UVB Light Stimulates Production of Reactive Oxygen Species

In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320–360 nm range with additional soret peaks around 400–410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer. In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320–360 nm range with additional soret peaks around 400–410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer. Chronic exposure to sunlight is a significant causative factor in the development of skin cancer. Modifications of DNA and other critical cellular macromolecules by the higher energy shorter solar wavelengths comprising the UVB spectra (290–320 nm) are the most damaging to the skin (for reviews, see Refs. 1Matsumura Y. Ananthaswamy H.N. Front. Biosci. 2002; 7: d765-d783Crossref PubMed Scopus (111) Google Scholar, 2Cleaver J.E. Crowley E. Front. Biosci. 2002; 7: d1024-d1043Crossref PubMed Google Scholar, 3Ullrich S.E. Front. Biosci. 2002; 7: d684-d703Crossref PubMed Google Scholar, 4de Gruijl F.R. van Kranen H.J. Mullenders L.H. J. Photochem. Photobiol. 2001; 63: 19-27Crossref PubMed Scopus (419) Google Scholar, 5Vink A.A. Roza L. J. Photochem. Photobiol. 2001; 65: 101-104Crossref PubMed Scopus (111) Google Scholar). Sunlight-induced cancer develops as a result of a complex cascade of events initiated by damage to DNA (2Cleaver J.E. Crowley E. Front. Biosci. 2002; 7: d1024-d1043Crossref PubMed Google Scholar, 4de Gruijl F.R. van Kranen H.J. Mullenders L.H. J. Photochem. Photobiol. 2001; 63: 19-27Crossref PubMed Scopus (419) Google Scholar, 6Decraene D. Agostinis P. Pupe A. de Haes P. Garmyn M. J. Photochem. Photobiol. 2001; 63: 78-83Crossref PubMed Scopus (56) Google Scholar, 7Alam M. Ratner D. N. Engl. J. Med. 2001; 344: 975-983Crossref PubMed Scopus (984) Google Scholar, 8Cadet J. Douki T. Pouget J.P. Ravanat J.L. Sauvaigo S. Curr. Probl. Dermatol. 2001; 29: 62-73Crossref PubMed Google Scholar). During this process several components of this cascade, including mutation of critical genes, can be mediated by the intracellular generation of reactive oxygen species (9Gocke E. J. Environ. Pathol. Toxicol. Oncol. 2001; 20: 285-292Crossref PubMed Google Scholar, 10Fu Y.C. Jin X.P. Wei S.M. Lin H.F. Kacew S. J. Toxicol. Environ. Health. 2000; 61: 177-188Crossref PubMed Scopus (37) Google Scholar, 11Scharffetter-Kochanek K. Wlaschek M. Brenneisen P. Schauen M. Blaudschun R. Wenk J. Biol. Chem. 1997; 378: 1247-1257PubMed Google Scholar). Reactive oxygen species are generated within mammalian tissues by redox reactions involving electron transfer groups such as quinolines, their phenolic precursors, metal complexes, aromatic nitro compounds, and conjugated imines (12Lee H.C. Wei Y.H. J. Biomed. Sci. 2000; 7: 2-15Crossref PubMed Google Scholar, 13Camhi S.L. Lee P. Choi A.M. New Horiz. 1995; 3: 70-82Google Scholar, 14Jackson A.L. Loeb L.A. Mutat. Res. 2001; 477: 7-21Crossref PubMed Scopus (516) Google Scholar, 15Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (838) Google Scholar). Oxidants, such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are also produced in cells during mitochondrial and microsomal electron transport and from NAD(P)H oxidases as well as lipo- and non-heme-containing dioxygenases (11Scharffetter-Kochanek K. Wlaschek M. Brenneisen P. Schauen M. Blaudschun R. Wenk J. Biol. Chem. 1997; 378: 1247-1257PubMed Google Scholar, 12Lee H.C. Wei Y.H. J. Biomed. Sci. 2000; 7: 2-15Crossref PubMed Google Scholar, 13Camhi S.L. Lee P. Choi A.M. New Horiz. 1995; 3: 70-82Google Scholar, 14Jackson A.L. Loeb L.A. Mutat. Res. 2001; 477: 7-21Crossref PubMed Scopus (516) Google Scholar, 15Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (838) Google Scholar). The unique electronic properties of excited oxygen electrons facilitates their interaction with numerous cellular molecules that regulate many biochemical processes (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 17Kovacic P. Jacintho J.D. Curr. Med. Chem. 2001; 8: 773-796Crossref PubMed Scopus (349) Google Scholar). These interactions can lead to altered cell growth and differentiation (13Camhi S.L. Lee P. Choi A.M. New Horiz. 1995; 3: 70-82Google Scholar, 16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 17Kovacic P. Jacintho J.D. Curr. Med. Chem. 2001; 8: 773-796Crossref PubMed Scopus (349) Google Scholar, 18Thiele J.J. Schroeter C. Hsieh S.N. Podda M. Packer L. Curr. Probl. Dermatol. 2001; 29: 26-42Crossref PubMed Google Scholar, 19Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar). When generated in excess, reactive oxygen species can also induce tissue injury and contribute to the development of skin cancer (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 17Kovacic P. Jacintho J.D. Curr. Med. Chem. 2001; 8: 773-796Crossref PubMed Scopus (349) Google Scholar, 18Thiele J.J. Schroeter C. Hsieh S.N. Podda M. Packer L. Curr. Probl. Dermatol. 2001; 29: 26-42Crossref PubMed Google Scholar, 19Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar). In the present studies we report that human skin expresses an enzyme that generates reactive oxygen species in response to UVB light; unexpectedly, this enzyme was identified as catalase. The conversion of DNA damaging solar radiation into less energetic oxidant species by catalase is a novel and previously unrecognized activity of this antioxidant enzyme. Cells, UVB Light Irradiation, Detection of Reactive Oxygen Species— PAM 212 keratinocytes were kindly provided by Dr. Stuart Yuspa (National Institutes of Health, Bethesda, MD). Human keratinocytes were from Clonetics (Gaithersburg, MD). HA-1 and catalase-overexpressing OC5 Chinese hamster fibroblasts were obtained from Dr. Douglas Spitz (University of Iowa, Iowa City, IO). Bovine and mouse liver catalase, twice crystallized bovine liver catalase, and all chemicals were from Sigma unless otherwise specified. Bovine catalase was processed as indicated below to confirm that the protein was purified to homogeneity. Repurified material was used to characterize the oxidant generating activity of the enzyme. Polyclonal anti-catalase antibodies were from Abcam Ltd. (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad. For anion exchange and size exclusion chromatography, an AKTA fast protein liquid chromatograph (Amersham Biosciences) fitted with a Mono Q HR 5/5 column and a Superose HR 10/30 column were used. Samples were applied to the columns in buffer containing 25 mm Tris-HCl, pH 8.0 supplemented with 5% glycerol. Catalase eluted at ∼0.3 m NaCl using a linear salt gradient on the anion exchange column. Catalase eluted from the size exclusion column with a molecular mass of 240 kDa, which corresponds to the tetramer form of the enzyme. Metal affinity chromatography is a highly efficient method to purify catalase (20Yang Q. DePierre J.W. Protein Expression Purif. 1998; 12: 277-283Crossref PubMed Scopus (11) Google Scholar), and we used a 1-ml HiTrap Chelating HP column (Amersham Biosciences) charged with Ni(NO3)2 according to the manufacturer's instructions for these studies. Catalase was eluted using a 100 mm linear imidazole gradient. Purified material was analyzed on 10% SDS-polyacrylamide gels and appeared as a single 60-kDa band following silver staining. For ultraviolet light treatment, cells or reaction mixtures were irradiated in uncovered 96-well tissue culture plates (Costar, Corning, NY) with UVB light emitted from two Westinghouse FS20 light tubes as described previously (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar). The UVB lights were calibrated with an IL 442A Phototherapy Radiometer (International Light, Newburyport, MA). To generate dose responses, reaction mixes were either treated with UVB for increasing periods of time at a fixed distances from the light source (25 cm) or by increasing the intensity of the light source. Results from both methods were expressed as mJ/cm2 and produced similar results. Unless otherwise indicated, intracellular reactive oxygen species were detected using 2′,7′-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR) in conjunction with flow cytometry as described previously (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar, 22Heck D.E. Laskin D.L. Gardner C.R. Laskin J.D. J. Biol. Chem. 1992; 267: 21277-21280Abstract Full Text PDF PubMed Google Scholar). For standard in vitro assays, reaction mixes contained 50 mm phosphate buffer, pH 7.4, and 4.3 μm catalase in a reaction volume of 100 μl. The reaction was initiated by the addition of the fluorescent probe (5 μm, final concentration). Fluorescence was quantified using an HTS 7000 plus bio-assay reader (PerkinElmer Life Sciences, Beaconsfield, Buckinghamshire, UK) with 495 nm excitation and 520 nm emission filters. In some assays, peroxides were detected using 1,2,3-dihydrorhodamine (1,2,3-DHR, Molecular Probes). To determine the effects of pH on UVB light-stimulated hydrogen peroxide production, the reaction was performed in buffers ranging in pH from 3.5 to 10.0 (sodium acetate, pH 3.5 and 4.5; sodium citrate, pH 5.5 and 6.0; potassium phosphate, pH 7.0 and 7.4; sodium borate, pH 8.0, 8.5, 9.0, and 10.0). Cytochrome c assay and nitro blue tetrazolium assays were used to measure superoxide anion production in reaction mixes (23Bors W. Saran M. Michel C. Oberly L. Superoxide Dismutase. II. CRC Press, Boca Raton, FL1982: 31-62Google Scholar, 24Weinberger B. Fakhrzadeh L. Heck D.E. Laskin J.D. Gardner C.R. Laskin D.L. Am. J. Respir. Crit. Care Med. 1998; 158: 931-938Crossref PubMed Scopus (50) Google Scholar). Catalase Variants, Transfections, Protein Sequencing, and Western Blotting—Protein was quantified using either the BCA protein reagent kit (Pierce) or the detergent-compatible (Dc) protein assay (Bio-Rad) with bovine serum albumin (BSA) 1The abbreviations used are: BSA, bovine serum albumin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; 1,2,3-DHR, 1,2,3-dihydrorhodamine; 3-AT, 3-amino-1,2,4-triazole; BSO, buthionine sulfoximine. as the standard. Western blots were run as previously described (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar, 22Heck D.E. Laskin D.L. Gardner C.R. Laskin J.D. J. Biol. Chem. 1992; 267: 21277-21280Abstract Full Text PDF PubMed Google Scholar). Briefly, lysates containing catalase were separated on 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. After blocking with 5% BSA in tTBS buffer (Tris-buffered saline with 0.1% Tween 20) for 1 h, membranes were incubated with anti-catalase antibodies overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Catalase-antibody complexes were visualized using enhanced chemiluminescence (ECL) reagents (PerkinElmer Life Sciences, Boston, MA). Protein fragments derived from tryptic digests of purified protein were analyzed using a Finigan LCQ DECA XP mass spectrometer interfaced with a Thermo separation system. Fragments were aligned and sequences compared using NCBI Blast algorithms (25Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Res. 1997; PubMed Scopus Google Scholar). Catalase were that contained to a were to the keratinocytes using an according to the manufacturer's In some were and using fluorescent In these of the cells contained The in conjunction with Dr. were to of the catalase In we analyzed the effects of UVB light on the generation of intracellular oxidants in human and mouse We found that UVB in the range of a marked increase in the of reactive oxygen species in both of these cell 1 and data response was on the dose of UVB light. To damaging cells possess and molecular molecules with antioxidant activity (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 19Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar, PubMed Scopus Google Scholar, Physiol. Google Scholar). intracellular as critical for cellular reactive oxygen intermediates is In addition to role as a for antioxidant this in the of and and functions to reactive species to (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, D. Biochem. J. 2001; PubMed Scopus Google Scholar, W. J.L. van Toxicol. Pharmacol. PubMed Scopus Google Scholar, S. A.A. Mutat. Res. 2001; PubMed Scopus Google Scholar). We found that keratinocytes of using buthionine an of intracellular levels of reactive oxygen species cells were to the effects of UVB light These that is critical for the of damaging reactive oxygen species in UVB-treated To by which UVB light the of reactive oxygen species in keratinocytes, we cells were for this by of mouse keratinocytes, were found to UVB oxidant generating activity. of the activity in cell was reduced to by higher of the oxidant generating activity was found in the cell was of and other intracellular including and the We found that the reactive oxygen species generating activity was non-dialyzable, by and suggesting that the material was a protein and data generating reactive oxygen intermediates in response to UVB light was purified using ion exchange, metal affinity, and size exclusion of the using an ion exchange column and a Western are in the size exclusion this activity eluted with a molecular mass of 240 and exhibited strong absorption in the range of 320–360 nm with additional soret peaks at 400–410 nm the presence of digests of the material were analyzed by liquid chromatography-ion trap mass spectrometry and found to fragments that were highly with the for catalase and of mouse catalase Institutes of these sequences were with catalase sequences from with human catalase, with bovine liver catalase, and with the of catalase the significant found in catalases and with a protein containing an heme. These data that the oxidant generating protein was catalase. was by Western of the column using antibodies A and catalase including twice crystallized bovine liver catalase and purified bovine and mouse liver catalase, produced reactive oxidant species stimulated by UVB light these data indicate that catalase is for the UVB reactive oxygen species generating activity in In we this activity using highly purified bovine liver catalase. We found that the of the enzyme to generate reactive oxidants was on the dose of light and the of catalase was also temperature- and and inhibited by and These were performed using as the probe for were also found using the peroxide probe In we found that peroxide production by UVB-treated catalase either or was used as a probe suggesting that oxygen was the of UVB used the superoxide anion be detected by c and nitro blue tetrazolium assays, suggesting that may be an in the reaction these we were to superoxide anion at of UVB that mJ/cm2 the present we the that the superoxide anion is in the reactions at that is observed in keratinocytes, the production of reactive oxygen species by purified catalase in response to UVB light was in the presence of reduced and The pH for oxidant generation was the reaction was additional were The and pH of the activity indicate that reactive oxygen species production is by the intracellular and the of the is well that catalase an activity for hydrogen peroxide the a process also to as catalatic activity B. N. Biochem. J. PubMed Scopus Google Scholar, N. R. B. Biochem. J. PubMed Scopus Google Scholar, A. A.L. Physiol. Rev. PubMed Scopus Google Scholar, New Scholar). In in the presence of of hydrogen peroxide catalase activity. In this oxygen containing electron such as and molecular phenolic are (for reviews, see Refs. N. R. B. Biochem. J. PubMed Scopus Google and Life Sci. PubMed Scopus Google Scholar, M. 2000; PubMed Scopus Google Scholar, E. 1997; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar, Rev. 20: PubMed Scopus Google Scholar). The the of catalase to in catalatic and reactions the of numerous B. N. Biochem. J. PubMed Scopus Google Scholar, Life Sci. PubMed Scopus Google Scholar, D.L. Curr. Chem. Biol. 2001; PubMed Scopus Google Scholar, N. B. Med. Biol. PubMed Scopus Google Scholar, Y. J. Mol. Biol. 2000; PubMed Scopus (349) Google Scholar, C. D.J. Biol. 2000; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). In the for in the protein was using which enzyme activity B. N. Y. Sci. PubMed Scopus Google Scholar). a of catalase that to the Y. J. Mol. Biol. 2000; PubMed Scopus (349) Google Scholar). A for both the catalatic and of the enzyme is a hydrogen peroxide functions to peroxide molecules for by catalase, an for both The enzyme to catalase by this Y. J. Mol. Biol. 2000; PubMed Scopus (349) Google Scholar, C. D.J. Biol. 2000; PubMed Scopus Google Scholar, S. J.L. M. J. Am. Chem. 2001; PubMed Scopus Google Scholar). studies indicate that catalatic and activity may also a that reaction intermediates and facilitates the of to regulate catalase activity at the metal by the of a and an at Y. J. Mol. Biol. 2000; PubMed Scopus (349) Google Scholar). electron by the presence of at the may to this interactions this and that are to for the of the enzyme by sodium A. A.L. Physiol. Rev. PubMed Scopus Google Scholar, Y. J. Mol. Biol. 2000; PubMed Scopus (349) Google Scholar). Our that the effects of UVB light on catalase were highly and additional that reactive oxygen species may be by the transfer of and interactions with molecular To this inhibitors of the catalatic and activity of catalase were used. We found that sodium a catalase activity at the generation of reactive oxygen species at higher In both sodium and enhanced UVB reactive oxygen species production The actions of these inhibitors on the of catalase that the UVB is The in production of reactive oxygen species at of sodium or in the presence of or sodium azide, may result from of a hydrogen peroxide activity of catalase, through of peroxide in the presence of higher of interaction of the anion with catalase is to to and to the activity of the both to in the production of reactive oxygen The generation of reactive oxygen species that is observed in the presence of and sodium that the presence of at the peroxide and the activity of the are both in UVB oxidant of to hypothesize that, similar to UVB effects the peroxide molecules to the In from molecules is also by of the as molecules a source for the generation of which with to generate reactive oxygen A as to the of catalase as a of oxidant production in keratinocytes to UVB light. We that catalase is in this then of the enzyme of reactive oxidants produced by the To this we reduced levels of catalase protein in cells by for catalase into PAM 212 We found that reduced both intracellular levels of catalase and the production of reactive oxygen species in response to UVB light was observed in cells that were or in cells with for the or with We also oxidant production in response to UVB light using a catalase cell and a to hydrogen peroxide that catalase Y. Res. 1998; Scopus Google Scholar). the and OC5 cells produced peroxides in response to UVB light of were produced by OC5 cells in response to UVB light. these data indicate that intracellular catalase mediates oxidant in cells to UVB light. Our that, in response to UVB catalase generates reactive oxygen intermediates is both novel and highly divergent from the well known antioxidant functions of this enzyme. We hypothesize that, on the intracellular oxidant catalase activity in response to UVB light can be either or through the actions of catalase, high energy ultraviolet light is absorbed by the enzyme and converted to reactive chemical intermediates that can be and detoxified by cellular antioxidant enzymes. antioxidant are UVB oxidants through the actions of catalase can cellular damage and tissue We that reactive peroxides produced in excessive or through the actions of catalase the to induce oxidative in keratinocytes, damage critical cellular molecules including and contribute to the development of skin cancer.