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Supranuclear Melanin Caps Reduce Ultraviolet Induced DNA Photoproducts in Human Epidermis

1998; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1998.00178.x

1523-1747

Nobuhiko Kobayashi, Akemi Nakagawa, Tsutomu Muramatsu, Yukio Yamashina, Toshihiko Shirai, Mitsumasa Hashimoto, Yasuhito Ishigaki, Takeo Ohnishi, Toshio Mori,

Circadian rhythm and melatonin

Melanin can form supranuclear caps in human epidermis, suggesting that intracellular melanin reduces ultraviolet transmission to underlying cell nuclei and inhibits the formation of ultraviolet induced DNA photoproducts. The purpose of this study was to determine the photoprotective effect of epidermal melanin. We irradiated normal human skin explants with ultraviolet B and determined the formation of cyclobutane pyrimidine dimers and (6–4)photoproducts in individual epidermal cells by indirect immunofluorescence and by laser cytometry using monoclonal antibodies specific for cyclobutane dimers or for (6–4)photoproducts. We found that epidermal cells with supranuclear melanin caps had significantly less DNA photoproducts (both types) than epidermal cells without supranuclear melanin caps. Moreover, the protection factor against both types of photolesions correlated with melanin concentration in epidermal cells. These results indicate that melanin reduces ultraviolet induced DNA photoproducts in human epidermis in a concentration dependent manner. Melanin can form supranuclear caps in human epidermis, suggesting that intracellular melanin reduces ultraviolet transmission to underlying cell nuclei and inhibits the formation of ultraviolet induced DNA photoproducts. The purpose of this study was to determine the photoprotective effect of epidermal melanin. We irradiated normal human skin explants with ultraviolet B and determined the formation of cyclobutane pyrimidine dimers and (6–4)photoproducts in individual epidermal cells by indirect immunofluorescence and by laser cytometry using monoclonal antibodies specific for cyclobutane dimers or for (6–4)photoproducts. We found that epidermal cells with supranuclear melanin caps had significantly less DNA photoproducts (both types) than epidermal cells without supranuclear melanin caps. Moreover, the protection factor against both types of photolesions correlated with melanin concentration in epidermal cells. These results indicate that melanin reduces ultraviolet induced DNA photoproducts in human epidermis in a concentration dependent manner. cyclobutane pyrimidine dimers (6–4) photoproducts Darker skinned individuals are much less susceptible to sun induced skin cancer than are fair skinned individuals (Kollias et al., 1991Kollias N. Sayre R.M. Zeise L. Chedekel M.R. Photoprotection by melanin.J Photochem Photobiol B. 1991; 9: 135-160Crossref PubMed Scopus (337) Google Scholar). Such a difference can be understood in terms of ultraviolet (UV) transmission of the epidermis (Kaidbey et al., 1979Kaidbey K.H. Agin P.P. Sayre R.M. Kligman A.M. Photoprotection by melanin – a comparison of black and Caucasian skin.J Am Acad Dermatol. 1979; 1: 249-260Abstract Full Text PDF PubMed Scopus (319) Google Scholar), because the epidermis of darker skin has more and larger melanosomes than that of fair skin (Montagna and Carlisle, 1991Montagna W. Carlisle K. The architecture of black and white facial skin.J Am Acad Dermatol. 1991; 24: 929-937Abstract Full Text PDF PubMed Scopus (153) Google Scholar). The absorption of light by melanin is nonspecific and extends through UV into visible ranges, but it is most pronounced toward the shorter end of the spectrum (Anderson and Parrish, 1981Anderson R.R. Parrish J.A. The optics of human skin.J Invest Dermatol. 1981; 77: 13-19Abstract Full Text PDF PubMed Scopus (1919) Google Scholar). Moreover, within individual keratinocytes, melanin can be located over nuclei to form supranuclear melanin caps, which are in the path of incoming UV photons (Montagna and Carlisle, 1991Montagna W. Carlisle K. The architecture of black and white facial skin.J Am Acad Dermatol. 1991; 24: 929-937Abstract Full Text PDF PubMed Scopus (153) Google Scholar). Two major types of DNA photoproducts (photoproduct type DNA damage), cyclobutane pyrimidine dimers (CPD) and (6–4)photoproducts (6–4PP), are induced in UV irradiated human skin, both of which are potentially carcinogenic (Setlow, 1978Setlow R.B. Repair deficient human disorders and cancer.Nature (London. 1978; 271: 713-717Crossref PubMed Scopus (379) Google Scholar; Muramatsu et al., 1992Muramatsu T. Kobayashi N. Tada H. Yamaji M. Shirai T. Mori T. Ohnishi T. Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6–4) photoproducts in organ-cultured normal human skin.Arch Dermatol Res. 1992; 284: 232-237Crossref PubMed Scopus (28) Google Scholar). These observations suggest that epidermal melanin pigment prevents UV induced skin cancer development by reducing UV transmission to underlying cell nuclei and preventing the formation of both types of DNA photoproducts. Intracellular melanin has been reported to reduce the formation of both CPD and 6–4PP in cultured human melanocytes and melanoma cells (Kobayashi et al., 1993Kobayashi N. Muramatsu T. Yamashina Y. Shirai T. Ohnishi T. Mori T. Melanin reduces ultraviolet-induced DNA damage formation and killing rate in cultured human melanoma cells.J Invest Dermatol. 1993; 101: 685-689Abstract Full Text PDF PubMed Google Scholar; Barker et al., 1995Barker D. Dixon K. Medrano E.E. et al.Comparison of the responses of human melanocytes with different melanin contents to ultraviolet B irradiation.Cancer Res. 1995; 55: 4041-4046PubMed Google Scholar). Tanning has also been reported to reduce DNA photoproducts induced by subsequent UV irradiation in human skin (Gange et al., 1985Gange R.W. Blackett A.D. Matzinger E.A. Sutherland B.M. Kochevar I.E. Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin.J Invest Dermatol. 1985; 85: 362-364Abstract Full Text PDF PubMed Scopus (87) Google Scholar; Eggset et al., 1986Eggset G. Krokan H. Volden G. UV-induced DNA damage and its repair in tanned and untanned human skin in vivo.Photobiochem Photobiophys. 1986; 10: 181-187Google Scholar; Young et al., 1991Young A.R. Potten C.S. Chadwick C.A. Murphy G.M. Hawk J.L.M. Cohen A.J. Photoprotection and 5-MOP photochemoprotection from UVR- induced DNA damage in humans: the role of skin type.J Invest Dermatol. 1991; 97: 942-948Abstract Full Text PDF PubMed Google Scholar; Potten et al., 1993Potten C.S. Chadwick C.A. Cohen A.J. Nikaido O. Matsunaga T. Schipper N.W. Young A.R. DNA damage in UV-irradiated human skin in vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.Int J Radiat Biol. 1993; 63: 313-324Crossref PubMed Scopus (50) Google Scholar), but this is accompanied by various epidermal changes, including epidermal thickening, in addition to melanization. Thus, it is inconclusive whether epidermal melanin itself is protective against photolesions. Ishikawa et al., 1984Ishikawa T. Kodama K. Matsumoto J. Takayama S. Photoprotective role of epidermal melanin granules against ultraviolet damage and DNA repair in guinea pig skin.Cancer Res. 1984; 44: 5195-5199PubMed Google Scholar determined the formation of DNA photoproducts in UV irradiated black and white guinea pig skin by measuring unscheduled DNA synthesis, but were unable to find any protective role of epidermal melanin; however, in guinea pigs, hair might serve as an effective sunscreen instead of melanin, and the autoradiographic technique used in that study might not be sensitive enough to discriminate differences in DNA photoproduct formation between pigmented and unpigmented areas. Thus, it is important to use normal human skin and a DNA photoproduct detection system with sufficient sensitivity to examine the photoprotective effect of epidermal melanin. We have established an in situ immunofluorescent laser cytometric method using monoclonal antibodies against DNA photoproducts (Mori et al., 1989Mori T. Wani A.A. D’Ambrosio S.M. Chang C.-C. Trosko J.E. In situ pyrimidine dimer determination by laser cytometry.Photochem Photobiol. 1989; 49: 523-526Crossref PubMed Scopus (21) Google Scholar, Mori et al., 1990Mori T. Matsunaga T. Chang C.-C. Trosko J.E. Nikaido O. In situ (6–4) photoproduct determination by laser cytometry and autoradiography.Mutation Res. 1990; 236: 99-105Crossref PubMed Scopus (21) Google Scholar; Muramatsu et al., 1992Muramatsu T. Kobayashi N. Tada H. Yamaji M. Shirai T. Mori T. Ohnishi T. Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6–4) photoproducts in organ-cultured normal human skin.Arch Dermatol Res. 1992; 284: 232-237Crossref PubMed Scopus (28) Google Scholar; Funayama et al., 1994Funayama T. Mitani H. Ishigaki Y. Matsunaga T. Nikaido O. Shima A. Photorepair and excision repair removal of UV-induced pyrimidine dimers and (6–4) photoproducts in the tail fin of the Medaka,.Oryzias Latipes. J Radiat Res. 1994; 35: 139-146Crossref Scopus (7) Google Scholar). This method allows the detection of DNA photoproducts (both CPD and 6–4PP) in individual epidermal cells after low doses of UV irradiation. In this study, we irradiated normal human skin explants with UVB and determined the formation of CPD and 6–4PP in epidermal cells, with or without supranuclear melanin caps, using in situ immunofluorescent laser cytometry. We found that supranuclear melanin caps reduced the formation of both types of photolesions in a melanin concentration dependent manner. Normal human skin samples were surgically obtained from the lateral side of the left upper arm of a 69 y old man and from the interscapulum of a 66 y old man, and were used for CPD and 6–4PP determination, respectively. Both individuals were skin phototype III and the skin samples were slightly pigmented (light brown) due to chronic sun exposure (Pathak, 1995Pathak M.A. Functions of melanin and protection by melanin.in: Zeise L. Chedekel M.R. Fitzpatrick T.B. Melanin: Its Role in Human Photoprotection. Valdenmar, Overland Park1995: 125-134Google Scholar). Informed consent was obtained prior to the surgery. The skin samples were sliced to a thickness of about 1 mm and cut into pieces ≈2 × 2 mm. Each piece was placed dermis side down on a Millipore filter (pore size, 0.45 μm; diameter, 13 mm) in a 35 mm dish (Falcon 3001), with Dulbecco’s modified Eagle’s medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Flow Laboratories, McLean, VA). The skin explants were incubated in humidified air with 5% CO2 at 37°C for 24 h and irradiated with UV. We have previously reported that epidermal cells remain viable for 24 h or more in this organ culture technique (Muramatsu et al., 1992Muramatsu T. Kobayashi N. Tada H. Yamaji M. Shirai T. Mori T. Ohnishi T. Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6–4) photoproducts in organ-cultured normal human skin.Arch Dermatol Res. 1992; 284: 232-237Crossref PubMed Scopus (28) Google Scholar). The skin explants were washed with 10 mM phosphate buffered saline (PBS) at pH 7.4, and were then UV irradiated with 11 health lamps (FL20SE, Toshiba, Tokyo, Japan; mainly UVB) equipped with a Kodacel TA407 sheet (Eastman Chemical Products, TN) to exclude wavelengths below 275 nm, at a dose rate of 7.14 J per m2 per s, monitored by a Topcon UV Radiometer (UVR-3036/S, Clinical Supply, Tokyo, Japan). Immediately after irradiation, explants were embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at –80°C. The formation of CPD and 6–4PP in UV irradiated normal human skin was detected by indirect immunofluorescence using monoclonal antibodies (TDM-2 and 64M-2) specific for CPD and 6–4PP, respectively (Mori et al., 1991Mori T. Nakane M. Hattori T. Matsunaga T. Ihara M. Nikaido O. Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6–4) photoproduct from the same mouse immunized with ultraviolet-irradiated DNA.Photochem Photobiol. 1991; 54: 225-232Crossref PubMed Scopus (379) Google Scholar; Muramatsu et al., 1992Muramatsu T. Kobayashi N. Tada H. Yamaji M. Shirai T. Mori T. Ohnishi T. Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6–4) photoproducts in organ-cultured normal human skin.Arch Dermatol Res. 1992; 284: 232-237Crossref PubMed Scopus (28) Google Scholar). For CPD detection, skin explants were sectioned to 3 μm thick slices at –25°C, mounted on silane coated glass slides (in quadruplicate), and fixed with cold methanol:acetic acid (3:1) on ice for 1 h. After dehydration through a graded ethanol series, slices were treated with 0.1% trypsin for 3 min at room temperature, and were then treated with 0.07 M NaOH in 70% ethanol for 15 min at room temperature to denature DNA. For 6–4PP detection, 4 μm thick slices were fixed and dehydrated as described above, and treated with 0.07 M NaOH for 3 min. All slices were then treated with 100 μg per ml of RNase A at 37°C for 30 min. They were subsequently incubated with 20% FBS at 37°C for 30 min, and then with TDM-2 at 1:10,000 dilution with 5% FBS at 4°C overnight or with 64M-2 at 1:50 dilution at 37°C for 30 min. The slides were incubated with goat anti-mouse immunoglobulin G (H + L) conjugated with biotin, F(ab′)2 fragment (Zymed, San Francisco, CA) at 37°C for 30 min at 1:50 dilution with 5% FBS for CPD detection or at 1:100 dilution for 6–4PP detection. They were then incubated with streptavidin conjugated with fluorescein isothiocyanate (FITC; Vector Laboratories, Burlingame, CA) at 37°C for 30 min at 1:50 dilution with 5% FBS for CPD detection or at 1:400 dilution for 6–4PP detection. Finally, slides were treated with 1 μg propidium iodide (PI; Sigma) per ml at 37°C for 15 min for CPD detection or 25 ng PI per ml for 5 min for 6–4PP detection, and covered by a coverslip with a drop of ProLongTM Antifade Kit (Molecular Probes, Eugene, OR). FITC and PI fluorescence was observed and analyzed using a Meridian INSIGHT laser microscope and computer system (Meridian Instruments, Okemos, MI). The software allows one to eliminate background level fluorescence and quantitate fluorescent intensity in each nucleus from original images. The formation of CPD or 6–4PP in each nucleus was calculated by dividing the intensity of FITC immunofluorescence by the intensity of PI fluorescence (FITC/PI), because nuclei in a section contain different amounts of DNA. We determined the formation of CPD or 6–4PP (FITC/PI) in 1–11 epidermal cells with supranuclear melanin caps and in 1–11 neighboring epidermal cells without supranuclear caps in 20 areas for each type of photolesion. In each area, we calculated mean values of the formation of CPD or 6–4PP among cells with or without supranuclear caps. We then determined the UV dose equivalent of the formation by comparing mean values with standard induction curves for both types of photolesions obtained in the UV dose–response study. We calculated the protection factor against CPD or 6–4PP formation by epidermal melanin by dividing the UV dose equivalent in cells without supranuclear caps by the equivalent in cells with supranuclear caps in each area. The INSIGHT microscope has an optical microscopic light source and the computer system measures transmitted light intensity (per μm2). The computer system can also permit inversion of data values and can estimate the absorbance of various parts of an image (Tajima et al., 1994Tajima Y. Kato K. Utsumi N. Hosoi K. Computer-aided image analysis applied to immunogold-silver staining: evaluation of proliferating cell nuclear antigen (PCNA) -reactive sites in paraffin sections.Histochem. 1994; 102: 177-181Crossref Scopus (6) Google Scholar). Thus, we measured the absorbance of supranuclear melanin caps and determined the melanin concentration in each area with an arbitrary unit by multiplying the absorbance value by 10–3. CPD and 6–4PP formation in UV irradiated normal human skin Figure 1 shows typical fluorescent images of UV irradiated normal human skin. FITC immunofluorescence, showing CPD or 6–4PP formation, was observed in nuclei that were counterstained with PI Figure 1a–a. We also recorded histologic images of the skin utilizing an optical microscopic light source to analyze the distribution and intracellular localization of melanin (Figure 1a,h). Supranuclear melanin caps were observed mainly in basal and suprabasal cells of the epidermis. Epidermal cells in upper layers had greater intensities of CPD and 6–4PP fluorescence than did cells in lower layers. Some dermal cells had greater intensities than did basal and suprabasal cells. These results suggest that UVB energy is attenuated while penetrating the epidermis and the dermis, and that melanin shields underlying cell nuclei from UV in basal and suprabasal layers. Thus, for the UV dose–response study, we measured the induction of both types of photolesions in more than 75 epidermal cells within five layers under the stratum corneum (not including basal or suprabasal layers) in 3–20 randomly selected areas per section. We found that the induction of CPD and 6–4PP increased in a UV dose dependent manner Figure 2.Figure 2The induction of CPD and 6–4PP in UV irradiated human epidermis. The induction of CPD (a) and 6–4PP (b) in epidermal cells after irradiation of human skin explants with UVB was detected by indirect immunofluorescence and was determined by dividing the intensity of FITC fluorescence for CPD or 6–4PP by the intensity of PI fluorescence for DNA obtained from the Meridian INSIGHT computer system (FITC/PI). Each point shows the mean ± SD of four sections.View Large Image Figure ViewerDownload (PPT) To examine the protective effect of melanin against UV induced DNA photoproducts, we compared the formation of CPD and 6–4PP in epidermal cells with or without supranuclear melanin caps after 8 kJ per m2 of UVB irradiation. Intensities of CPD or 6–4PP immunofluorescence in cells with supranuclear caps were weaker than intensities in cells without supranuclear caps, whereas intensities of DNA fluorescence by PI were similar in both types of cells Figure 3. This result suggests that cells with melanin had less CPD and 6–4PP than cells without melanin. We determined the formation of CPD and 6–4PP as UV dose equivalents in epidermal cells with or without supranuclear caps located in the same layer in 20 areas, because epidermal cells in the same layer should have received similar doses of UV. We then calculated the protection factor against CPD or 6–4PP formation by epidermal melanin in each area. Figure 4(a,b) shows the photoprotective effect of epidermal melanin against CPD and 6–4PP formation as a function of melanin concentration in epidermal cells. Each protection factor value was greater than 1 (1.15–3.92 against CPD, 1.11–5.24 against 6–4PP), indicating that in each area melanin was photoprotective and that epidermal cells with melanin had less CPD and 6–4PP than cells without melanin. Moreover, the protection factor correlated well with melanin concentration. These results suggest that supranuclear melanin caps reduce UV induced DNA photoproducts in a melanin concentration dependent manner.Figure 4Supranuclear melanin caps reduce UV induced DNA photoproducts in a melanin concentration dependent manner in human epidermis. The relationship between protection factor against CPD (a) or 6–4PP (b) formation and melanin concentration among 20 areas for each type of photolesion is shown. Protection factor against CPD or 6–4PP formation by supranuclear melanin caps was calculated by dividing the formation of CPD or 6–4PP (as UV dose equivalent) in cells without supranuclear caps by that in cells with supranuclear caps in the same layer of the epidermis after irradiation of human skin explants with 8 kJ per m2 of UVB. Melanin concentrations of supranuclear caps were determined as arbitrary units by measuring their absorbance using the INSIGHT microscope and computer system utilizing an optical microscopic light source.View Large Image Figure ViewerDownload (PPT) It has been generally assumed that melanin protects epidermal cells against UV induced DNA photoproducts in human skin; however, previously published results designed to test this hypothesis are inconclusive, although melanin induction for photoprotection has begun to find clinical application (Levine et al., 1991Levine N. Sheftel S.N. Eytan T. et al.Induction of skin tanning by subcutaneous administration of a potent synthetic melanotropin.JAMA. 1991; 266: 2730-2736Crossref PubMed Scopus (193) Google Scholar; Young et al., 1991Young A.R. Potten C.S. Chadwick C.A. Murphy G.M. Hawk J.L.M. Cohen A.J. Photoprotection and 5-MOP photochemoprotection from UVR- induced DNA damage in humans: the role of skin type.J Invest Dermatol. 1991; 97: 942-948Abstract Full Text PDF PubMed Google Scholar; Potten et al., 1993Potten C.S. Chadwick C.A. Cohen A.J. Nikaido O. Matsunaga T. Schipper N.W. Young A.R. DNA damage in UV-irradiated human skin in vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.Int J Radiat Biol. 1993; 63: 313-324Crossref PubMed Scopus (50) Google Scholar; Kinley et al., 1997Kinley J.S. Brunborg G. Moan J. Young A.R. Photoprotection by furocoumarin-induced melanogenesis against DNA photodamage in mouse epidermis in vivo..Photochem Photobiol. 1997; 65: 486-491Crossref Scopus (10) Google Scholar). An appropriate assay system for the simultaneous analysis of UV induced DNA photoproducts and histology is necessary to demonstrate the photoprotective effect of melanin in multilayered epidermis. Thus, we have established a DNA photoproduct detection system using in situ immunofluorescent laser cytometry (Figures 1, 2). We found that supranuclear melanin caps reduced the formation of CPD and 6–4PP in UV irradiated human epidermal cells in a melanin concentration dependent manner (Figures 3, 4). We also found that protection factors against CPD and 6–4PP formation were 2.14 ± 0.77 and 2.77 ± 1.39 (mean ± SD), respectively, whereas melanin concentrations in the skin explants for CPD and 6–4PP determination were 2.00 ± 1.24 and 3.52 ± 1.54 arbitrary units (mean ± SD), respectively. This result suggests that skin explants used for 6–4PP detection had a higher melanin content than explants used for CPD detection, and that melanin protected epidermal cells against CPD and 6–4PP formation with similar efficiency. The protection factor values were similar to those achieved by tanning (1.1–4.2; Gange et al., 1985Gange R.W. Blackett A.D. Matzinger E.A. Sutherland B.M. Kochevar I.E. Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin.J Invest Dermatol. 1985; 85: 362-364Abstract Full Text PDF PubMed Scopus (87) Google Scholar; Young et al., 1991Young A.R. Potten C.S. Chadwick C.A. Murphy G.M. Hawk J.L.M. Cohen A.J. Photoprotection and 5-MOP photochemoprotection from UVR- induced DNA damage in humans: the role of skin type.J Invest Dermatol. 1991; 97: 942-948Abstract Full Text PDF PubMed Google Scholar; Potten et al., 1993Potten C.S. Chadwick C.A. Cohen A.J. Nikaido O. Matsunaga T. Schipper N.W. Young A.R. DNA damage in UV-irradiated human skin in vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.Int J Radiat Biol. 1993; 63: 313-324Crossref PubMed Scopus (50) Google Scholar). Photoprotection by tanning is due to the combined effect of melanization and epidermal thickening. Thus, the photoprotective effect of supranuclear melanin caps seems to be very effective. Recent evidence has suggested that UV induced DNA photoproducts and/or their excision repair stimulates melanin synthesis (Gilchrest et al., 1993Gilchrest B.A. Zhai S. Eller M.S. Yarosh D.B. Yaar M. Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation.J Invest Dermatol. 1993; 101: 666-672Abstract Full Text PDF PubMed Google Scholar; Eller et al., 1994Eller M.S. Yaar M. Gilchrest B.A. DNA damage and melanogenesis.Nature. 1994; 372: 413-414Crossref PubMed Scopus (168) Google Scholar, Eller et al., 1996Eller M.S. Ostrom K. Gilchrest B.A. DNA damage enhances melanogenesis.Proc Natl Acad Sci USA. 1996; 93: 1087-1092Crossref PubMed Scopus (232) Google Scholar). Melanin induced by DNA photoproducts and/or by DNA repair may protect epidermal cells against further UV induced DNA photoproduct formation, providing an efficient photoprotection system by melanin in the skin. In this study, we irradiated skin explants with relatively high doses of UVB (up to 8 kJ per m2). Supranuclear melanin caps were most abundant in basal and suprabasal cells. The formation of CPD and 6–4PP was lower in basal and suprabasal cells compared with epidermal cells in upper layers Figure 1. Moreover, supranuclear caps reduced the formation of both types of photolesions (Figures 3, 4). Therefore, we needed relatively high UV doses to measure low numbers of CPD and 6–4PP induced in basal and suprabasal cells with supranuclear caps. We found that protection factors against CPD and 6–4PP formation correlated with melanin concentration Figure 4; however, the correlation was not very high (the correlation coefficient was 0.59 for CPD and 0.72 for 6–4PP). The most likely explanation for this variation may be that the melanin concentration measured did not correctly reflect the actual amount of intracellular melanin responsible for shielding the nucleus from UV irradiation, because intracellular melanin observed was not always located just over the nucleus in the path of UV irradiation, depending on the cutting angle. It has been reported that more highly melanotic cell lines in tissue culture are not always protected more efficiently by melanin than are less melanotic cell lines against UV induced DNA photoproducts (Chalmers et al., 1976Chalmers A.H. Lavin M. Atisoontornkul S. Mansbridge J. Kidson C. Resistance of human melanoma cells to ultraviolet radiation.Cancer Res. 1976; 36: 1930-1934PubMed Google Scholar; Hill and Setlow, 1980Hill H.Z. Setlow R.B. Postreplication repair in three murine melanomas, a mammary carcinoma, and a normal mouse lung fibroblast line.Cancer Res. 1980; 40: 1867-1872PubMed Google Scholar; Huselton and Hill, 1990Huselton C.A. Hill H.Z. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells.Environ Mol Mutagen. 1990; 16: 37-43Crossref PubMed Scopus (30) Google Scholar; Niggli, 1990Niggli H.J. Comparative studies on the correlation between pyrimidine dimer formation and tyrosinase activity in Cloudman S91 melanoma cells after ultraviolet-irradiation.Photochem Photobiol. 1990; 52: 519-524Crossref PubMed Scopus (24) Google Scholar; Schothorst et al., 1991Schothorst A.A. Evers L.M. Noz K.C. Filon R. van Zeeland A.A. Pyrimidine dimer induction and repair in cultured human skin keratinocytes or melanocytes after irradiation with monochromatic ultraviolet radiation.J Invest Dermatol. 1991; 96: 916-920Crossref PubMed Scopus (32) Google Scholar; Yohn et al., 1992Yohn J.J. Lyons M.B. Norris D.A. Cultured human melanocytes from black and white donors have different sunlight and ultraviolet A radiation sensitivities.J Invest Dermatol. 1992; 99: 454-459Abstract Full Text PDF PubMed Google Scholar; Cieszka et al., 1997Cieszka K. Hill H.Z. Xin P. et al.Survival of Cloudman mouse melanoma cells after irradiation by solar wavelengths of light.Pigment Cell Res. 1997; 10: 193-200Crossref Scopus (7) Google Scholar). This may result from the fact that only a small amount of melanin can be located over the nucleus because of the flattened morphology of cells in culture, and dispersed intracellular melanin cannot protect against DNA photoproduct formation as efficiently as supranuclear melanin caps observed in human epidermis in vivo (Kobayashi et al., 1993Kobayashi N. Muramatsu T. Yamashina Y. Shirai T. Ohnishi T. Mori T. Melanin reduces ultraviolet-induced DNA damage formation and killing rate in cultured human melanoma cells.J Invest Dermatol. 1993; 101: 685-689Abstract Full Text PDF PubMed Google Scholar). Further, melanin in cultured cells that is located in the same plane as the nucleus may actually absorb UV energy and generate melanin free radicals, which potentially serve as photosensitizers (Pathak and Stratton, 1968Pathak M.A. Stratton K. Free radicals in human skin before and after exposure to light.Arch Biochem Biophys. 1968; 123: 468-476Crossref PubMed Scopus (146) Google Scholar). Thus, cultured cells with different types of melanin are good experimental models for examining the effect of melanin on UV induced oxidative DNA damage and mutation (Menon et al., 1983Menon I.A. Persad S. Ranadive N.S. Haberman H.F. Effects of ultraviolet-visible irradiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells.Cancer Res. 1983; 43: 3165-3169PubMed Google Scholar; Huselton and Hill, 1990Huselton C.A. Hill H.Z. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells.Environ Mol Mutagen. 1990; 16: 37-43Crossref PubMed Scopus (30) Google Scholar; Li and Hill, 1997Li W. Hill H.Z. Induced melanin reduces mutations and cell killing in mouse melanoma.Photochem Photobiol. 1997; 65: 480-485Crossref PubMed Scopus (27) Google Scholar), whereas human skin is more suitable for determining protective effects of melanin against DNA photoproducts. In this study, we have determined the photoprotective effect of epidermal melanin against CPD and 6–4PP formation; however, because normal human skin samples used in this study were obtained from only two individuals, similar experiments are in progress using skin samples from different individuals with different phenotypes (skin phototypes, amounts and types of melanin such as eu- and pheomelanin, etc.) to more completely characterize this effect. We would like to thank Dr. B. Carrier for providing Kodacel sheets. We would also like to thank Ms. Y. Nakata for her assistance with the immunofluorescence. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, The Japanese Dermatological Association, and The Cosmetology Research Foundation.