Role of Reduced Glutathione Efflux in Apoptosis of Immortalized Human Keratinocytes Induced by UVA

Abstract

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation. We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation. multidrug resistance-associated protein buthionine sulfoximine cyclosporin A 5,5′-dithiobis(2-nitrobenzoic acid) lactate dehydrogenase phosphatidylserine verapamil phosphate-buffered saline 7-amino-4-trifluoromethyl coumarin Solar UV irradiation reaching the surface of the earth, consisting mainly of UVB (280–315 nm) and UVA (315–400 nm), presents a major environmental challenge to the skin, contributing not only to photo-aging but also to carcinogenesis (1Longstreth J. de Gruijl F.R. Kripke M.L. Abseck S. Arnold F. Slaper H.I. Velders G. Takizawa Y. van der Leun J.C. J. Photochem. Photobiol. B. Biol. 1998; 46: 20-39Google Scholar, 2de Gruijl F.R. Eur. J. Cancer. 1999; 35: 2003-2009Google Scholar, 3Pourzand C. Tyrrell R.M. Photochem. Photobiol. 1999; 70: 380-390Google Scholar). UVA has long been considered less of a causative factor in skin carcinogenesis than UVB due to its negligible absorption by DNA. However, the greater abundance of UVA in solar UV irradiation and deeper penetration of UVA into the actively dividing basal layer of the skin increases the relative importance of UVA as compared with UVB (4Robert C. Muel B. Benoit A. Dubertret L. Sarasin A. Stary A. J. Invest. Dermatol. 1996; 106: 721-728Google Scholar). UVA has been shown to be a risk factor for melanoma in fish (5Setlow R.B. Grist E. Thompson K. Woodhead A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6666-6670Google Scholar) and could be also in humans (6Wang S.Q. Setlow R. Berwick M. Polsky D. Marghoob A.A. Kopf A.W. Bart R.S. J. Am. Acad. Dermatol. 2001; 44: 837-846Google Scholar, 7Runger T.M. Brocker E.B. Hautarzt. 1995; 46: 394-399Google Scholar, 8Aubin F. Humbey O. Humbert P. Laurent R. Mougin C. Presse Med. 2001; 30: 546-551Google Scholar). Fortunately, the carcinogenic effects of UV irradiation may be decreased by apoptosis, programmed cell death, which eliminates DNA-damaged or potentially mutated cells. Much more is known about UVB-induced apoptosis than about apoptosis induced by UVA in both normal keratinocytes and immortalized keratinocytes (9Schwarz A. Bhardwaj R. Aragane Y. Mahnke K. Riemann H. Metze D. Luger T.A. Schwarz T. J. Invest. Dermatol. 1995; 104: 922-927Google Scholar, 10Henseleit U. Zhang J. Wanner R. Haase I. Kolde G. Rosenbach T. J. Invest. Dermatol. 1997; 109: 722-727Google Scholar, 11Gniadecki R. Hansen M. Wulf H.C. J. Invest. Dermatol. 1997; 109: 163-169Google Scholar, 12Parat M.O. Richard M.J. Pollet S. Hadjur C. Favier A. Beani J.C. J. Photochem. Photobiol. B. Biol. 1997; 37: 101-106Google Scholar, 13Tron V.A. Trotter M.J. Tang L. Krajewska M. Reed J.C. Ho V.C. Li G. Am. J. Pathol. 1998; 153: 579-585Google Scholar, 14Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. J. Invest. Dermatol. 1999; 112: 769-774Google Scholar, 15Zhuang L. Wang B. Shinder G.A. Shivji G.M. Mak T.W. Sauder D.N. J. Immunol. 1999; 162: 1440-1447Google Scholar, 16Assefa Z. Vantieghem A. Garmyn M. Declercq W. Vandenabeele P. Vandenheede J.R. Bouillon R. Merlevede W. Agostinis P. J. Biol. Chem. 2000; 275: 21416-21421Google Scholar, 17Mammone T. Gan D. Collins D. Lockshin R.A. Marenus K. Maes D. Cell Biol. Toxicol. 2000; 16: 293-302Google Scholar, 18Petit-Frere C. Capulas E. Lyon D.A. Norbury C.J. Lowe J.E. Clingen P.H. Riballo E. Green M.H. Arlett C.F. Carcinogenesis. 2000; 21: 1087-1095Google Scholar, 19Tsuru K. Horikawa T. Budiyanto A. Hikita I. Ueda M. Ichihashi M. J. Dermatol. Sci. 2001; 26: 209-216Google Scholar). However, Godar and co-workers (20Godar D.E. Lucas A.D. Photochem. Photobiol. 1995; 62: 108-113Google Scholar, 21Godar D.E. Photochem. Photobiol. 1996; 63: 825-830Google Scholar, 22Godar D.E. J. Invest. Dermatol. 1999; 112: 3-12Google Scholar) have reported that UVA-induced apoptosis in lymphoma and Jurkat cells involves mechanisms that differ from those seen with UVB. Oxidative stress is presently considered to be involved in UVA-induced apoptosis (3Pourzand C. Tyrrell R.M. Photochem. Photobiol. 1999; 70: 380-390Google Scholar). In support of this involvement, a protective role is played by tea polyphenols against both cytotoxicity and apoptosis induced by UVA in rat keratinocytes; this protection correlates well with the ability of the polyphenols to quench reactive oxygen species (23Fu Y.C. Jin X.P. Wei S.M. Lin H.F. Kacew S. J. Toxicol. Environ. Health. 2000; 61: 177-188Google Scholar). Nevertheless, the mechanisms involved in UVA-induced apoptosis of human keratinocytes are still poorly understood. There is evidence that protection of human skin cells against a wide range of solar UV radiation damage, including UVA, involves endogenous glutathione (24Tyrrell R.M. Pidoux M. Photochem. Photobiol. 1988; 47: 405-412Google Scholar). This thiol is involved in many biological processes, including the regulation of gene expression, apoptosis, and membrane transport (25Lomaestro B.M. Malone M. Ann. Pharmacother. 1995; 29: 1263-1273Google Scholar, 26Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Google Scholar, 27Hammond C.L. Lee T.K. Ballatori N. J. Hepatol. 2001; 34: 946-954Google Scholar). Glutathione is considered to be the most prevalent and most important intracellular non-protein thiol-disulfide redox buffer in mammalian cells (25Lomaestro B.M. Malone M. Ann. Pharmacother. 1995; 29: 1263-1273Google Scholar, 26Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Google Scholar, 27Hammond C.L. Lee T.K. Ballatori N. J. Hepatol. 2001; 34: 946-954Google Scholar). The higher glutathione content in immortalized HaCaT cells is expected to confer resistance to UVA irradiation as compared with normal keratinocytes (28Leccia M.T. Richard M.J. Joanny-Crisci F. Beani J.C. Eur. J. Dermatol. 1998; 8: 478-482Google Scholar). In apoptosis, the role of glutathione is controversial and dependent on cell types and pro-apoptotic stimuli. (i) High intracellular reduced GSH levels have been found to prevent Fas-induced cell apoptosis in the Fas-resistant variant CEM2D1R (29Chiba T. Takahashi S. Sato N. Ishii S. Kikuchi K. Eur. J. Immunol. 1996; 26: 1164-1169Google Scholar), whereas decreased GSH levels enhance Fas-induced apoptosis in the Fas-resistant variant CEM2D1R and the human hepatoma cell line HepG (2de Gruijl F.R. Eur. J. Cancer. 1999; 35: 2003-2009Google Scholar, 29Chiba T. Takahashi S. Sato N. Ishii S. Kikuchi K. Eur. J. Immunol. 1996; 26: 1164-1169Google Scholar, 30Yang C.F. Shen H.M. Ong C.N. Arch. Biochem. Biophys. 2000; 374: 142-152Google Scholar). (ii) Low intracellular GSH levels have also been shown to prevent apoptosis by compromising caspase activation in mouse hepatocytes (31Hentze H. Kunstle G. Volbracht C. Ertel W. Wendel A. Hepatology. 1999; 30: 177-185Google Scholar, 32Hentze H. Gantner F. Kolb S.A. Wendel A. Am. J. Pathol. 2000; 156: 2045-2056Google Scholar). Depletion of intracellular GSH prevented CD95-triggered apoptosis upstream of caspase-8 activation in T and B cells (33Hentze H. Schmitz I. Latta M. Krueger A. Krammer P.H. Wendel A. J. Biol. Chem. 2002; 277: 5588-5595Google Scholar). Furthermore, cells undergoing apoptosis also appear to export GSH into the extracellular space (34Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Google Scholar, 35van den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google Scholar, 36Oda T. Sadakata N. Komatsu N. Muramatsu T. J. Biochem. (Tokyo). 1999; 126: 715-721Google Scholar, 37Pullar J.M. Hampton M.B. J. Biol. Chem. 2002; 277: 19402-19407Google Scholar). However, neither the mechanism involved in the transport of GSH nor the functional benefit of this process is known. In this study we have found that in immortalized HaCaT cells, UVA irradiation induces the active efflux of GSH. We have surveyed the mechanisms involved in this efflux, focusing on the possible roles of MRP1 transporter protein. We also found that GSH export is closely associated with UVA-induced apoptosis. Reduced GSH, GSSG, verapamil (VP), cyclosporin A (CsA), 5-sulfosalicylic acid glutathione reductase, and buthionine sulfoximine (BSO) were purchased from Sigma. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), 2-vinylpyridine, EDTA, glucose, NADPH, and triethanolamine were purchased from Aldrich. The spontaneously immortalized human keratinocyte cell line HaCaT (38Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Google Scholar), obtained from Prof. N. Fusenig was in in in with and HaCaT keratinocytes were in or for h to UVA cells were GSH was to the and the cells were for h to UVA this on cell The was and cells were with the addition of or of glucose, the cells were with with the The UVA was with a UV with a UVA were in the the the was or as and the cells were with the apoptosis was and the cells were and cells were and to apoptosis In cells were with GSH or for multidrug resistance-associated protein for to irradiation. GSH and were a for glutathione in Scholar, A. Biochem. Scholar). of the cells in were with and with of acid and were and for and for of the were of which were to for protein the of 5-sulfosalicylic acid was and the was and on for to protein. for of were by the addition of and into 2 for of and GSH, was to of the to a concentration of The was by of or of of of NADPH, of and of glutathione was into The was into the and the absorption The GSH or levels were as protein or as compared with without UVA extracellular GSH and GSSG, cell were with or without followed by the The of DNA was by cell were in buffer and on for h with A and h with the cell was with DNA was with and with DNA in were by on or with and UV were cells were in and the cell was for h with buffer of the caspase for for and for The of was on a and of the was in the caspase buffer without to and of The were as of the to a that is upon the increase in plasma membrane is by cytotoxicity of was to the were of was to well of the and the was for in the The was by of and was by the by a The was by to the cells. The were as was to the translocation of phosphatidylserine in UVA-induced apoptosis. or cells including and cells were and by for were with and with to the for were by a are as of to The was for A of was considered In to the of UVA on the efflux of GSH, we HaCaT cells in with UVA, with and without We found that in the of glucose to provide UVA induced efflux of GSH The glutathione in HaCaT cells in normal is protein, of which more than is in the reduced compared with normal cells in intracellular GSH GSSG, of cells in the decreased to and protein for and glucose GSH levels also whereas levels of the GSSG, increased HaCaT cells were with UVA a of in the of glucose intracellular glutathione levels and GSH decreased from whereas levels the in glutathione to be due to a in GSH. GSH was into the extracellular were and GSH, GSSG, and glutathione levels were In the the of glucose on the levels of extracellular GSH, GSSG, or the of the intracellular and extracellular glutathione was compared with intracellular glutathione in normal cells, the of intracellular glutathione was in the of GSH caused a in intracellular GSH. In the of glucose, was GSH efflux, and the intracellular GSH depletion was due to the to A and HaCaT cells were with of UVA GSH efflux increased with the UVA There was GSH efflux but GSH efflux increased to and with A and GSH efflux was of UVA irradiation in the of the of glucose, GSH efflux was in the of of glucose UVA irradiation GSH was in a glucose These that glucose is for GSH efflux induced by UVA irradiation. the efflux of GSH UVA irradiation is influenced by GSH we exogenous GSH to increase extracellular GSH levels or a known to intracellular GSH. was and with cells to UVA irradiation. In the exogenous GSH with cells for did not affect intracellular GSH or in the of glucose 2 cells were with of UVA in the of glucose, GSH was into the extracellular However, the addition of exogenous GSH reduced the in intracellular GSH and thus the UVA-induced efflux The did not with or without exogenous GSH 2 was to the cell h to UVA intracellular glutathione decreased cytotoxicity was not was the with cells, the UVA-induced in intracellular glutathione in cells was from the extracellular 2 Although GSH efflux was induced by UVA irradiation GSH depletion by the efflux process due to the in intracellular GSH. the possible transport involved in UVA-induced GSH efflux, for multidrug resistance-associated protein were with cells to irradiation. In the or and or caused not cells with verapamil or were with UVA, UVA in the of caused less of a GSH efflux as compared with UVA of or more inhibition on GSH efflux. These that verapamil and both the efflux of GSH, with more The inhibition by verapamil could be due to the that verapamil activity and may inhibition on the of GSH with as compared with cyclosporin A, a of However, irradiation, UVA did not GSH efflux as compared with the verapamil nor cyclosporin A the GSH efflux. This that UVA-induced GSH efflux the irradiation but not irradiation. the role of UVA-induced GSH efflux, we the of GSH efflux inhibition on UVA-induced apoptosis. HaCaT cells were with or without cyclosporin A, or GSH and to UVA irradiation. DNA was by the of and were with the plasma membrane permeability was by and the externalization of on the cell surface was by in with shown in A UVA irradiation caused a and increase in DNA in HaCaT cells. However, UVA-induced DNA was not by of HaCaT cells with cyclosporin A, or GSH UVA irradiation and in a UVA irradiation and were as as However, with cyclosporin A, or GSH did not affect the activation of or h Furthermore, the of the cells with the on UVA-induced GSH efflux There was activation not GSH efflux occurred UVA suggesting that GSH efflux induced by UVA the of GSH efflux has on the activation of and by A, UVA the cells were for The caspase activity was in the in a the cells were with or GSH and with UVA The caspase activity was h UVA the cells were the as in and the caspase activity was h UVA irradiation. the cells were with and UVA irradiation the were and glutathione levels were are the of to UVA also caused a gradual increase in in HaCaT cells in a and The of cyclosporin A or verapamil or or GSH or the It is that GSH is more in than or These that GSH efflux induced by UVA is involved in the increased in plasma membrane Cell in the of apoptosis are In to GSH efflux is involved in the translocation of a membrane in the or of apoptosis and the in the recognition of cells by the of V.A. J. Immunol. Scholar), was to on the membrane surface of the shown in UVA induced a increase in translocation as as h However, the with or GSH translocation both and h UVA irradiation. These that GSH efflux to UVA-induced translocation in HaCaT cells. It is that inhibition of MRP1 is not the only of verapamil and cyclosporin A. Verapamil is known to be a and apoptosis in rat J.M. M. Toxicol. 1997; Scholar). A increased Y.C. J.C. Biochem. Biophys. 1999; Scholar) and B.M. A.D. 1995; 12: Scholar). Although the of verapamil or cyclosporin A on DNA or caspase the for apoptosis, was in This is the with the addition of exogenous GSH. the was by the of cyclosporin A with exogenous GSH, which GSH efflux and we that the of and verapamil on apoptosis was due to the inhibition of have that types of cells undergoing apoptosis induced by pro-apoptotic as and to GSH and into the extracellular space (34Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Google Scholar, 35van den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google Scholar, 36Oda T. Sadakata N. Komatsu N. Muramatsu T. J. Biochem. (Tokyo). 1999; 126: 715-721Google Scholar, 37Pullar J.M. Hampton M.B. J. Biol. Chem. 2002; 277: 19402-19407Google Scholar). However, neither the mechanisms involved in the transport of GSH nor the biological functional of this GSH is known. to this study the of GSH efflux in apoptosis of keratinocytes induced by UVA irradiation, an important environmental stress was also In the we have the mechanisms involved in GSH efflux and apoptosis of HaCaT cells induced by We have shown that of HaCaT cells to UVA irradiation increased the transport of reduced GSH into the extracellular and that UVA-induced apoptosis was closely associated with this efflux. It is that UVA irradiation caused glutathione efflux in HaCaT cells the decreased intracellular glutathione and increased extracellular that an was cells in the the efflux of intracellular GSH was resulting from the cells were from normal to or glucose den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google A. Proc. Natl. Acad. Sci. U. S. A. Scholar). We found that UVA-induced GSH efflux on the of the transport of GSH into the extracellular space is an active process and den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google Scholar, 2001; Scholar) and is not due to as a of the of membrane UVA irradiation, only about of cells were and membrane not It that cells could be for an increase in GSH efflux of glucose, UVA irradiation did not a greater GSH efflux than in suggesting that GSH efflux is due to of for the of glucose not only the for active transport of GSH but also intracellular levels and higher GSH levels UVA stress by a of that is for the of to GSH P. M. Sci. 1988; Scholar, K. H. K. N. T. K. Scholar). In UVA irradiation in the of glucose, the of both intracellular and extracellular GSH to was found to be the for the depletion of intracellular GSH (24Tyrrell R.M. Pidoux M. Photochem. Photobiol. 1988; 47: 405-412Google Scholar, Photochem. Photobiol. 1997; Scholar). GSH efflux was as was the in The efflux of GSH was by exogenous addition of GSH and by GSH depletion with This that the transport process is by the concentration gradient of GSH across the cell membrane. as the mechanisms and involved in GSH transport are it is known that the multidrug resistance-associated MRP1 and are for GSH efflux in the 2001; Scholar, Ballatori N. Am. J. 2000; Scholar, H. G. Lee K. J. 2001; Scholar). the of is to and whereas MRP1 is found in many T. M.T. K. M. Chem. Med. 2000; Scholar), it is to that in HaCaT MRP1 may be the transporter protein that is by UVA irradiation to GSH efflux. Furthermore, the rapid efflux of GSH UVA irradiation 2 that this active transport process may be due to the activation of pre-existing transporter by UVA in the of the glucose, than the of transporter by UVA irradiation or oxidative stress Y. M. R. R.T. T. M.T. J. Biol. Chem. 1998; Scholar). Verapamil and for prevent the efflux of GSH induced by UVA irradiation, which the of MRP1 in the transport process of GSH in HaCaT cells. The rapid and active efflux of GSH induced by UVA irradiation in HaCaT cells could be an important biological of keratinocytes to UVA-induced Although is known about the biological of GSH efflux in we have found that apoptosis of HaCaT cells that occurred GSH efflux is closely to the GSH export GSH efflux has on caspase activation and DNA in However, the increases in plasma membrane permeability and translocation apoptosis of HaCaT cells are associated with GSH efflux. of GSH efflux by the MRP1 verapamil and also the of plasma membrane and The of exogenous GSH has a to and B. R. D.J. J. Immunol. 1999; Scholar, Cell 2000; Scholar) have shown that translocation is of both activity F. S. S. F. M. S. S. E. Cell Biol. 1998; Scholar) and caspase It most likely that in HaCaT cells GSH efflux is involved in membrane than caspase activation or DNA in UVA-induced apoptosis. a of in apoptosis of Jurkat induced by GSH efflux was apoptosis, but inhibition of this process did not affect DNA den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google Scholar). of GSH efflux has been shown to cells from apoptosis induced by in cells but not in cells by DNA (34Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Google Scholar). in the of cells (34Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Google Scholar), UVA-induced GSH efflux in HaCaT cells DNA DNA is not by inhibition of GSH efflux. It that cells stimulated by UVA to apoptosis of GSH the irradiation in to the of the GSH efflux apoptosis a mechanism to GSH in the cells than GSH to GSSG, which the oxidative without intervention of reactive oxygen species (34Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Google Scholar, 35van den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Google Scholar). The increased oxidative stress has been shown to in the phosphatidylserine that phosphatidylserine externalization in apoptosis A.A. V.A. K. 2000; Scholar, A.A. K. V.A. C. J. Invest. Dermatol. 2002; Scholar). phosphatidylserine as an important for recognition and of cells by V.A. J. Immunol. Scholar), the phosphatidylserine translocation increased the macrophage recognition and phagocytosis of the cells in and thus potential to invoke inflammation or neoplastic transformation. These may be important for skin and the of UVA-induced skin We and for about the transporter We are to for with cell We also for this