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UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin¹

TAKETSUGU TADOKORO^*, NOBUHIKO KOBAYASHI^*, BARBARA Z. ZMUDZKA, SHOSUKE ITO, KAZUMASA WAKAMATSU, YUJI YAMAGUCHI^*, KATALIN S. KOROSSY, SHARON A. MILLER, JANUSZ Z. BEER and VINCENT J. HEARING^*,2

^* Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;
Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland, USA; and
Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192 Japan

²Correspondence: Laboratory of Cell Biology, Building 37, Room 1B25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: hearingv{at}nih.gov

SPECIFIC AIMS

DNA damage induced by UV radiation is a critical event in skin photocarcinogenesis, but the role of racial/ethnic origin in determining individual UV sensitivity remains unclear. This study examined the relationships between melanin content, its changes, and DNA damage induced in situ in normal human skin of different racial/ethnic groups, phototypes, and UV sensitivities after exposure to a relatively low UV dose of 1 minimal erythema dose (MED).

PRINCIPAL FINDINGS

1. Formation of DNA photoproducts and melanin content in UV irradiated human skin
Constitutive skin pigmentation dramatically affects the incidence of skin cancer, and the photoprotective function of melanin in the skin is highly significant. In the U.S., rates of basal and squamous cell carcinomas are 50 times higher in Caucasians than in African Americans and African Americans have a 13-fold lower incidence of melanoma than do Caucasians. Whether the photoprotection is due solely to the role of melanin as a UV filter or whether other properties of melanins or skin are also involved remains to be resolved. Cyclobutane pyrimidine dimers (CPD) and (6–4) photoproducts (64PP), the two major types of DNA lesions resulting from UV damage, are potentially carcinogenic. However, there are significant gaps in our knowledge about the relationships between DNA damage/repair and the different types, quantities, and distribution of melanin in the skin of individuals with different racial/ethnic origins and different skin phototypes.

Induction and removal of DNA photoproducts in the skin after UV exposure of 1 MED, the smallest dose that causes skin reddening, was quantified for 37 subjects differing in racial origin (the six official U.S. census groupings are American Indian or Alaska Native, Asian, Black or African American, Native Hawaiian or Pacific Islander, White, and Hispanic or Latino), Fitzpatrick phototype (ranging from phototype 1, which burns easily and never tans, to phototype 6, which never burns and tans black), and UV sensitivity (we characterized this by MED ranges from very sensitive a, 0–225 J/m² through b, 226–300, c, 301–400, d, 401–600, e, 601–800 to very resistant f, >800 J/m².

Typical examples of DNA damage for one subject (S26, a White male, phototype 4.5, UV sensitivity c) are shown in Fig. 1 . DNA damage measured as CPD or 64PP was extensive immediately after UV exposure and colocalized with nuclear DNA. Examination of images from unexposed controls and from those taken at various times (7 min, 1 day, and 7 days) after UV exposure indicates that DNA damage was maximal immediately after irradiation and gradually reduced over time.

View larger version (73K): [in this window] [in a new window]   Figure 1. Immunohistochemical detection of DNA photoproducts. Typical fluorescence images of DNA damage detected by binding of antibodies reactive with DNA photoproducts by FITC (green, left column) and nuclei detected by PtdIns (red, middle column) in UV irradiated skin. Representative specimens from one subject (S26, White male, skin phototype 4.5, UV sensitivity group c) are shown. The column to the right shows the merged images; colocalization of DNA over the nuclei is seen as the yellow color. Rows 1 and 3 show skin before UV radiation, rows 2 and 4 show skin immediately after (7 min) UV radiation. Rows 1 and 2 are stained with TDM-2 (to detect CPD), rows 3 and 4 are stained with 64M-2 (to detect 64PP).

2. DNA damage and melanin content before and after UV irradiation
Figure 2 summarizes DNA damage for all subjects exposed to 1 MED UV sorted by racial/ethnic origin, phototype, and MED range. DNA damage was significantly greater in the lighter, more UV-sensitive skin types in each classification and significantly lower in the darker, more UV-resistant skin. In other subjects, we examined whether different skin types exposed to the same physical UV dose (180–200 J/m²) showed comparable kinetics of DNA damage removal. The curves generated had the same shape as those generated after exposure to 1 MED (only the curves sorted by MED range are shown).

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of different skin characteristics and exposures on DNA damage. DNA damage (as CPD) was measured before and after exposure to UV at 1 MED or 200 J/m2 and expressed as the ratio of the intensities of the FITC fluorescence for DNA damage and that of the PtdIns fluorescence for nuclear DNA. The data are grouped by racial/ethnic origin, phototype, and MED range. Results are reported as means of each group and the error bars reflect SE. The bottom right panel shows data for the group subjected to 200 J/m2 of UV.

Skin specimens from a subset of 15 of our subjects were analyzed chemically for melanin content and Fontana-Masson staining (data not shown). There was a relatively good correlation between eumelanin content measured as PTCA and melanin content determined by Fontana-Masson staining (R²=0.6237, P<0.05) but no correlation with pheomelanin content (R²=0.0647, NS).

Figure 3 shows the melanin content in subjects UV irradiated at 1 MED and sorted by racial/ethnic origin. White and Asian skin generally had similarly low constitutive contents of melanin whereas Black or African American skins had levels ranging from two- to fourfold higher. Although amounts of melanin measured in the various skin types was consistent with expectations based on visible phenotype, only the darker, more UV-resistant group had appreciable increases in melanin content after UV irradiation within the 7 day time course of these experiments. Although the melanin content of lighter, more UV-sensitive skins showed little change within 1 wk of a single 1 MED exposure, the skin of these subjects usually became darker (i.e., a visible tan developed).

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic showing the induction and molecular effects of UV in different racial/ethnic groups. Data reported in the boxes are the means ± SE except for MED, which is reported as the median, the bar showing the range of values; n=17 for White, 3 for Asian, and 6 for Black or African American.

The MED was lowest in Whites and highest in Black or African Americans (Fig. 3) . DNA damage immediately after UV exposure was significantly higher in the erythemally more UV-sensitive groups and significantly lower in the less UV-sensitive groups. This difference is even more striking when one considers that the groups with less immediate DNA damage were receiving significantly more UV to achieve their MED than were the less UV-sensitive groups.

Levels of DNA photoproduct removal were calculated as % removal of damage during or over 7 days after the UV exposure vs. maximal damage seen immediately after UV radiation. We found no correlation between melanin content or racial/ethnic origin and the efficiency of DNA damage removal. Some subjects in each group were able to repair virtually 100% of the DNA damage within 1 wk; others were very inefficient at doing that, with levels of repair as low as 40%.

CONCLUSIONS AND SIGNIFICANCE

Here, for the first time, we report the effects of melanin on UV responses in different racial/ethnic groups. We compared levels of DNA damage (and their removal) as well as melanin content in skin of human subjects representing five racial/ethnic origins, different phototypes, and/or UV sensitivities. Key observations, as summarized in Fig. 3 , were 1) DNA damage in all subjects was greatest immediately after UV exposure and was gradually repaired thereafter; 2) rates and efficiencies of removal of DNA lesions differed dramatically between subjects in all groupings; 3) whereas DNA damage was most severe in the light skin, UV-sensitive skin types, even the darkest, incurred significant DNA damage at levels 1 MED; and 4) there was a significant inverse correlation between baseline skin pigmentation and the extent of DNA damage. Thus, even very low UV exposures cause measurable damage to DNA in all types of skin, further underscoring there is no such thing as totally UV-resistant human skin.

It is clear from these results that melanin does afford significant protection against DNA damage in underlying skin cells, a concept previously supported by tissue culture and organ model systems. Melanin can absorb UV efficiently at most wavelengths; an important consideration is whether the protective benefits of melanin derive solely from its function as an optical filter. The absolute amount of melanin in the skin is an important factor, but its distribution plays at least as important a role in determining visible skin color and UV absorption (and photoprotection of underlying cells).

An important factor identified in this study is the highly variable rate of DNA damage removal/repair among subjects even within individual groups. Some subjects were highly efficient at repairing DNA lesions and no DNA damage was evident 1 wk after UV exposure; other subjects were inefficient and repaired < 50% of the initial UV damage within 1 wk. It may turn out that though the initial amount of DNA damage is critical to ensuing photocarcinogenesis, the efficiency of DNA repair may be at least as important in controlling the passage of critical mutations to daughter cells.

Epidemiologic evidence has indicated UV irradiation as a major cause of skin cancer in humans. Racial/ethnic origin and UV sensitivity are important in determining individual UV damage, and nucleotide excision repair efficiency is an important factor in quickly repairing that damage. Delays in accurately repairing UV-induced DNA lesions are thought to be closely correlated with mutations passed onto daughter cells that eventually lead to skin carcinogenesis. It is clear from this study that characterization of skin according to its erythemal response (MED range) is the most accurate way to predict levels of DNA damage after UV radiation; in turn, this response correlates with melanin content in the skin. Future study will further evaluate parameters important to the UV-induced damage and subsequent repair of DNA lesions and the photoprotective role of melanin in various races.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0865fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0865fje

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