Results

IPD and persistent pigmentation after UVA irradiation

The kinetics and dose–response curves of UVA-induced skin darkening and persistent pigmentation are shown in Figure 1. In Japanese subjects, grayish black IPD was observed during and after irradiation for several minutes at UVA doses of 5.6 to 22.0 J/cm² using the Solar UV simulator. Even after the grayish darkening (IPD) had faded, brownish black pigmentation remained for several weeks in skin exposed to high doses of UVA radiation. This persistent pigmentation was observed after a single UVA irradiation of greater than 16.8 J/cm², which corresponds to approximately 1 h of UVA exposure at midday (full sun) in the summer in Yokohama, Japan.

Figure 1.

Kinetics and dose–response curves of IPD and persistent pigmentation after UVA irradiation. The untanned inside of the forearms of seven healthy Japanese men were exposed to UVA radiation at 5.6, 11.2, 16.8, or 22 J/cm² using the Solar UV simulator. The degree of skin pigmentation was measured in terms of M, the change in the melanin index M, which was calculated as the M of a preexposure reading over the M at each measurement time after exposure.

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Histologic examination

First, we histologically observed pigmentary changes after exposure to UV radiation. Areas on the backs of three Japanese subjects with light or intermediate skin type (III or IV) were irradiated with UVA (30 J/cm²) or UVB (2 MED) using the Solar UV simulator, and unexposed areas were used as unirradiated controls. After 3 h of UVB irradiation, the skin on the exposed sites turned slightly red, and those exposed to UVA radiation exhibited brownish black persistent pigmentation without sunburn, after IPD had completely faded. Skin biopsies were obtained 3 h after irradiation. Light microscopic and immunohistochemical examination of UVA irradiated skins revealed that the brownish black pigmentation occurred outside of melanocytes at the basal layer and partially at the suprabasal layer of the epidermis (Figure 2, Figure 3). The pigmentation induced in the skin specimens by UVA did not fade. Furthermore, there was no significant morphologic change in either the melanocytes or the surrounding keratinocytes. In the UVB-irradiated skins, no notable pigmentary change was histologically apparent at 3 h after irradiation Figure 2.

Figure 2.

Light microscopic image of skin sections obtained after back exposure to UVA or UVB irradiation. Cryosections of skin 3 h after irradiation and unirradiated skin from untanned backs of Japanese male volunteers. (a) Unirradiated control; (b) UVA irradiation (a single dose of 30 J/cm²); (c) UVB irradiation (2 MED). Increases in the amount and density of pigment at the basal and suprabasal layer of the epidermis are apparent only in UVA-irradiated skin.

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Figure 3.

Immunofluorescence image of melanocytes located in the basal layer of the epidermis. Cryosection of skin 1 d after UVA irradiation (a single dose of 30 J/cm²). (a) Light microscopic image (arrows, melanocytes). (b) Immunofluorescence image of fluorescence isothiocyanate-labeled antibody against tyrosinase-related protein-1, which is a melanocyte-specific antigen (arrows, melanocytes).

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Effect of direct UVA irradiation on excised specimens

To confirm that this pigmentation was not the result of a cellular response, freshly excised skin specimens were exposed to UVA irradiation. As in the case of in vivo UVA exposure, the pigmentation was observed in the basal layer and partially in the suprabasal layer of the epidermis. Pigmentation occurring outside of melanocytes at the basal and suprabasal layers of the epidermis was also observed histologically on freshly excised skin specimens immediately after UVA irradiation and did not appear to fade Figure 4.

Figure 4.

Cryosection of freshly excised skin specimens obtained from the unirradiated backs of Japanese men. (a) Before UVA irradiation; (b) after UVA irradiation (a single dose of 20 J/cm²).

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Pigment formation from colorless melanogenic precursors by UVA radiation

We examined whether photooxidation of the colorless melanogenic precursors, DHI and DHICA, and the further metabolites, 6H5MICA and 5H6MICA, participates in UVA-induced persistent pigmentation Figure 5b, c. Although no change was observed in color in tyrosine or dopa exposed to UVA radiation, the hydroxyindole carboxylic acid derivatives, DHICA and 6H5MICA, readily produced irreversible brownish black pigments upon UVA irradiation in vitro Figure 5a. Pigment formation from DHICA and 6H5MICA by UVA radiation increased concentration-dependently up to 50 J/cm² Figure 6a and also increased concentration-dependently in the concentration range of 0.0001 to 1 mM Figure 6b. 5H6MICA showed some darkening by UVA radiation, but to a lesser extent than DHICA and 6H5MICA. Although DHI also showed some darkening in the photograph, this was because it was not stable in the solution for more than a few minutes. Moreover, it was temperature-sensitive and quickly blackened with increase of temperature. In contrast, the solution of DHICA was comparatively stable and did not become brownish black during experiments. In addition, the spectral characteristics of the hydroxyindole carboxylic acid derivative darkened by UVA radiation were similar to those of skin with persistent pigmentation after UVA radiation Figure 7.

Figure 5.

(a) Pigment formation from tyrosine, dopa, DHI, DHICA, and its metabolite (6H5MICA) exposed to UVA radiation (a single dose of 5 J/cm²). (b) The putative formation pathway and the chemical structures of these melanogenic precursors. (c) The absorbance spectra of these melanogenic precursors..

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Figure 6.

Energy and concentration dependence of pigment formation from DHICA or 6H5MICA by UVA radiation. (a) Energy dependence of pigment formation from DHICA or 6H5MICA by UVA radiation. (b) Concentration dependence of pigment formation from DHICA or 6H5MICA by UVA radiation.

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Figure 7.

The spectral characteristics of UVA-induced persistent pigmentation and UVA-induced pigment formation from DHICA..

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Effect of hydrogen peroxide on excised skin specimens and on pigment formation from DHICA and 6H5MICA by UVA radiation

Delay in the use of excised skin specimens, or storage, gradually weakened and even abolished the reaction to UVA. So, the dependence of UVA-induced persistent pigmentation on oxidative status was studied. The pigmentation induced by UVA irradiation at the basal layer of epidermis in the excised skin specimens was stimulated by local application of hydrogen peroxide. In the presence of hydrogen peroxide, the skin specimens were darkened by UVA irradiation in a short time, and the extent of pigmentation increased. DHICA and its O-methyl metabolite (6H5MICA) readily changed into dark-colored pigment upon UVA irradiation in the presence of hydrogen peroxide in vitro Figure 8. The pigment formation increased linearly in the presence of hydrogen peroxide from 0.0001% to 0.1%. No similar influence of superoxide anion or singlet oxygen in tissues or in vitro was noted (data not shown).

Figure 8.

Effect of hydrogen peroxide on UVA-induced pigment formation from DHICA and 6H5MICA in vitro. (a) Pigment formation from DHICA exposed to UVA radiation (a single dose of 1 J/cm²) with or without hydrogen peroxide. (b) Pigment formation from 6H5MICA exposed to UVA radiation (a single dose of 1 J/cm²) with or without hydrogen peroxide.

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Determination of colorless melanogenic precursors

We determined the presence of colorless melanogenic precursors in excised skin specimens and supernatants of cultured human melanocytes by making use of their fluorescence properties. The DHICA metabolite 6H5MICA was detected in the excised skin specimens and also abundantly in the supernatant of cultured human melanocytes Figure 9. In melanocyte cultures, the supernatant, in which 6H5MICA accumulated, became brownish black upon direct exposure to UVA radiation, as in the case of UVA irradiation of 6H5MICA in vitro Figure 10a, b.

Figure 9.

Determination of 6H5MICA and 5H6MICA in the excised skin specimens..

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Figure 10.

Components of the supernatant of cultured human melanocytes after UVA irradiation. (a) The concentrations of DHICA and the O-methyl metabolite of DHICA (6H5MICA) in the cells and the supernatant of cultured human melanocytes. (b) Effect of UVA irradiation (a single dose of 5 J/cm²) on the supernatant of cultured human melanocytes.

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Discussion

Human skin is routinely exposed to solar UVA and UVB. UVA causes the skin to darken immediately and causes tanning without sunburn, as is often seen in people taking part in outdoor activities in summer, especially in people with skin types II to IV. These phenomena are thought to result from photooxidation of preexisting melanin occurring in preformed (stage IV) melanosomes, and this is generally believed to be the mechanism of IPD (^{Miescher and Minder, 1939;Pathak and Stratton, 1968;Beitner and Wennersten, 1985}). Our results suggest that melanogenic precursors already exist at the basal and suprabasal layers of the epidermis, are immediately darkened by UVA, and remain so for several weeks. These immediate but persistent pigment changes are not thought to result from photooxidation of preexisting melanin, which is generally believed to be the mechanism of IPD (^{Miescher and Minder, 1939;Pathak and Stratton, 1968;Beitner and Wennersten, 1985}), which is reversible.

Meirowsky discovered in 1902 that pieces of fresh human skin showed irreversible darkening of the epidermal melanin when heated (^{Meirowsky, 1909}). Later investigators showed that this type of heat-induced darkening was oxygen-dependent (^{Findlay and Merwe, 1966}). Researchers, however, neglected Meirowsky's observations for nearly 30 years, because the biologic role of the phenomenon remained unrecognized. We observed that fresh skin specimens excised from living bodies also darken irreversibly on exposure to UVA irradiation, and this irreversible darkening is not thought to be the same reaction as IPD. Irreversible darkening, or persistent pigmentation, after UVA irradiation was first described by^{Henschke and Schulz in 1939}, and^{Kaidbey and Kligman (1978a)} reported that at larger doses (18 J/cm²) IPD gave way, without fading, to persistent pigmentation in Caucasians. The few ultrastructural and morphologic alterations observed histologically in persistent pigmentation did not seem to explain the clinically visible tanning of UVA-induced persistent pigmentation (^{Mutzhas et al, 1981;Ryckmanns et al, 1987}). The distribution of dark-colored pigment in the skin during the 21-d period of the kinetic study of UVA-induced persistent pigmentation was not clarified in this study, because skin biopsy was not carried out. The brownish black pigmentation remained apparent at the end of the 21-d period. Because the turnover time of epidermis was reported to be 45 or 39 d (^{Bergstresser and Taylor, 1977;Weinstein et al, 1984}), the pigment in the epidermis would not have been completely excreted within 21 d. The skin pigmentation was no longer apparent by 2 mo after exposure.

Following exposure of adult male Asians to UVB of 2 MED, pigmentation increased gradually with a peak at 1 wk and remained for several months or more. The melanocytes were activated and melanogenesis was stimulated after irradiation. Nevertheless, UVA-induced persistent pigmentation has distinct effects that are wavelength-dependent: UVA irradiation between 340 and 400 nm increases melanin density localized at the basal layer of epidermis, whereas UVA irradiation between 320 and 340 nm increases the synthesis and transfer of melanized melanosomes to keratinocytes (^{Fitzpatrick, 1986}). The amount of UVA required to cause melanocyte activation and melanogenesis stimulation is more than 100 J/cm² in skin type III and IV (^{Beitner, 1986}). This amount of UVA is five or more times the dose at which UVA-induced persistent pigmentation occurred in this study. It corresponds to more than 8 h of sunlight in midsummer. Therefore, it is unlikely that the UVA-induced persistent pigmentation involves melanogenesis.

It appears possible that photochemical reactions of colorless melanogenic precursors found in unmelanized melanosomes, which are transferred from melanocytes to keratinocytes, or of precursors which are released from melanocytes and taken up by keratinocytes may contribute to UVA-induced persistent pigmentation. The number of melanized melanosomes (stage IV) in the cytoplasmic area in keratinocytes is much higher than in melanocytes (^{Beitner and Wennersten, 1985}). Unmelanized melanosomes, which are stage II and III by ultrastructural estimation, also exist in the keratinocytes of unexposed skin (^{Jimbow and Pathak, 1974}). These unmelanized melanosomes contain various melanogenic precursors in addition to completely polymerized melanin (^{Hatta et al, 1988}). Melanin is produced from the amino acid tyrosine, in a series of reactions catalyzed by tyrosinase through dopa, dopaquinone, and various melanogenic precursors such as DHI and DHICA. DHICA is also O-methylated to produce 6H5MICA and 5H6MICA by catechol-O-methyl transferase present in the cytoplasm of melanocytes (^{Duchon and Matous, 1967;Smit et al, 1990}).

Hydrogen peroxide is a strong oxidant, which can be decomposed into water and oxygen by enzymatic and nonenzymatic routes (^{Neyens and Baeyens, 2003}). It was shown that application of hydrogen peroxide promoted UVA-induced pigment formation from DHICA and 6H5MICA in vitro and UVA-induced brownish black persistent pigmentation in tissue specimens. Photooxidation of melanogenic precursors, such as DHICA and 6H5MICA, may occur, leading to the formation of a quinone moiety, thus inducing a red shift in the absorption spectrum, and resulting in the conversion of dimer, trimer, or polymer to brownish black pigment. This result suggests that the observed brownish black persistent pigmentation is a photochemical reaction involving colorless substances already present in the basal layer of the epidermis, not a cellular response. The chemical structure of the dark-colored pigment produced in the photochemical reaction by UVA irradiation may be a polymer(s) of DHICA or 6H5MICA, different from biologically formed melanin. The UVA-induced tanning appears to differ functionally from pigmentation induced by shorter wavelengths; it is less protective than UVB-induced tanning against subsequent UVB exposure (^{Kaidbey and Kligman, 1978b}).

In summary, we conducted in vivo and in vitro experiments to identify a new mechanism of pigment production outside of melanocytes after UVA irradiation and found that DHICA and its O-methyl metabolite (6H5MICA) rapidly undergo an oxidative reaction on exposure to UVA radiation to produce brownish black pigment. The UVA-induced pigment formation from 6H5MICA, at least, at the basal and suprabasal layers of the epidermis is presumed to be involved in the mechanism of persistent skin pigmentation, without concurrent reddening, that is observed immediately after exposure to sunlight.

Materials and Methods

Subjects

Healthy Japanese men with Fitzpatrick skin type III and IV, who had no history of photosensitivity and were taking no medication, participated in this study. The untanned back and untanned inside of the forearm were used for the study. This study was performed after the ethics committee approval and informed consent and was conducted according to the Declaration of Helsinki Principle.

Light source

The UVA source used in this study was a 300 W xenon solar UV simulator (Multiport 601, Solar Light Company, Philadelphia, PA) or a FL20S BLB fluorescent tube (Toshiba Electric Inc., Tokyo, Japan). The solar UV simulator was filtered with a dichroic mirror, a Schott WG335, 3-mm thick short cutoff filter to minimize the UVB contribution and a UG11, 1-mm thick filter to minimize the visible, and infrared contributions to the spectral output. The FL20S BLB fluorescent tube was filtered with a Schott WG335 glass, and its emission spectrum extended from 320 to 410 nm with a maximum around 365 nm. The UVB source was a FL20S E/DMR fluorescent tube (Toshiba Electric Inc.), the emission spectrum of which extended from 280 to 370 nm with a maximum around 305 nm. The total energy dose was measured with a UV radiometer (UVR-305/365D (II), Topcon, Tokyo, Japan).

Kinetics and dose–response curves of UVA-induced skin darkening and persistent pigmentation

The kinetics and dose–response curves of UVA-induced skin darkening and persistent pigmentation were studied in seven healthy Japanese subjects (24–40 y old). Their inside forearms were irradiated with the solar UV simulator for different times and with different UVA doses (single doses of 5.6–22 J/cm²). The degree of skin pigmentation was measured in terms of the skin melanin index 'M' using a Mexameter MX16 (Courage-Khazaka, Köln, Germany) before and 5 min, 20 min, 3 h, 1 d, 3 d, 7 d, 14 d, and 21 d after irradiation. M, the change in the melanin index M, was calculated as the M of a pre-exposure reading over the M at each measurement time after exposure.

Histologic examination

Portions of untanned back skin were exposed to UVA (30 J/cm²) or UVB (2 MED) radiation. The exposure sites were biopsied 3 h after irradiation under local anesthesia. Skin samples were embedded in OCT compounds and snap-frozen. The microscopic images of cryosections (5 m) were captured using a microscope fitted with a CCD camera (Vanox-T, Olympus Optical, Tokyo, Japan). Fresh and stored (-20°C) excised skin specimens (10 m) were exposed to UVA radiation at a dose of 20 J per cm².

Immunohistochemistry

Cryosections were processed in the following solutions: 2% skim milk powder and 0.1% (vol/vol) Triton X-100 in phosphate-buffered saline (PBSMT) for 20 min at room temperature; rat anti-tyrosinase-related protein-1 (TMH-2) 1:10 overnight at 4°C and fluorescence isothiocyanate-conjugated rat anti-IgG (Biosource international, Camarillo, CA) (1:500) for 1 h at room temperature. The specimens were washed three times with PBSMT between steps and then mounted with PermaFluor aqueous mounting medium (ThermoShandon, Pittsburgh, PA). Images were captured using the microscope with the CCD camera.

Pigment formation from colorless melanogenic precursors by UVA radiation

5,6-Dihydroxyindole-2-carboxylic acid (DHICA) and its O-methyl metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), were synthesized by a published method (^{Benigni and Minnis, 1965;Wakamatsu and Ito, 1988}). 5,6-Dihydroxyindole (DHI) and 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MICA) were provided by Dr Wakamatsu (Fujita-Gakuen Health University, Toyoake, Japan). Tyrosine, dopa, DHI, DHICA, 6H5MICA, and 5H6MICA (1 mM) were dissolved in 100 mM phosphate buffer (pH 7.4), and 200 L aliquots were distributed into a 96 well UV microplate (Corning Incorporated, Corning, NY). These melanin precursors were exposed to UVA (5 J/cm²) using the FL20S BLB fluorescent tube filtered with a Schott WG335 glass, and then the absorbance at 500 nm was measured using a microplate reader (Spectra MAX250, Molecular Devices Corporation, Sunnyvale, CA). The energy and concentration dependence of UVA-induced pigment formation from DHICA and 6H5MICA were examined by increasing the UVA radiation dose up to 50 J/cm² and their concentrations from 0.0001 to 1 mM.

Effect of hydrogen peroxide on excised skin specimens and on dark-colored pigment formation from DHICA by UVA radiation

The stored skin specimens (10 m) were exposed to a UVA radiation dose of 30 J/cm² in the presence or absence of 0.01% hydrogen peroxide. Pigment formation from DHICA (1 mM, 0.05 mM) and 6H5MICA (1 mM, 0.05 mM) by UVA radiation (1 J/cm²) was also examined with or without 0.01% hydrogen peroxide.

The response to a broadband light source with varying wavelengths eliminated

The spectral characteristics of UVA-induced persistent pigmentation and UVA-induced pigment formation from DHICA were estimated using 320, 335, 340, 340, 370, and 390 nm short cutoff filters to minimize the effect of short wavelengths. Irradiation was administered at 12 J/cm². The degree of pigmentation was measured using the Mexameter after 1 d of exposure to UVA radiation through a short cutoff filter to evaluate the persistent pigmentation. Further, the UVA-induced pigment formation from DHICA after exposure to UVA through a short cutoff filter was measured with a colorimeter as described above.

Melanocyte cultures

Human melanocytes obtained from Asian neonatal foreskins were cultured for about 2 wk in Medium 154S (Kurabou, Osaka, Japan) containing 0.2% (vol/vol) bovine pituitary extract, 0.5% (vol/vol) fetal bovine serum, 3 ng/mL recombinant basic fibroblast growth factor, 5 g/mL insulin, 5 g/mL transferrin, 10 ng/mL phorbol 12-myristate 13-acetate, 3 g/mL heparin, and antibiotics. The cultured melanocytes were placed in 75 cm² dishes at a density of 110⁴ cells/cm². The supernatant of 1-d-old culture was collected and divided into two portions for UVA irradiation (a single dose of 5 J/cm²) or nonirradiation as a control.

Determination of melanogenic precursors

Melanin precursors were determined by measuring the concentrations of DHICA, 6H5MICA, and 5H6MICA in culture supernatant as previously described (^{Maeda and Fukuda, 1996}). A biopsy skin specimen (3 mm punch, 40 m section) was sonicated in 10 L of 0.4 M HClO₄, and then the supernatant was measured by HPLC (Nanospace Sl-1, Shiseido Co. Ltd., Tokyo, Japan) on a CapcellPak C18 ODS column (UG120, 1.5 mm diameter250 mm, 5 m; Shiseido Co., Ltd.) using isocratic elution with 5 mM H₃PO₄, pH 2.3, containing 10% (vol/vol) methanol at a flow rate of 100 L/min. Carboxylated indoles were monitored using a fluorescence detector (320/405 nm, Shiseido Co., Ltd.) and identified by comparison with authentic DHICA, 6H5MICA, and 5H6MICA. The supernatant (90 L) of cultured human melanocytes was sonicated with 10 L of 4 M HClO₄, and then the concentrations in the assay mixture (5 L) were determined by means of the above HPLC methods.

References

References

Acknowledgments

We thank Drs K. Wakamatsu and S. Ito (Fujita-Gakuen Health University) for providing the DHI and 5H6MICA and Ms Naomi Oka for technical assistance.