Original Article

Journal of Investigative Dermatology (1998) 110, 806–810; doi:10.1046/j.1523-1747.1998.00178.x

Supranuclear Melanin Caps Reduce Ultraviolet Induced DNA Photoproducts in Human Epidermis

Nobuhiko Kobayashi, Akemi Nakagawa, Tsutomu Muramatsu, Yukio Yamashina, Toshihiko Shirai, Mitsumasa W Hashimoto‡, Yasuhito Ishigaki§, Takeo Ohnishi* and Toshio Mori†

  1. Departments of Dermatology Biology, and Radioisotope Center, Nara Medical University, Kashihara, Nara, Japan
  2. *Biology, and Radioisotope Center, Nara Medical University, Kashihara, Nara, Japan
  3. †Radioisotope Center, Nara Medical University, Kashihara, Nara, Japan
  4. ‡Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan
  5. §Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

Correspondence: Dr Toshio Mori, Radioisotope Center, Nara Medical University, Kashihara, Nara 634, Japan

Received 12 April 1997; Revised 8 January 1998; Accepted 20 January 1998.

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Abstract

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.

Keywords:

cyclobutane pyrimidine dimer, DNA damage, photoprotection, (6–4)photoproduct

Abbreviations:

CPD, cyclobutane pyrimidine dimers; 6–4PP, (6–4) photoproducts

Darker skinned individuals are much less susceptible to sun induced skin cancer than are fair skinned individuals (Kollias et al. 1991). Such a difference can be understood in terms of ultraviolet (UV) transmission of the epidermis (Kaidbey et al. 1979), because the epidermis of darker skin has more and larger melanosomes than that of fair skin (Montagna and Carlisle 1991). 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 1981). 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 1991). 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 1978;Muramatsu et al. 1992). 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. 1993;Barker et al. 1995). Tanning has also been reported to reduce DNA photoproducts induced by subsequent UV irradiation in human skin (Gange et al. 1985;Eggset et al. 1986;Young et al. 1991;Potten et al. 1993), 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. (1984) 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. 1989,1990;Muramatsu et al. 1992;Funayama et al. 1994). 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.

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MATERIALS AND METHODS

Normal human skin

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 1995). 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 times 2 mm. Each piece was placed dermis side down on a Millipore filter (pore size, 0.45 mum; 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. 1992).

UV irradiation
 

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.

Immunofluorescence
 

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. 1991;Muramatsu et al. 1992). For CPD detection, skin explants were sectioned to 3 mum 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 mum 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 mug 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 mug 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).

DNA photoproduct determination
 

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.

Protection factor
 

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.

Melanin concentration in epidermal cells
 

The INSIGHT microscope has an optical microscopic light source and the computer system measures transmitted light intensity (per mum2). The computer system can also permit inversion of data values and can estimate the absorbance of various parts of an image (Tajima et al. 1994). 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.

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RESULTS

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 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Typical fluorescent images of CPD and 6–4PP formation in UV irradiated normal human skin. The formation of CPD (a) and 6–4PP (b) was detected by indirect immunofluorescence using monoclonal antibodies specific for either CPD or 6–4PP in individual epidermal and dermal cells after irradiation of human skin explants with 8 kJ per m2 of UVB. Nuclear DNA was counterstained with PI for CPD detection (c) and for 6–4PP detection (d). The FITC fluorescence of (a) and (b) was combined with the PI fluorescence of (c) and (d), respectively (e, f). Parts (g) and (h) show images with transmitted light that correspond to parts (a), (c), and (e) and (b), (d), and (f), respectively. Scale bar, 40 mum.

Full figure and legend (152K)

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The 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 plusminus SD of four sections.

Full figure and legend (12K)

Photoprotection by supranuclear melanin caps

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 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Epidermal cells with supranuclear melanin caps had less CPD and 6–4PP than cells without supranuclear caps in UV irradiated human skin. The formation of CPD and 6–4PP in individual epidermal cells was detected by indirect immunofluorescence after irradiation of human skin explants with 8 kJ per m2 of UVB. Intensities of nuclear CPD (a) and 6–4PP (b) immunofluorescence by FITC in epidermal cells with supranuclear caps were weaker than in cells without supranuclear caps, whereas intensities of nuclear DNA fluorescence by PI were similar in both types of cells (c, CPD;d, 6–4PP). Each fluorescent image is superimposed on the corresponding image obtained with transmitted light. Parts (e) and (f) show schemata of parts (a) and (c) and (b) and (d), respectively. Scale bar, 20 mum.

Full figure and legend (250K)

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Supranuclear 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.

Full figure and legend (15K)

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DISCUSSION

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. 1991;Young et al. 1991;Potten et al. 1993;Kinley et al. 1997). 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 plusminus 0.77 and 2.77 plusminus 1.39 (mean plusminus SD), respectively, whereas melanin concentrations in the skin explants for CPD and 6–4PP determination were 2.00 plusminus 1.24 and 3.52 plusminus 1.54 arbitrary units (mean plusminus 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. 1985;Young et al. 1991;Potten et al. 1993). 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. 1993;Eller et al. 1994,1996). 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. 1976;Hill and Setlow 1980;Huselton and Hill 1990;Niggli 1990;Schothorst et al. 1991;Yohn et al. 1992;Cieszka et al. 1997). 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. 1993). 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 1968). 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. 1983;Huselton and Hill 1990;Li and Hill 1997), 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.

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Acknowledgments

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.

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