Blue Light Induces Mitochondrial DNA Damage and Free Radical Production in Epithelial Cells

Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390–550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis. Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390–550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis. It is well recognized that non-ionizing radiation can react photochemically with biological chromophores, producing end products that are toxic and/or mutagenic in mammalian cells. Most studies have concentrated on the role of UV irradiation due to its high energy, photoreactivity, wide range of biological chromophores, specific cellular responses, and association with pathologies such as skin melanoma and cataract (1Jones C.A. Huberman E. Cunningham M.L. Peak M.J. Radiat. Res. 1987; 110: 244-254Crossref PubMed Scopus (122) Google Scholar, 2Peak J.G. Peak M.J. Mutat. Res. 1991; 246: 187-191Crossref PubMed Scopus (102) Google Scholar, 3Peak J.G. Peak M.J. Photochem. Photobiol. 1995; 61: 484-487Crossref PubMed Scopus (16) Google Scholar, 4Peak J.G. Peak M.J. Sikorski R.S. Jones C.A. Photochem. Photobiol. 1985; 41: 295-302Crossref PubMed Scopus (92) Google Scholar). However, the role of visible light has been less extensively investigated, even though studies have demonstrated that visible light can induce cellular dysfunction and cell death both in vitro and in vivo (1Jones C.A. Huberman E. Cunningham M.L. Peak M.J. Radiat. Res. 1987; 110: 244-254Crossref PubMed Scopus (122) Google Scholar, 2Peak J.G. Peak M.J. Mutat. Res. 1991; 246: 187-191Crossref PubMed Scopus (102) Google Scholar, 3Peak J.G. Peak M.J. Photochem. Photobiol. 1995; 61: 484-487Crossref PubMed Scopus (16) Google Scholar, 5Tyrrell R.M. Werfelli P. Moraes E.C. Photochem. Photobiol. 1984; 39: 183-189Crossref PubMed Scopus (49) Google Scholar, 6Setlow R.B. Grist E. Thompson K. Woodhead A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6666-6670Crossref PubMed Scopus (599) Google Scholar, 7Setlow R.B. Woodhead A.D. Mutat. Res. 1994; 307: 365-374Crossref PubMed Scopus (79) Google Scholar). The blue region (400–500 nm) of the visible spectrum is likely to be particularly important because it has a relatively high energy, can penetrate tissue(s), and is associated with the occurrence of malignant melanoma in animal models (6Setlow R.B. Grist E. Thompson K. Woodhead A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6666-6670Crossref PubMed Scopus (599) Google Scholar, 7Setlow R.B. Woodhead A.D. Mutat. Res. 1994; 307: 365-374Crossref PubMed Scopus (79) Google Scholar). Surprisingly, despite its potential for damage, the use of blue light blockers in sunscreens and spectacle lenses has, until recently, received only limited attention. Studies have shown that irradiation of mammalian cells with visible light induces cellular damage primarily via reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; RPE, retinal pigment epithelium; QPCR, quantitative PCR; nDNA, nuclear DNA; mtDNA, mitochondrial DNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. (8Peak M.J. Peak J.G. Photodermatol. 1989; 6: 1-15PubMed Google Scholar). ROS such as the hydroxyl radical, superoxide anion, and singlet oxygen can be produced when visible light excites cellular photosensitizers (9Peak M.J. Peak J.G. Photochem. Photobiol. 1990; 51: 649-652Crossref PubMed Scopus (76) Google Scholar, 10Cunningham M.L. Krinsky N.I. Giovanazzi S.M. Peak M.J. J. Free Radic. Biol. Med. 1985; 1: 381-385Crossref PubMed Scopus (104) Google Scholar). Whereas photosensitizers such as melanin and lipofuscin in pigmented cells and retinoids in photoreceptor cells have been identified, the identity and location of photosensitizers in non-pigmented cells remain largely unknown. However, a number of options exist, including flavin-containing oxidases, the cytochrome system, heme-containing proteins, and tryptophan-rich proteins. The interaction of these chromophores with light can generate ROS, which in turn can damage lipids, proteins, and DNA. This is emphasized by the study of Hockberger et al. (11Hockberger P.E. Skimina T.A. Centonze V.E. Lavin C. Chu S. Dadras S. Reddy J.K. White J.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6255-6260Crossref PubMed Scopus (265) Google Scholar), who found that violet-blue light stimulated H2O2 production from peroxisomes and mitochondria in cultured 3T3 and CV1 mammalian cells. Hydrogen peroxide production was enhanced by overexpression of flavin-containing oxidases, which proposes that violet-blue light initiates photoreduction of flavins, which activate flavin-containing oxidases in mitochondria and peroxisomes, resulting in H2O2 production. Furthermore, the mechanism by which photosensitization leads to cellular dysfunction is unclear but may center on DNA damage. As reviewed by Goyns (12Goyns M.H. Mech. Ageing Dev. 2002; 123: 791-799Crossref PubMed Scopus (52) Google Scholar), the role of DNA damage in aging mammals appears to be pivotal, and there is increasing evidence that oxidative damage is an important factor in producing mutations in genes, shortening telomeres, and damaging mitochondrial DNA. To support a role for visible light in DNA damage, Pflaum et al. (13Pflaum M. Kielbassa C. Garmyn M. Epe B. Mutat. Res. 1998; 408: 137-146Crossref PubMed Scopus (102) Google Scholar) have previously shown that oxidative damage induced by visible light does yield DNA modifications. The aim of this study was to establish whether blue light is able to cause differential damage to the mitochondrial and nuclear genome and to determine whether this occurs via ROS production at mitochondria. Our results demonstrate that exposure of non-pigmented epithelial cells to blue light causes mitochondrial dysfunction and mtDNA damage and that such effects are mediated by the action of reactive oxygen species. We have further identified that the ROS primarily responsible for blue light-induced mtDNA damage is the superoxide radical. Ham's F-10 and Ham's SF10PF cell culture media were obtained from Invitrogen (Paisley, UK). Fetal calf serum was obtained from TCS Biologicals Ltd. (Buckingham, UK). Sucrose, EDTA, and trichloroacetic acid were obtained from BDH (Poole, UK). Trypsin, antibiotics, fungizone, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), phosphate-buffered saline tablets, mannitol, cytochrome c (horse heart, type III), superoxide dismutase (bovine erythrocytes), 4-nitroso-N,N-dimetylaniline, histidine, salicylic acid, catalase (bovine liver), and sodium azide were from Sigma. Qiagen genomic tip 20G kit was from Qiagen (Valencia, CA). Recombinant Thermus thermophilus DNA polymerase was from PerkinElmer Life Sciences, and the primers for PCR were synthesized by Invitrogen (Rockville, MD). Taxifolin and MitoPBN were from Merck Biosciences Ltd., UK. All other chemicals used were of highest purity analytical grade. Human RPE cells were isolated and cultured as previously described (14Shamsi F.A. Boulton M. Investig. Ophthalmol. Vis. Sci. 2001; 42: 3041-3046PubMed Google Scholar). Human eyes (Bristol Eye Bank, Bristol, UK) were obtained from donors between the ages of 40 and 70 years. The corneas had been used for transplantation, and permission had been given to use the poles for research. The RPE cells were grown in Ham's F-10 medium supplemented with 20% fetal calf serum and antibiotics, and cultures were maintained at 37 °C and 5% CO2. RPE cells were grown to confluence in 24-well plates or 75-cm2 culture flasks as required. Depigmented cells between passage 3 and 5 were used for all experiments (14Shamsi F.A. Boulton M. Investig. Ophthalmol. Vis. Sci. 2001; 42: 3041-3046PubMed Google Scholar). At confluence, growth medium was replaced with basal Ham's F-10 medium supplemented with 2% fetal calf serum and maintained for an additional 24 h. Prior to light exposure, the basal medium was replaced with SF10PF medium, which lacks the photosensitizers phenol red, tryptophan, riboflavin, and folic acid (15Schutt F. Davies S. Kopitz J. Holz F.G. Boulton M.E. Investig. Ophthalmol. Vis. Sci. 2000; 41: 2303-2308PubMed Google Scholar). For each set of experiments, one set of RPE cultures was wrapped in a black sheet of paper (dark maintained), and the other set was left uncovered (light exposed). Both light and dark cultures were exposed to 390–550-nm light (referred to as blue light by a light UV Ltd., with for h. The was 2.8 and the cells were maintained at a of 37 light exposure and lipofuscin cellular DNA was isolated using a Qiagen genomic tip 20G kit as described by the DNA by this results in genomic for cellular DNA were by bromide with an with an at nm and an at nm using DNA as a DNA was assessed by to As previously described B. C.A. Eye Res. 1999; PubMed Scopus Google Scholar), was by of a in the mtDNA and an in the genes and and with the that yield were in a PCR with the kit Life of genomic DNA as The for the have been previously described for the mtDNA and B. C.A. Eye Res. 1999; PubMed Scopus Google Scholar, B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The a for the number of cells in a given It is on the of a mitochondrial from cells to the tetrazolium of the and dark blue that are largely to cell resulting in its cells. The of the is to the number of cells in the were in 24-well plates at a of and at 37 the medium was replaced with were at and At each cell was using the as described previously (15Schutt F. Davies S. Kopitz J. Holz F.G. Boulton M.E. Investig. Ophthalmol. Vis. Sci. 2000; 41: 2303-2308PubMed Google Scholar). medium was from the and of was each The cells were for 3 h at 37 which the was and of was to the blue were to a and was at a test of nm and a of nm on a were using the as a of of of cells are the cells the of the human RPE cells from 75-cm2 flasks were at for 5 at The cell was in of and to a The cells were to for 5 and with of the of mannitol, and EDTA, was to of and the the was at for 5 at °C to cells, and The mitochondria from the were by at for at The mitochondrial was in 5 of mannitol, 70 EDTA, and 5 and on a mitochondrial was to the that of of and by at for at The mitochondria at the were The was with phosphate-buffered and the mitochondria were at for at The was with phosphate-buffered and the mitochondria were in phosphate-buffered saline for ROS production anion production was by the of cytochrome c P.E. J. 1990; PubMed Scopus Google Scholar, J. 1993; PubMed Scopus Google Scholar). A cytochrome c III), and The was by the of isolated mitochondria and exposure of the to blue light or in the the at nm was at on a anion production was confirmed by of production by superoxide dismutase (bovine oxygen was in by the of and E. Photochem. Photobiol. Scopus Google Scholar), with modifications. The of and 5 a of The was exposed to blue light, and were in the of peroxide of with singlet oxygen results in the of the of which is at of singlet oxygen in the was by the with sodium by mitochondria in the of light or dark was by the of et al. B. J. A. PubMed Scopus Google Scholar). The the EDTA, and The was by the of mitochondria and of the blue light or in the The was by the of of and of The products were by which was to at 40 and the were in of The were in the trichloroacetic acid in sodium and of sodium the for 5 at was and the was at of hydroxyl in the was by the with catalase (bovine were isolated and from human donors as described by Boulton et al. M. A. J. M. J. Photochem. Photobiol. B. 1993; PubMed Scopus Google Scholar), that RPE cells were by of The of the was using a were with lipofuscin in basal medium (14Shamsi F.A. Boulton M. Investig. Ophthalmol. Vis. Sci. 2001; 42: 3041-3046PubMed Google Scholar). The cultures were maintained for which the basal medium was cells were by blue light as described were each set of of superoxide and MitoPBN were in SF10PF medium at a of the of the the was to the medium to a of cells were by blue light as described All were a of using of was used to determine and test was to between of were The of Light on determine whether blue light is able to cause cell the was to mitochondrial respiratory as an of the cell was in mitochondrial the 3 h of irradiation compared with cells maintained in the dark However, a small but of was observed at 6 h in cells. cells to an increasing loss of at h was in in cells maintained in the of blue light The of Light on DNA investigate the mechanism of cell loss blue light the damage to mtDNA and was assessed with a which is on the that oxidative DNA lesions DNA B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). DNA damage was by the of of of DNA kb for the mtDNA and kb for the and this to the of which have a of damaged B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). DNA damage was observed for mtDNA in cells damage was observed at 3 h (0.7 lesion/10 kb by 6 the number of DNA lesions was decreased to lesion/10 kb mtDNA, that DNA was lesions were in cells exposed to blue light at of the was there damage to mitochondrial or nuclear DNA for cells maintained in the ROS determine whether blue light-induced mtDNA lesions and cell loss were associated with production of reactive oxygen species from the ROS were in isolated mitochondria exposed to blue mitochondria a increase in the of ROS superoxide anion, hydroxyl radical, and singlet A and increase in the of superoxide was observed h of exposure and until 6 h end of were found in the of hydroxyl and singlet oxygen of each ROS was confirmed by the of the and sodium azide catalase for superoxide anion, hydroxyl radical, and singlet oxygen, each the ROS were production of ROS was observed in mitochondria maintained in the dark and with which may the effects of of on and have previously shown that lipofuscin, an intracellular can light-induced damage via ROS in the RPE cells M. J. Boulton M.E. J. Biol. 1995; PubMed Scopus Google Scholar, U. A. K. Free Radic. Biol. Med. PubMed Scopus Google Scholar, S. M.H. E. M. F.A. Boulton M.E. Free Radic. Biol. Med. 2001; PubMed Scopus Google Scholar). We used this as a to that a biological intracellular ROS generator can cause DNA damage and to determine whether the mtDNA damage was from the mitochondria or from chromophores in the cells, DNA damage was observed for both mtDNA and For mtDNA, damage was observed at 3 h lesion/10 kb by 6 the number of DNA lesions had decreased to lesion/10 kb mtDNA, that DNA was DNA demonstrated a increase in DNA lesions in cells. At 3 observed lesion/10 kb nDNA, which to lesion/10 kb at 6 h cells maintained in the dark damage. However, mtDNA in RPE cells with lipofuscin and in a in at all The of Taxifolin and MitoPBN on DNA the role of specific ROS in blue light-induced mtDNA damage, the superoxide of and the of the MitoPBN in cells, mtDNA was observed at and 6 h compared with cells in the of which damage at 3 and 6 h. cells mtDNA the blue light and cells maintained in the dark mtDNA damage at of the light damage is an of the skin and This is because both be UV through and or are exposed to the visible spectrum, of which the blue light region is the the blue light damage is photochemical in and to largely from chromophores and the the is less but is likely to chromophores associated with mitochondria and that in the blue region of the visible spectrum to contribute significantly to this have demonstrated that blue light is able to ROS from isolated mitochondria. This that mitochondria blue Studies have shown that blue light is able to cause damage and cell death in RPE cells J. S. Ophthalmol. 1998; PubMed Scopus Google Scholar, J. J. Ophthalmol. 1999; PubMed Scopus Google Scholar, A. E. Photochem. Photobiol. PubMed Scopus Google Scholar), which that other chromophores, lipofuscin, in the RPE cells these light It has been that blue light-induced retinal damage is mediated by mitochondrial respiratory Ophthalmol. 1993; PubMed Scopus Google Scholar, Eye Res. 1994; PubMed Scopus Google Scholar), and it has been shown that the mitochondrial respiratory ROS A. E. Photochem. Photobiol. PubMed Scopus Google Scholar, Med. 41: PubMed Scopus Google Scholar). This is further by the that H2O2 production is significantly in epithelial cells flavin-containing oxidases (11Hockberger P.E. Skimina T.A. Centonze V.E. Lavin C. Chu S. Dadras S. Reddy J.K. White J.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6255-6260Crossref PubMed Scopus (265) Google Scholar), which are concentrated in mitochondria J. 1989; Google Scholar). oxidases the spectrum, with at and it has been demonstrated that the of cytochrome c was in the RPE cells exposure to blue light E. B. Eye Res. PubMed Scopus Google Scholar). c is an important mitochondrial respiratory in oxidative It has at nm in the S. J. Photochem. Photobiol. B. PubMed Scopus Google Scholar). these the of mitochondrial respiratory in the of singlet oxygen, superoxide anion, and the hydroxyl radical, all of which are relatively reactive which are likely to with in the mtDNA and proteins. This is by the that ROS from the mitochondrial respiratory are responsible for the basal of oxidative observed in of mammalian cells S. Epe B. Free Radic. Biol. Med. PubMed Scopus Google Scholar). ROS be in to the of ROS generated as a of oxidative Biol. Med. 2002; PubMed Scopus Google Scholar). We have demonstrated that mtDNA damage exposure to blue DNA lesions were at 3 h exposure with evidence of at that there is an to with the oxidative Davies 1994; PubMed Scopus Google Scholar). the damage it be because there was damage to the nuclear and it be in with studies on ROS by isolated mitochondria. This of the mtDNA to light damage compared with be due to a number of mtDNA is to the mitochondrial the ROS are mtDNA is by as is the for B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google mitochondria a number of chromophores that in the blue light region and J. 1989; Google Scholar, E. B. Eye Res. PubMed Scopus Google Scholar, PubMed Scopus Google the of the ROS a intracellular blue generator of ROS including singlet oxygen, superoxide anion, hydroxyl and mtDNA damage but cause damage in This that mtDNA damage is a and in the of damage, it is the generated that are able to the Our that mtDNA in RPE cells with lipofuscin and in the dark a in at all may well a to due to of DNA The of RPE cells with lipofuscin may have caused DNA damage, DNA We have previously shown that lipofuscin a of ROS dark M. A. J. M. J. Photochem. Photobiol. B. 1993; PubMed Scopus Google Scholar). It has been demonstrated that oxidative can induce of DNA such as and DNA polymerase in RPE cells B. C.A. Eye Res. 1999; PubMed Scopus Google Scholar, Eye Res. 2001; PubMed Scopus Google Scholar). as lipofuscin in the RPE cells with aging, light-induced ROS production by lipofuscin and cell damage may a role in aging of these cells. The that mtDNA had less damage 6 h of light irradiation compared with that 3 h that DNA is by 3 h. it was that the mitochondria an and DNA to that nDNA, et al. Mutat. Res. 1999; PubMed Scopus Google Scholar) have demonstrated that mitochondria are of oxidative DNA damage to damage to and Whereas mitochondria have an lesions by Res. Biol. 2001; PubMed Scopus Google Scholar, Res. Biol. 2001; PubMed Google Scholar). the mtDNA appears relatively in because the mitochondrial even at the 6 and h of these lesions in a dysfunction in oxidative a of mtDNA damage B. Mutat. Res. 2002; PubMed Scopus Google Scholar). It is that the mitochondrial to oxidative on the of oxidative can in damage or the of cell death assessed the effect of ROS damage that caused mtDNA lesions but lead to cell dysfunction via this mechanism is likely to in decreased respiratory and the of mtDNA ROS damage to mitochondria the M. A. J. 2001; 1: PubMed Scopus Google Scholar, M.J. J. Sci. 2002; PubMed Scopus Google Scholar, M.J. J. Res. PubMed Scopus Google Scholar, A. A. M. Centonze V.E. B. Res. PubMed Scopus Google Scholar). of ROS by can lead to mitochondrial M.J. Acad. Sci. PubMed Scopus Google Scholar), mitochondrial M.J. J. Res. PubMed Scopus Google Scholar), and and mitochondrial and A. A. M. Centonze V.E. B. Res. PubMed Scopus Google Scholar). To there is evidence that the damage can be via growth and factor (12Goyns M.H. Mech. Ageing Dev. 2002; 123: 791-799Crossref PubMed Scopus (52) Google Scholar, J. M. S. A. PubMed Scopus Google Scholar). A study by et al. J. M. S. A. PubMed Scopus Google Scholar) demonstrated that oxidative mtDNA damage in The biological activity of has been previously identified in studies Biol. Med. PubMed Scopus Google Scholar, M. Biol. PubMed Scopus Google Scholar). are of with to the in blue light-induced mtDNA damage. We indicating that the superoxide anion may be a primary ROS in mtDNA damage blue light It is that the of superoxide a wide of mitochondrial such as flavins, and which have all been shown to generate superoxide C. J. 2002; PubMed Scopus Google Scholar, J. Biol. 1994; PubMed Google Scholar, C.A. J. Biol. 1998; PubMed Scopus Google Scholar). Furthermore, have previously demonstrated that dismutase is to oxidative damage, the superoxide of the mitochondria Boulton M. Free Radic. Biol. Med. Google Scholar). To further support for these used a which is the superoxide J. K. F. S. J. Biol. PubMed Scopus Google Scholar). This which is and to mtDNA this that these relatively that are generated in mitochondrial to the genome are the primary of mtDNA damage in this the of mtDNA to ROS in to blue light a role for visible irradiation in cellular dysfunction be in such as skin and which are exposed to blue light and ROS V.E. K. Acad. Sci. 2002; PubMed Scopus Google Scholar, M.L. A. C. A. C. Acad. Sci. PubMed Scopus Google Scholar). The of oxidative damage in mitochondria a number of of aging in which the a role Biol. Med. 2002; PubMed Scopus Google Scholar, A. J. 2002; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, A. 2001; PubMed Scopus Google Scholar). Furthermore, the aging is likely to contribute to a of age-related pathologies such as skin and age-related We and for