intracellular ROS generation. In vitiligo melanocyte cultures
we demonstrated an intracellular hyperproduction of ROS, associated with membrane lipoperoxidation, impaired CxI activity and increased susceptibility to oxidant-induced apoptosis. Moreover, we observed a modified transmembrane distribution of CL, the major membrane lipid envelope of ETC complexes. It should be noted that the specific modification of the CL pattern was observed in the absence of any peroxidative treatment or senescence in culture, demonstrating that before any experimental manipulation, cultured vitiligo melanocytes possess an altered membrane structure that could affect some mitochondrial activities. We speculate that the intrinsic alteration of the membranes precedes the changes of CxI activity and is the basis for the susceptibility to the cell by chemical oxidants (Maresca et al., 1997; Yang and Boissy, 1999; Boissy and Manga, 2004; Kroll et al., 2005). The lipid composition of the cellular membranes affects the structure, the function, and the spatial transmembrane orientation of the proteins (Haucke and Schatz, 1997; Rafique et al., 2001; Gohil et al., 2004; Zhang et al., 2005). CL is an acidic dimeric phospholipid consisting of four fatty acyl chains (Haines and Dencher, 2002; Gohil et al., 2004). The defective CL function could be subsequent to the oxidation www.jidonline.org 1229
ML Dell’Anna et al. Membrane Damage and Vitiligo
∆ ETC proteins housing
e-
Membrane defect
ee-
ATPase hsp 70 expression CxII
CxI
CxIII Succinate CoQ reductase
CxIV
ADP
ATP
Cyt.c oxidase
ROS
ROS
NADH CoQ oxidore ductase
CoQ cyt.c oxidore ductase
Membrane peroxidation
Immune system activaion
Oxidant suspectibility
Figure 5. The possible pathogenetic mechanism. A schematic picture of the causal and temporal sequence leading to the melanocyte functional impairment during vitiligo.
by the high intracellular levels of H2O2, leading in turn to the propagation of the membrane lipo-lipoperoxidation and to mitochondrial impairment (Petrosillo et al., 2001). However, the oxidative process did not affect transmembrane distribution of the CL that could be due to a defective fatty acid composition or to the remodelling process (Haines and Dencher, 2002; Broquet et al., 2003; Valianpour et al., 2003; Gohil et al., 2004). In any case, the observed defective content of CL in the outer surface of the inner mitochondrial membrane leads us to hypothesize that the initial defect is mitochondrial, mainly at the level of the membrane harboring the ETC complexes where H2O2 was generated. Finally, CL oxidation modulates the assembly and the catalytic activity of CxI, CxIII, and CxIV (Ugalde et al., 2004). In vitro exposure of vitiligo melanocytes to the depigmenting agent 4-tert-butyl-phenol leads to a high release of the lipid-bound hsp70, capable of activating dendritic cells and possibly triggering and maintaining the immune recognition of the melanocytes (Kroll et al., 2005). It is strictly associated with lipid rafts and participates in the mitochondrial transport inner membrane complex. Along with CL it regulates the import of some ETC proteins (Haucke and Schatz, 1997; Broquet et al., 2003; Xu et al., 2003; D’Silva et al., 2004; Hood and Joseph, 2004). Thus, the altered lipid composition could account for the increased release of hsp70 and then for the switch-on immune response. Finally, the depletion, owing to synthesis/recycling defects, or the oxidation of the CL, owing to increased ROS generation, could lead to the release of cytochrome c, as reported in other in vitro models, accounting at least in part for the melanocyte loss in vivo and 1230 Journal of Investigative Dermatology (2007), Volume 127
for increased cell death during oxidative challenge (Shidoji et al., 1999; Petrosillo et al., 2001; Ott et al., 2002). Moreover, CxI is a possible target for dopamine damage, an altered, systemic, or local catecholamine metabolism which has been described in vitiligo patients (Morrone et al., 1992; Schallreuter et al., 1994; Le Poole et al., 1994; Salzer and Schallreuter, 1995; Cucchi et al., 2000; Ben-Shachar et al., 2004). Our data are in agreement with the involvement of the mitochondrial respiratory chain in vitiligo previously indicated by the increased expression of the mitochondrial malate dehydrogenase. This accounts for an augmented intramitochondrial nicotinamide adenine dinucleotide (reduced form) level and thus for a normal ATP production despite impaired CxI activity, at least in basal conditions (Dell’Anna et al., 2003). At least four potential mechanisms linking vitiligo to oxidative stress have been identified so far: (a) altered metabolism of catechols and biopterins with subsequent production of toxic intermediates for melanocytes, (b) polymorphism of the catalase gene giving rise to an inadequate antioxidant defense, (c) toxicity of the melanine intermediates acting directly inside the melanocyte, (d) inhibition of the transcription factor microphthalmia-associated transcription factor by excessive H2O2 or by insufficient growth factors (stem cell factor) which affect both melanin production and cell survival (Casp et al., 2001; Jimenez-Cervantes et al., 2001; Hasse et al., 2004; Lee et al., 2005). In this study, we indicate the primitive membrane defect as a possible basis for some factors, at least, from the above list.
ML Dell’Anna et al. Membrane Damage and Vitiligo
On the basis of our results, a possible pathogenetic hypothesis could be that a defective arrangement of the membrane lipids, with an initial altered CL composition and function, leads to impaired ETC activity. These structural and functional failings are in turn associated with an increased ROS production, overcoming the antioxidant defense; the last events could be ROS-mediated membrane peroxidation, with a possible further CL oxidation, and increased susceptibility to stressful agents (Figure 5). MATERIALS AND METHODS
acquisition was carried out. In all, 10,000 total events were acquired and MFI of FL3 was evaluated. MFI of higher NAO concentration has been considered as 100% fluorescence, corresponding to the overall CL saturation; the other MFI are reported as a percentage of the maximum MFI and plotted versus NAO concentration. When all CL binding sites are saturated, red fluorescence shows a plateau. NAO can thus cross the membrane and binds CL in the matrix side with a further increase in FL3 fluorescence. Finally, when the inner leaflet is completely occupied a second plateau is reached. Each plateau was mathematically calculated by GraFit 3.0 software using the second derivative of the fitting curve, indicating the inflection point.
Subjects and cells A skin punch-biopsy (4 mm diameter) was obtained from axillary or gluteal areas from five vitiligo subjects with active (on the basis of the progression or appearance of lesions in the last 3 months) nonsegmental disease and five normal subjects, sex and age matched. Autoimmune diseases or familiarity were not present in any vitiligo subjects. The epidermis was separated from the dermis (dispase overnight, 41C) and cells were disaggregated (0.05% Trypsin 0.02% EDTA, 12 minutes, 371C under gentle agitation). Epidermal melanocytes were seeded in 254 conditioned medium (Cascade Biologics, Mansfield, UK), containing fetal bovine serum (0.5%), bovine insulin (5 mg/ml), bovine transferrin (5 mg/ml), basic fibroblast growth factor (3 ng/ml), hydrocortisone (0.18 mg/ml), heparin (3 mg/ml), phorbol 12-myristate 13-acetate (10 ng/ml), and analyzed in subconfluent phase at passages 0–2. For flow cytometric methods FACSCalibur (Becton Dickinson, San Diego, CA; argon laser 488 nm/15 mW and red diode 635 nm), and CellQuest and Flo-Jo software were used. The MFI analysis was performed in a linear scale. In all, 5,000 total events were acquired (when not specified differently). The study was approved by the San Gallicano Dermatological Institute ethics committee and all patients provided informed consent before inclusion in the study. The Declaration of Helsinki Principles were followed.
Membrane peroxidation Adherent melanocytes were stained in 254 conditioned medium with the fluorescent fatty acid analog C11-BODIPY581/591 (2 mM, 20 minutes, 371C 5% CO2) (Molecular Probes, CA) and immediately analyzed by confocal microscope (LSM5 Pascal, Zeiss, Germany) (Brouwers et al., 2005). The shift of the fluorescence from red to green (520 nm) correlates well to the formation of phospholipid peroxides and cholesterol oxidation products (Brouwers and Gadella, 2003). The fluorescence of C11-BODIPY581/591 was also evaluated by FACSCalibur. Owing to the different sensibilities of the two instruments, the probe used here was at 0.1 mM.
ROS production Cells (2.5 105) were loaded with 0.5 mM dihydrorhodamine 123 (Molecular Probes), that passively diffuses across cell membranes where it is oxidized by ROS, mainly H2O2, to cationic rorhodamine 123 which localizes into the mitochondria (Sastre et al., 1996).
Transmembrane CL distribution Melanocytes (5 105) were fixed in paraformaldehyde 1% (15 minutes, 221C) and stained with NAO (Molecular Probes). A wide range of NAO was used: 0.1, 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, and 35 mM (Fernandez et al., 2002). After extensive washes the FACS
Melanocyte CxI activity Melanocytes were seeded on CultureSlides (Becton Dickinson) in 254 conditioned medium. The semiquantitative analysis of CxI activity was performed by the histochemical method, based on the CxI-mediated reduction of 3(4,5-dimethylthiazol-2yl)2,5 diphenyltetrazolium bromide dye to the black colored product formazan (de Halac et al., 2000). The specificity of the reaction was evaluated by the sensitivity to the inhibitor rotenone. The cells were exposed to 0.6 g/ml rotenone (in 50% ethanol), or to 50% ethanol alone and then incubated with buffer A pH 7.0 (2 mg/ml 3(4,5-dimethylthiazol2yl)2,5 diphenyltetrazolium bromide, 0.2 M Tris pH7.4, 0.5 M CoCl2, 0.05 M MgCl2, and 2 mg/ml nicotinamide adenine dinucleotide (reduced form)). The cells were fixed (paraformaldehyde 1%) and analyzed by contrast-phase microscope (Axioscop 2 plus, Zeiss). A semiquantitative analysis was performed by a dedicated macro that allows the evaluation of the brightness of the white in 50 different areas of the slide for each sample analyzed. The brightness of the white inversely correlates with activity of the CxI.
Stress treatments Melanocytes were treated for 60 minutes with 7 and 20 mM CuH (Sigma, Milano, Italy). Apoptosis was assayed after 6 hours using Annexin V and propidium iodide (Becton Dickinson). The test was performed in 254 conditioned medium supplemented with specific melanocyte growth factors.
Statistical analysis Student’s t-test was used and a Po0.05 was assumed as significant. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS The work was supported by a grant of the Ministero della Salute (Italy).
REFERENCES Agrawal D, Shajil EM, Marfatia YS, Begum R (2004) Study of the antioxidant status of vitiligo patients of different age groups in Baroda. Pigment Cell Res 17:289–94 Akyol M, Celik VK, Ozcelik S, Polat M, Marufihah M, Atalay A (2002) The effects of vitamin E on the skin lipid peroxidation and the clinical improvement in vitiligo patients treated with PUVA. Eur J Dermatol 1:24–6 Ben-Shachar D, Zuk R, Gazawi H, Ljubuncic P (2004) Dopamine toxicity involves mitochondrial complex I inhibition: implications to dopamine-related neuropsychiatric disorders. Biochem Pharmacol 67:1965–74
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ML Dell’Anna et al. Membrane Damage and Vitiligo
Boissy RE, Manga P (2004) On the etiology of contact/occupational vitiligo. Pigment Cell Res 17:208–14
Le Poole IC, Das PK, van den Wijngaard RM, Bos JD, Westerhof W (1993) Review of the etiopathomechanism of vitiligo: a convergence theory. Exp Dermatol 2:145–53
Broquet AH, Thomas G, Masliah J, Trugnan G, Bachelet M (2003) Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278:21601–6
Le Poole IC, van den Wijngaard RM, Smit NP, Oosting J, Westerhof W, Pavel S (1994) Catechol-O-methyltransferase in vitiligo. Arch Dermatol Res 286:81–6
Brouwers JF, Silva PFN, Gadella BM (2005) New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freezing–thawing. Theriogenology 63:458–69
Lee YA, Kim NH, Choi WI, Youm YH (2005) Less keratinocyte-derived factors related to more keratinocyte apoptosis in depigmented than normally pigmented suction-blistered epidermis may cause passive melanocyte death in vitiligo. J Invest Dermatol 124:976–83
Brouwers JFHM, Gadella BM (2003) In situ detection and localization of lipid peroxidation in individual bovine sperm cells. Free Rad Biol Med 35:1382–91
Maresca V, Roccella M, Roccella F, Camera E, Del Porto G, Passi S et al. (1997) Increased sensitivity to peroxidative agents as possible pathogenic factor of melanocyte damage in vitiligo. J Invest Dermatol 109:310–3
Casp CB, She JX, McCormack WT (2001) Genetic association of the catalase gene (CAT) with vitiligo susceptibility. Pigment Cell Res 15: 62–6
Morrone A, Picardo M, De Luca C, Terminali O, Passi S, Ippolito F (1992) Cathecolamines and vitiligo. Pigment Cell Res 5:65–9
Cucchi ML, Frattini P, Santagostino G, Orecchia G (2000) Higher plasma catecholamine and metabolite levels in the early phase of nonsegmental vitiligo. Pigment Cell Res 13:28–32
Ott M, Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S (2002) Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci USA 99:1259–63
D’Silva P, Liu Q, Walter W, Craig EA (2004) Regulated interactions of mtHsp70 with Tim44 at the translocon in the mitochondrial inner membrane. Nat Struct Mol Biol 11:1084–91
Petrosillo G, Ruggiero FM, Pistolese M, Paradies G (2001) Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. FEBS Lett 509:435–8
de Halac IN, Bacman SR, de Kremer RD (2000) Histoenzymolgy of oxidases and dehydrogenases in peripheral blood lymphocytes and monocytes for the study of mitochondrial oxidative phosphorylation. Histochem J 32:133–7
Rafique R, Schapira AHV, Cooper M (2001) Sensitivity of respiratory chain activities to lipid peroxidation: effect of vitamin E deficiency. Biochem J 357:887–92
Dell’Anna ML, Maresca V, Briganti S, Camera E, Falchi M, Picardo M (2001) Mitochondrial impairment in peripheral blood mononuclear cells during the active phase of vitiligo. J Invest Dermatol 117:908–13
Rokos H, Beazley WD, Schallreuter KU (2002) Oxidative stress in vitiligo: photo-oxidation of pterins produces H2O2 and pterin-6-carboxylic acid. Biochem Biophys Res Commun 292:805–11
Dell’Anna ML, Urbanelli S, Mastrofrancesco A, Camera E, Iacovelli P, Leone G et al. (2003) Alterations of mitochondria in peripheral blood mononulcear cells of vitiligo patients. Pigment Cell Res 16: 553–9
Salzer BA, Schallreuter KU (1995) Investigation of the personality structure in patients with vitiligo and a possible association with impaired catecholamine metabolism. Dermatol 190:109–15
Fernandez MG, Troiano L, Moretti L, Nasi M, Pinti M, Salvioli S et al. (2002) Early changes in intramitochondrial cardiolipin distribution during apoptosis. Cell Growth Diff 13:449–55 Gauthier Y, Cario-Andre´ M, Taieb A (2003) A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy? Pigment Cell Res 16:322–32 Gohil VM, Hayes P, Matsuyama S, Schagger H, Schlame M, Greenberg ML (2004) Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent. J Biol Chem 279:42612–8 Haines TH, Dencher NA (2002) Cardiolipin: a proton trap for oxidative phosphorylation. FEBS Lett 26508:1–5 Hasse S, Gibbons NCJ, Rokos H, Marles LK, Schallreuter KU (2004) Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H2O2 stress. J Invest Dermatol 122:307–13
Sastre J, Pallardo` FV, Pla` R, Pellı`n A, Juan G, O’Connor J et al. (1996) Aging of the liver: age-associated mitochondrial damage in intact hepatocytes. Hepatology 24:1199–205 Schallreuter KU, Chiuchiarelli G, Gemeli E, Elwary SM, Gillbro JM, Spencer JD et al. (2006) Estrogens can contribute to hydrogen peroxide generation and quinone-mediated DNA damage in peripheral blood lymphocytes from patients with vitiligo. J Invest Dermatol 126:1036–42 Schallreuter KU, Moore J, Wood JM, Beazley WD, Gaze DC, Tobin DJ et al. (1999) In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Investig Dermatol Symp Proc 4:91–6
Haucke V, Schatz G (1997) Reconstitution of the protein insertion machinery of the mitochondrial inner membrane. EMBO J 16:4560–7
Schallreuter KU, Moore J, Wood JM, Beazley WD, Peters EMJ, Marles LK et al. (2001) Epidermal H2O2 accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH4-dependent processes? J Invest Dermatol 116:167–74
Hood DA, Joseph AM (2004) Mitochondrial assembly: protein import. Proc Nutr Soc 63:293–300
Schallreuter KU, Wood JM, Berger J (1991) Low catalase levels in the epidermis of patients with vitiligo. J Invest Dermatol 97:1081–5
Jimbow K, Chen H, Park JS, Thomas PD (2001) Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinaserelated protein in vitiligo. Br J Dermatol 144:55–65
Schallreuter KU, Wood JM, Ziegler I, Lemke KR, Pittelkow MR, Lindsey NJ et al. (1994) Defective tetrahydrobiopterin and catecholamine biosynthesis in the depigmentation disorder vitiligo. Biochim Biophys Acta 1226:181–92
Jimenez-Cervantes C, Martinez-Esparza M, Perez C, Daum N, Solano F, Garcia-Borron J (2001) Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphtalmia transcription factor. J Cell Sci 114:2335–44 Koca R, Armutcu F, Altinyazar HC, Gurel A (2004) Oxidant-antioxidant enzymes and lipid peroxidation in generalized vitiligo. Exp Dermatol 29:406–9 Kroll TM, Bommiasamy H, Boissy RE, Hernandez C, Nickoloff BJ, Mestril R et al. (2005) 4-tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing: relevance to vitiligo. J Invest Dermatol 124:798–806
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Schallreuter KU, Zschiesche M, Moore J, Panske A, Hibberts NA, Herrmann FH et al. (1998) In vivo evidence for compromised phenylalanine metabolism in vitiligo. Biochem Biophys Res Commun 243:395–9 Shidoji Y, Hayashi K, Komura S, Ohishi N, Yagi K (1999) Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation. Biochem Biophys Res Commun 264:343–7 Taieb A (2000) Intrinsic and extrinsic pathomechanisms in vitiligo. Pigment Cell Res 13:41–7 Tobin DJ, Swanson NN, Pittelow MR, Peters EM, Schallreuter KU (2000) Melanocytes are not absent in lesional skin of long duration vitiligo. J Pathol 191:407–16
ML Dell’Anna et al. Membrane Damage and Vitiligo
Ugalde C, Janssen RJRJ, van den Heuvel LP, Smeitink JAM, Nijtmans LGJ (2004) Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency. Hum Mol Genet 13:659–67 Valianpour F, Wanders RJA, Overmars H, Vaz FM, Barth PG, van Gennip AH (2003) Linoleic acid supplementation of Barth syndrome fibroblasts rerstores cardiolipin levels: implications for treatment. J Lipid Res 44:560–6 Wrona M, Patel K, Wardman P (2005) Reactivity of 20 ,70 -dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals. Free Radic Biol Med 38:262–70
Xu Y, Kelley RI, Blanck TJJ, Schlame M (2003) Remodeling of cardiolipin by phospholipid transacylation. J Biol Chem 278:51380–5 Xu Y, Sutachan JJ, Plesken H, Kelley RI, Schlame M (2005) Characterization of lymphoblast mitochondria from patients with Barth syndrome. Lab Invest 85:823–30 Yang F, Boissy RE (1999) Effects of 4-tertiary butylphenol on the tyrosinase activity in human melanocytes. Pigment Cell Res 12: 237–45 Zhang W, Campbell HA, King SC, Dowhan W (2005) Phospholipid as determinants of membrane protein topology. J Biol Chem 280: 26032–8
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