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Keywords:

  • eumelanin;
  • pheomelanin;
  • melanin;
  • hair;
  • UVA;
  • hydrogen peroxide

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

Eumelanin is photoprotective while pheomelanin is phototoxic to pigmented tissues. Ultraviolet A (UVA)-induced tanning seems to result from the photooxidation of pre-existing melanin and contributes no photoprotection. However, data available for melanin biodegradation remain limited. In this study, we first examined photodegradation of eumelanin and pheomelanin in human black hairs and found that the ratio of Free (formed by peroxidation in situ) to Total (after hydrogen peroxide oxidation) pyrrole-2,3,5-tricarboxylic acid (PTCA) increases with hair aging, indicating fission of the dihydroxyindole moiety. In red hair, the ratio of thiazole-2,4,5-tricarboxylic acid (TTCA) to 4-amino-3-hydroxyphenylalanine (4-AHP) increases with aging, indicating the conversion from benzothiazine to benzothiazole moiety. These photodegradation of melanins were confirmed by UVA (not UVB) irradiation of melanins from mice and human hairs and synthetic eumelanin and pheomelanin. These results show that both eumelanin and pheomelanin degrade by UVA and that Free/Total PTCA and TTCA/4-AHP ratios serve as sensitive indicators of photodegradation.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

In contrast to extensive biosynthetic studies on melanin pigments, biodegradation of eumelanin and pheomelanin remain little known. Immediate pigment darkening induced by UVA can now be attributed to oxidation of dihydroxyindole to the o-indolequinone moiety, which is then cleaved to form Free PTCA (photobleaching) in eumelanin. Pheomelanin behaves differently, with conversion of benzothiazine to benzothiazole moiety accompanied by chain elongation. The UVA-induced oxidative degradation of eumelanin and pheomelanin can be estimated by the Free/Total PTCA and TTCA/4-AHP ratio, respectively. For pheomelanin, 4-AHP/3-AHP and TTCA/PTCA ratios also provide valuable information. It is likely that the UVA irradiation actually reduces photoprotection by melanins.

Introduction

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

The ultraviolet radiation (UVR) that reaches the Earth’s surface consists mainly of ∼95% UVA (320–400 nm) and ∼5% UVB (280–320 nm). Exposure of human skin to the solar UVR promotes melanin production in epidermal melanocytes, which protects skin from DNA damage (Brenner and Hearing, 2008). UVB causes DNA photodamage, which leads to transcriptional activation of melanogenic enzymes, resulting in delayed tanning 2–3 days after UV exposure. In contrast, UVA causes oxidative damage and leads to immediate pigment darkening within minutes. UVB is much more effective in promoting pigmentation in delayed tanning that is photoprotective, whereas UVA-induced tanning seems to result from the photooxidation of pre-existing melanin and its precursors and contributes no photoprotection (Miyamura et al., 2011; Wolber et al., 2008). UVB has long been considered to be carcinogenic. Evidence that has accumulated in recent years suggests that UVA also causes skin cancers including melanoma (Coelho and Hearing, 2010; Wood et al., 2006).

Melanocytes produce two chemically distinct types of melanin: the insoluble, black to brown eumelanin and the alkaline-soluble, yellow to reddish-brown pheomelanin (Simon and Peles, 2010; Simon et al., 2009). It is generally accepted that eumelanin is photoprotective, whereas pheomelanin is phototoxic, to pigmented tissues (Brenner and Hearing, 2008; Simon and Peles, 2010). Melanin pigments are produced within the specific cytosolic organelle melanosomes in the melanocytes, and the melanosomes are then transferred to the surrounding keratinocytes in the epidermis. Both eumelanin and pheomelanin are derived from the common precursor dopaquinone that is produced from tyrosine by the action of tyrosinase (Hearing, 2011; Ito and Wakamatsu, 2008). The availability of cysteine determines the ratio of eumelanin to pheomelanin produced in the melanosomes (Ito and Wakamatsu, 2011). Cysteine reacts rapidly and quantitatively with dopaquinone to produce 5-S-cysteinyldopa (5SCD) and 2-S-cysteinyldopa (2SCD) in a ratio of 5.3:1 (Ito and Prota, 1977). 5SCD and 2SCD are then oxidized by dopaquinone to give benzothiazine intermediates via the o-quinone forms. The benzothiazines gradually polymerize to form pheomelanin pigment. In the late stage of pheomelanin production, the benzothiazine moiety is gradually converted to benzothiazole (Wakamatsu et al., 2009). It was shown that UVR also promotes the conversion to benzothiazole moiety (Greco et al., 2009). When cysteine is depleted in the melanosomes, dopaquinone spontaneously reacts to give, via dopachrome, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The production of DHICA is accelerated by dopachrome tautomerase or certain metal ions. The dihydroxyindoles DHI and DHICA are then further oxidized to produce the eumelanin polymer.

The quantity and quality of eumelanin and pheomelanin in tissue samples can be analyzed as a number of specific degradation products. Alkaline hydrogen peroxide oxidation of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) as a specific degradation product of the DHICA moiety (Figure 1; Ito and Fujita, 1985; Ito et al., 2011). Pheomelanic pigment can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) after reductive hydrolysis with hydroiodic acid (Ito and Fujita, 1985; Wakamatsu et al., 2002). 4-AHP and 3-AHP arise from the 5SCD- and 2SCD-derived benzothiazine moiety in pheomelanin, respectively. Benzothiazole moiety is analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA) after the alkaline hydrogen peroxide oxidation (Ito et al., 2011). The total amount of melanin (TM) can be estimated spectrophotometrically by analyzing absorbance at 500 nm after solubilization of melanin in Soluene-350 plus water (Ozeki et al., 1996).

image

Figure 1.  UVA-induced structural modification of eumelanin and pheomelanin and their markers. DHICA moiety in eumelanin gives PTCA (Total PTCA) upon alkaline hydrogen peroxide oxidation, whereas UVA-indiuced peroxidative degradation of the same moiety liberates PTCA (Free PTCA). Benzothiazine moiety in pheomelanin gives 4-AHP (and 3-AHP; not shown) upon reductive hydrolysis with hydroiodic acid originated from 5SCD and 2SCD. UVA induces the conversion of the benzothiazine to the benzothiazole moiety. The latter units give TTCA upon alkaline hydrogen peroxide oxidation.

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In contrast to the biosynthetic pathway of pigmentation and melanosome biogenesis, which has been extensively described (Borovanský and Riley, 2011; Hearing, 2011), the data available for melanin and melanosome biodegradation remain limited and ambiguous (Borovanský and Elleder, 2003). This unfavorable situation leaves many essential questions unanswered: (i) are eumelanin and pheomelanin in the skin, hair, and eyes photodegraded in structure (or photobleached in appearance), and how; and (ii) what are the consequences of photodegradation of eumelanin and pheomelanin? In the latter connection, it is likely that the photoprotective effect of eumelanin may deteriorate if eumelanin is degraded by UVR, whereas the phototoxic effect of pheomelanin may be increased by UVR.

In this study, we first analyzed changes of melanin markers in hair samples that varied in length from the base to identify the structural markers for photodegradation of eumelanin and pheomelanin. To follow the oxidative degradation of eumelanin, we found that the PTCA level obtained with H2O2 oxidation in K2CO3 (referred to as Total PTCA) decreases with increasing length from the base, whereas the PTCA level obtained with K2CO3 extraction under reducing conditions (Free PTCA) increases (Figure 1). Thus, the Free/Total PTCA ratio can serve as a sensitive marker for oxidative degradation of eumelanin structure. We also found that the TTCA/4-AHP and TTCA/PTCA ratios increase toward the tip of the pheomelanic hair, whereas the 4-AHP/3-AHP ratio decreases. With proper markers in hand to be followed, we irradiated suspensions of mouse and human eumelanic and pheomelanic hairs with UVA to follow changes in the structural markers in eumelanin and pheomelanin. Finally, structural modifications of irradiated synthetic soluble melanins were examined, which confirmed the structural changes observed with hair melanins and the phenomena of immediate pigment darkening and photobleaching of eumelanin.

Results

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

(Photo)degradation of eumelanin and pheomelanin in intact human hair

We first examined whether any (photo)degradation takes place in situ in human hair. The possible photodegradation of eumelanin was examined by analyzing human black hair samples (n = 11) that were cut at a 4-cm length for TM, Total PTCA and Free PTCA levels. As shown in Figure 2(A), TM remained almost constant from the base (0–4 cm) to the tip (16–20 cm). Total PTCA showed a slight decrease of 14% (not significant) (Figure 2B). Free PTCA showed a clear increase toward the middle of the hair (8–12 cm) and then remained almost constant (Figure 2C). As a result, the Free/Total PTCA ratio increased 1.6-fold, from 7.2% at the base to 11.6% at the middle (Figure 2D). Interestingly, among the 11 subjects examined, six showed a constant increase in Free PTCA from the base to the tip, whereas the other five showed either no changes or an increase to the middle followed by decline (Supporting Information Figure S1). Thus, it appears that in some subjects Free PTCA stayed on the hair, whereas in others the compound may have been washed away, possibly by repeated shampooing.

image

Figure 2.  (Photo)degradation of eumelanin in black hair (n = 11). Black hair samples were cut to 4-cm lengths. (A) TM levels, (B) Total PTCA levels, (C) Free PTCA levels, (D) Free/Total PTCA ratios. Data are mean ± SE. * and ** denote P < 0.05 and P < 0.01 from the base, respectively.

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Next, human red hair samples (n = 6) were analyzed for TM, TTCA and PTCA, and 4-AHP and 3-AHP levels. As shown in Figure 3(A), TM showed a slight increase (not significant). 4-AHP showed a dramatic decrease, whereas 3-AHP remained almost constant (Figure 3B). TTCA showed a constant increase, whereas PTCA showed a constant decrease (Figure 3C). As a result, the TTCA/4-AHP ratio increased fivefold, from 0.39 at the base to 2.1 at the tip, whereas the 4-AHP/3-AHP ratio decreased one-third, from 3.3 to 1.2 (Figure 3D). The TTCA/PTCA ratio also increased twofold, from 1.9 to 3.9.

image

Figure 3.  (Photo)degradation of pheomelanin in red hair (n = 6). Red hair samples were cut to 4-cm lengths. (A) TM levels, (B) 4-AHP and 3-AHP levels, (C) TTCA and PTCA levels, (D) TTCA/4-AHP, 4-AHP/3-AHP, and TTCA/PTCA ratios. Data are mean ± SE. * and ** denote P < 0.05 and P < 0.01 from the base, respectively. In (D), P values are <0.01 except for the TTCA/PTCA ratio between 8–12 cm and 12–16 cm (P < 0.05).

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UVA-induced degradation of natural eumelanin and pheomelanin

We next examined whether the (photo)degradation observed with human hair melanins in situ actually proceeds by UVA irradiation of hair melanin in suspension. We did not isolate melanins (or melanosomes) to avoid possible structural alteration. Eumelanin-containing suspensions of homogenized and sonicated black mouse and human hair were irradiated with a 4.0 mW/cm2 dose of UVA for up to 7 days. As shown in Figure 4(A), changes in TM in mouse black hair were minimal, with a small decrease at 7 days. Total PTCA decreased slightly, whereas Free PTCA showed a 34% increase at 7 days (Figure 4B). As a result, the Free/Total PTCA ratio increased from 5.2 to 7.8% after 7 days’ irradiation. TM in human black hair showed a gradual decrease of 22% at 7 days (Figure 4C). Total PTCA decreased by 21% at 7 days, whereas Free PTCA increased 2.6-fold (Figure 4D). As a result, the Free/Total PTCA ratio increased from 4.6 to 15.1%. We also examined TTCA in human black hair, as this pheomelanin marker was produced, albeit at low yields, possibly from protein-bound forms of dopa and cysteinyldopas (Ito et al., 2011). However, no changes in TTCA were observed during 7 days’ irradiation (data not shown).

image

Figure 4.  UVA-induced degradation of hair eumelanin. (A) TM levels and (B) Total and Free PTCA levels in eumelanin from mouse black hair. (C) TM levels and (D) Total and Free PTCA levels in eumelanin from human black hair. The Free/Total PTCA ratios are also shown. Suspensions of hair melanin (10 mg/ml water) were irradiated with 4.0 mW/cm2 UVA for the indicated time (days). Marker analyses were performed in duplicate and averages are shown. The irradiation experiment was repeated once with similar results.

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Pheomelanic yellow mouse hair and red human hair suspensions were next irradiated with the same 4.0 mW/cm2 dose of UVA. TM in mouse yellow hair did not change much during 7 days’ irradiation (Figure 5A). 4-AHP showed a rather sharp decrease, whereas 3-AHP showed a slower decrease (Figure 5B). TTCA increased 2.2-fold at 7 days and PTCA decreased 2.4-fold. As a result, the TTCA/4-AHP ratio increased from 0.24 to 2.2 in 7 days, whereas the 4-AHP/3-AHP ratio decreased from 3.6 to 2.9 (Figure 5C). The TTCA/PTCA ratio increased from 1.0 to 5.4. TM in human red hair showed a clear >50% decrease at 7 days (Figure 5D). 4-AHP showed a dramatic decrease (>10-fold) , whereas 3-AHP showed a much slower decrease (3.6-fold) at 7 days. TTCA showed only a slight increase, whereas PTCA showed a clear 2.3-fold decrease (Figure 5E). As a result, the TTCA/4-AHP ratio increased from 0.70 to 10.0 in 7 days, whereas the 4-AHP/3-AHP ratio decreased from 2.3 to 0.70 (Figure 5F). The TTCA/PTCA ratio also increased from 2.0 to 5.6. It is noteworthy that after the 7 days’ irradiation, 4-AHP decreased to a level below 3-AHP, indicating a nearly complete degradation of the 5SCD-derived benzothiazine moiety in pheomelanin structure.

image

Figure 5.  UVA-induced degradation of hair pheomelanin. (A) TM levels, (B) marker levels, and (C) marker ratios in pheomelanin from mouse yellow hair. (D) TM levels, (E) marker levels, and (F) marker ratios in pheomelanin from human red hair (hairs from six females combined; the samples were taken from the base). Suspensions of hair melanin (10 mg/ml water) were irradiated with 4.0 mW/cm2 UVA for the indicated time (days). Marker analyses were performed in duplicate and averages are shown. The irradiation experiment was repeated once with similar results.

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Structural modifications in UVA-induced degradation of eumelanin and pheomelanin

Photodegradation of eumelanin and pheomelanin was confirmed with melanins from eumelanic and pheomelanic hairs. The next question addressed was how structural features of eumelanin and pheomelanin were modified during the UVA-induced degradation. For this purpose, we used synthetic soluble melanins – DHICA-melanin as a model of eumelanin and Dopa + Cys-melanin as a model of pheomelanin. We also examined the effect of melanin concentration on the course of photodegradation. The irradiation times were reduced to one-third those used for irradiating hair melanins.

When a 0.2 mg/ml concentration of synthetic eumelanin was irradiated with a 4.0 mW/cm2 dose of UVA, Total PTCA decreased constantly from 72 to 52 μg/mg in 56 h, whereas the Free PTCA level increased from 3.3 to 21 μg/mg (Figure 6A). As a result, the percent ratio of Free/Total PTCA increased dramatically from 4.6 to 41% after 56 h of irradiation. A similar type of degradation also occurred when a 0.02 mg/ml solution was irradiated, but the degradation proceeded ca. twofold faster than with the 0.2 mg/ml solution (Figure 6B). Control experiments with unirradiated solution showed some (thermal)degradation in 56 h. But the rates of degradation were approximately one-tenth those with UVA irradiation.

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Figure 6.  UVA-induced degradation of synthetic eumelanin and pheomelanin. (A) Total and Free PTCA levels in DHICA-melanin (0.2 mg/ml) and (B) Total and Free PTCA levels in DHICA-melanin (0.02 mg/ml). The Free/Total PTCA ratios are also shown. (C) Marker levels and (D) marker ratios in Dopa + Cys-melanin (0.2 mg/ml), and (E) marker levels and (F) marker ratios in Dopa + Cys-melanin (0.02 mg/ml). Solutions of melanin were irradiated with 4.0 mW/cm2 UVA for the indicated time (h) except for the control, which was kept close to the irradiated samples for 56 h but was sealed with aluminum foil. Marker analyses were performed in duplicate and averages are shown. The irradiation experiments were repeated once with similar results.

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When a 0.2 mg/ml concentration of Dopa + Cys-melanin was irradiated with the same dose of UVA, 4-AHP decreased constantly from 169 to 49 μg/mg at 56 h, whereas the TTCA level increased from 17 to 33 μg/mg (Figure 6C). 3-AHP also decreased at a slightly slower rate than 4-AHP. As a result, the TTCA/4-AHP ratio increased from 0.10 to 0.66 after 56 h irradiation, whereas the 4-AHP/3-AHP ratio showed a slight decrease from 6.0 to 4.7 (Figure 6D). The TTCA/PTCA ratio increased threefold, from 3.0 to 10.9. Interestingly, the changes of ratios in the synthetic pheomelanin (Figure 6D) showed a similar pattern to those observed with mouse hair pheomelanin (Figure 5C). The photodegradation in a 0.02 mg/ml solution proceeded about twofold faster (Figure 6E,F). Control experiments with unirradiated solution showed some (thermal)degradation in 56 h. But the rates of degradation were approximately one-tenth those with UVA irradiation.

The structural modifications observed with the synthetic soluble melanins upon UVA irradiation were similar to what we observed with hair melanins. We then examined changes in UV-visible absorption spectra, fluorescence spectra, and molecular size by taking advantage of soluble properties. Changes in UV-visible absorption spectra of DHICA-melanin (0.02 mg/ml) showed a biphasic change (Figure 7A): (i) DHICA chromophore absorbing at 323 nm disappeared at 8 h of UVA irradiation concomitant with an increase in visible absorption between 380 and 600 nm (pigment darkening) and (ii) this change was followed by a progressive decrease (68% decrease at 600 nm after 56 versus 0 h irradiation) in absorption of the entire visible region at 24 and 56 h (photobleaching). DHICA-melanin at 0.2 mg/ml showed a similar pattern of changes at a much slower rate (Supporting Information Figure S2A). Changes in the spectra of Dopa + Cys-melanin showed a peculiar phenomenon depending on concentration. Dopa + Cys-melanin at 0.02 mg/ml did not show much change until a 24% decrease of absorbance at 600 nm after 56 h (Figure 7B). However, at 0.2 mg/ml, Dopa + Cys-melanin showed the opposite pattern of changes, with a small increase between 270 and 600 nm (Figure S2B).

image

Figure 7.  UVA-induced changes in UV-visible absorption spectra of synthetic melanins. (A) DHICA-melanin (0.02 mg/ml) and (B) Dopa + Cys-melanin (0.02 mg/ml). Solutions of melanin were irradiated as described for Figure 6. Spectra were taken with a JASCO V-520 UV-VIS spectrophotometer (JASCO Co., Tokyo, Japan).

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Changes in fluorescence spectra were also examined. The fluorescence intensity of DHICA-melanin (0.2 mg/ml) increased 16-fold against 0 h control (Figures 8A, Supporting Information S3A), whereas Dopa + Cys-melanin showed a 2.1-fold increase (Figures 8B, S3B). The change of DHICA-melanin also occurred thermally at a much slower rate. It is interesting that the fluorescence intensity of DHICA-melanin did not increase during the first 8 h of irradiation, although pigment darkening occurred during this period (Figure 8A).

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Figure 8.  UVA-induced changes in fluorescence spectra of synthetic melanins. (A) DHICA-melanin (0.2 mg/ml) and (B) Dopa + Cys-melanin (0.2 mg/ml). Solutions of melanin were irradiated as described for Figure 6. Spectra were taken with a Hitachi F-7000 spectrofluorometer (Hitachi High-Tech, Tokyo, Japan) after 15-fold dilution with the pH 6.8 buffer. DHICA-melanin and Dopa + Cys-melanin solutions were excited at 320 and 280 nm, respectively. Fluorescence intensities were calculated from emission spectra between 380 and 800 nm by weighing spectra (see Figure S3) cut with scissors.

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Size exclusion HPLC analysis was performed on synthetic melanins (Table 1 and Supporting Information Figure S4A, B). DHICA-melanin showed a slight, but constant, decrease in molecular size by UVA irradiation, as evidenced by a progressive increase in retention time from 8 h to 24 and to 56 h of irradiation. Conversely, Dopa + Cys-melanin showed a clear increase in molecular size. By using DHICA oligomers as molecular weight markers (Figure S4C), the average molecular weights of synthetic melanins were calculated to be 1410 and 1210 Da for DHICA-melanin (0.2 mg/ml) and 1100 and 1950 Da (the 7.125 min peak) for Dopa + Cys-melanin (0.2 mg/ml), respectively, for 0 h and 56 h of UVA irradiation, provided that there was no adsorption of melanin molecules on the gel matrix.

Table 1.   Size exclusion HPLC analysis of synthetic melanins irradiated with UVAa
SampleRetention time (min)
  1. aRetention times of DHICA and its oligomers were 14.392, 10.775, 9.692, 9.200 and 8.242 min for monomer (193 Da), dimers (384 Da), trimers (575 Da), tetramers (766 Da) and hexamers (1148 Da), respectively. For the preparation of mixtures of DHICA oligomers, see the Methods. Note that the retention time of DHICA monomer is longer than expected for the molecular weight due to adsorption to the column matrix. Based on a calibration curve using data for DHICA oligomers, retention times of 7, 8, and 9 min correspond to molecular weight of 2040, 1290, and 800 Da, respectively.

  2. bRetention times of pheomelanin monomers and a related dimeric trichochrome were 16.042, 16.050 and 11.183 min for BZ (238 Da), ODHBT (268 Da) and decarboxytrichochrome C (516 Da), respectively. For the preparation of BZ and ODHBT, see the Methods. Note that these molecules were retarded due to adsorption.

  3. cTwo values with + represents two peaks (see Figure S4B).

DHICA-melanina0.2 mg/ml0.02 mg/ml
0 h7.7927.933
8 h UVA7.7257.867
24 h UVA7.7927.983
56 h UVA7.9428.142
56 h No UVA7.7507.892
Dopa + Cys-melaninb0.2 mg/ml0.02 mg/ml
0 h8.3838.417
8 h UVA8.2258.292
24 h UVA8.0678.167
56 h UVA7.125 + 7.908c8.175
56 h No UVA8.2008.358

Finally, we examined whether UVB also elicits similar pattern of changes to UVA. DHICA-melanin and Dopa + Cys-melanin at 0.2 mg/ml showed similar patterns of changes at much slower rates as compared to those with UVA (Supporting Information Figure S5). The dose of UVB applied in this study was about 20% that of UVA. When we consider the 5% content of UVB in solar UV radiation, photodegradation of eumelanin and pheomelanin observed in this study can be attributed mostly to UVA in solar radiation, although UVB might cause a stronger degradation than UVA at the same dose. Chemical analysis of irradiated melanin solutions confirmed that UVB-induced changes in the melanin structure were indeed much slower (ca. 1/3) than with UVA (Supporting Information Figure S6).

Discussion

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

The role of melanins (eumelanin and pheomelanin) against UV-induced oxidative stress appears to be rather complex. Miyamura et al. (2011) has shown that UVA tanning confers no photoprotection, and all types of UV-induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis. Wood et al. (2006) have shown, using a genetically melanoma-susceptible Xiphophorus fish model, that the action spectrum of melanin photosensitized oxidant production is identical to that for melanoma induction in the 300–440 nm wavelength range. Swalwell et al. (2012) have recently presented evidence for dual roles of melanin, where melanin protects against mitochondrial superoxide generation and mtDNA damage but acts as a direct photosensitizer of mtDNA damage during UVA irradiation of human melanoma cells. Fernández et al. (2012) have examined effects of UVA-visible light on photodamage of human red hair, showing a small increase in melanin free radical at a low dose but a large decrease at a high dose due to photobleaching. Despite these studies of the photochemical behaviors of melanins, how eumelanin and pheomelanin are degraded by UVA radiation has been little known until the present study.

UVA-induced degradation of eumelanin

Total PTCA liberated by alkaline hydrogen peroxide oxidation serves as a marker to quantify DHICA-derived units in eumelanin (Ito et al., 2011). Ward et al. (2008) showed that various melanosomes from Sepia, human black hair, and bovine choroid and iris contain water-soluble PTCA (Free PTCA) at levels one-tenth those of Total PTCA. Free PTCA may be produced by oxidative degradation of DHICA-derived eumelanin in melanosomes in situ (Figure 1). In our hands, we could not detect PTCA by a simple extraction of black hair homogenates with water. However, the alkaline condition of K2CO3 used for the alkaline H2O2 oxidation (but omitting H2O2 and adding reducing agent Na2SO3) was found to be sufficient to extract the pre-existing PTCA (Free PTCA). In this study, we analyzed human black hair samples that vary in length from the base for Total and Free PTCA levels and found a significant increase in the Free/Total PTCA ratio depending on the length from the base. UVA (4.0 mW/cm2) irradiation of black hair eumelanin from mice and humans induced time-dependent increases in the Free/Total PTCA ratio. Thus, the Free/Total PTCA ratio can serve as a marker to estimate photo-induced oxidative degradation (of DHICA-derived units) in eumelanin. A problem with the Free/Total PTCA ratio is that its UVA-induced change is rather small compared with the robust change in the pheomelanin marker ratio TTCA/4-AHP. In this regard, the TTCA/PTCA ratio could serve as an alternative marker for UVA-induced degradation of eumelanin (and pheomelanin) as most natural melanins are a copolymer of both melanins (Ito and Wakamatsu, 2003). Another merit of the TTCA/PTCA ratio is that it can be obtained in a single assay of oxidation products.

Then what would be the implications of UVA-induced degradation of eumelanin? The liberation of PTCA in situ (Free PTCA) suggests degradation of eumelanin polymer to smaller fragments (decrease in molecular weight). The decrease in molecular weight was confirmed using size exclusion HPLC of DHICA-melanin exposed to UVA (Table 1 and Figure S4). The UVA-induced degradation of DHICA-melanin also led to a 16-fold increase in fluorescence intensity (Figure 8A). In this connection, it should be added that photo-induced melanin degradation gave rise to an intense fluorescence in the epidermis (Elleder and Borovanský, 2001) as a result of hydrogen peroxide production (Felix et al., 1978; Sarna and Sealy, 1984).

The mechanism of immediate pigment darkening induced by UVA is not fully understood. However, it certainly involves peroxidative process of preexisting eumelanin and possibly of its precursors (Maeda and Hatao, 2004). From the results of the present study, it can be inferred that UVA irradiation of eumelanin leads to photooxidation (pigment darkening) of dihydroxyindole to o-indolequinone moiety in the existing eumelanin in the early phase of irradiation (i.e. at 8 h irradiation in Figure 7A) but this darkening is followed gradually by photobleaching (i.e. 56 h irradiation in Figure 7A) of the pigment due to fission of the o-indolequinone moiety accompanied by fluorescence production. The photooxidation seen in the present study is consistent with the spectrophotometric study by Ou-Yang et al. (2004) showing that synthetic soluble eumelanin increased absorbance around 450 nm with a prominent decrease around 330 nm after 70 J/cm2 UVA irradiation, which is a similar dose as our 8 h of irradiation. The fission of the o-indolequinone moiety results in the production of not only Free PTCA but also (photo)degraded eumelanin (Figure 1). Interestingly, the latter structure resembles that of benzophenone, a well known photosensitizer/UV absorber, both being biarylketones. Also, the fission of o-indolequinone moiety during photobleaching of eumelanin is consistent with the study by Slawinska and Slawinski (1982) demonstrating that the common exergonic step of the excitation of melanin is the oxidative opening of the six-membered ring of indolequinone. Another example of the fission of the o-indolequinone moiety is provided by Napolitano et al. (1996) in oxidative degradation of eumelanin with hydrogen peroxide. It should be emphasized that the photobleaching would attenuate photoprotective effect of eumelanin.

Differential photoreactivity of DHI and DHICA moieties also needs to be considered. Among the DHI and DHICA units, the latter appears to be more potent in protecting mouse epidermis against UVA-induced oxidative stress and in scavenging hydroxyl radicals (Jiang et al., 2010). Thus, it would be possible that DHICA moiety is the labile site in the eumelanin polymer, giving rise to the production of Free PTCA.

UVA-induced degradation of pheomelanin

4-AHP and 3-AHP are produced by reductive hydrolysis with HI of 5SCD- and 2SCD-derived benzothiazine moiety of pheomelanin, respectively (Figure 1; Wakamatsu et al., 2002). On the other hand, TTCA is produced from benzothiazole moiety of pheomelanin upon alkaline hydrogen peroxide oxidation (Wakamatsu et al., 2009; Ito et al., 2011). In this study, we analyzed human red hair samples that vary in length from the base and found a constant and sharp decrease in 4-AHP depending on the length. 3-AHP showed a smaller decrease, whereas TTCA level increased constantly. UVA irradiation of pheomelanins from mouse yellow hair and human red hair showed similar trends of change. Thus, the TTCA/4-AHP and the 4-AHP/3-AHP ratios can serve as markers for photo-induced oxidative degradation of pheomelanin. The increase in the TTCA/4-AHP ratio reflects a degradative conversion of benzothiazine to benzothiazole moiety, whereas the decrease in the 4-AHP/3-AHP ratio may be indicative of the extent of photodegradation of pheomelanin. The increase in the TTCA/PTCA ratio may reflect a degradation of both pheomelanin (increase in TTCA) and eumelanin (decrease in PTCA). Which of the TTCA/4-AHP and 4-AHP/3-AHP ratios would be more useful in evaluating photodegradation of pheomelanin is difficult to conclude. It should be mentioned, however, that the TTCA/4-AHP increases more robustly than the 4-AHP/3-AHP ratio while the latter has a merit that it is more precise because this ratio can be obtained in a single analysis of HI hydrolysate.

Using another set of pheomelanin markers, 6-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA) and isomeric 7-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA-2), Greco et al. (2009) found the preferential degradation of 5SCD-derived benzothiazine units in pheomelanin as evidenced by a decrease in the BTCA/BTCA-2 ratio upon UV irradiation. They also noted changes in the 4-AHP/3-AHP and the TTCA/4-AHP ratios that were similar to our results. The present results have confirmed those previous findings in more details and have shown that those changes are UVA-dependent.

What would be the implications of UVA-induced degradation of pheomelanin? In human red hair samples, pheomelanin in the tip (16–20 cm) appears to consist mostly of benzothiazole moiety (Figure 3). Likewise, pheomelanin from mouse yellow hair and human red hair may be converted to benzothiazole structure after 7 days’ irradiation (Figure 5). The UV-induced conversion of a benzothiazine structure to benzothiazole was proved at a monomer level by Costanti et al. (1994). This conversion appears to be accompanied by chain elongation in pheomelanin structure, as evidenced by size exclusion HPLC (Table 1 and Figure S4A, B). We propose that the benzothiazole conversion and the chain elongation proceed simultaneously. These two processes account not only for the UVA-induced decrease in 4-AHP (and BTCA) and the increase in TTCA but also for the increase in molecular size. The position 5 (next to the hydroxy group) over the position 7 is likely the position at which the chain elongation takes place because of the less steric hindrance (Figure 1). The decrease in BTCA during (photo)degradation of pheomelanin observed in our previous study (Greco et al., 2009) can be explained by the chain elongation at the benzothiazole moiety (Figure 1) because such a cross-linked benzothiazole cannot give rise to BTCA. However, it would be interesting to confirm the increase in molecular weight of irradiated pheomelanin by using matrix-assisted laser desorption/ionization mass spectral analysis.

Chedekel et al. (1978, 1980) showed that UV irradiation of pheomelanin leads to an oxygen-dependent production of superoxide radical anion and hydrogen peroxide with the photoionization threshold of pheomelanin being around 325 nm. In more recent years, renewed interests in the phototoxicity of pheomelanin led to the confirmation and expansion of this earlier finding (Brenner and Hearing, 2008; Simon and Peles, 2010). This threshold ionization potential was confirmed using modern physical techniques by Ye et al. (2006). Wenczl et al. (1998) demonstrated that DNA single strand breaks are greater in human cultured melanocytes containing more pheomelanin when irradiated by UVA. An in vivo study by Takeuchi et al. (2004) using dorsal skin of congenic black, yellow and albino mice showed that UV-irradiated melanin, particularly pheomelanin, photosensitizes adjacent cells to apoptosis.

In the eye, ocular melanin exerts photoprotective effects (Hu et al., 2008; Borovanský and Riley, 2011). We showed that ocular melanin in light-colored eyes contains similar or even greater levels of pheomelanin compared with dark-colored eyes (Wakamatsu et al., 2008). Melanin in the iris is subjected to the life-long exposure to UVR. Thus, the phototoxic effect of pheomelanin appears to contribute to the higher incidence of ocular melanoma and age-related macular degeneration (Hu et al., 2008). Phototoxicity of retinal melanosomes from aged animals including humans has been extensively studied, showing that aged retinal melanosomes can be phototoxic to human retinal pigment epithelium (Różanowski et al., 2008). However, in the adult eye, only visible light (>390 nm) reaches the retina, and thus a deleterious effect of UVA does not need to be considered for retinal melanin.

Conclusions

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

The present study has shown that UVA irradiation induces oxidative fission of o-indolequinone moiety in eumelanin polymer and conversion of benzothiazine to benzothiazole moiety in pheomelanin polymer accompanied by chain elongation. These structural changes lead to photobleaching of melanins, especially eumelanin. The UVA-induced photodegradation is concentration-dependent, and thus it is possible that in the epidermis of Caucasian skin, melanins (both eumelanin and pheomelanin) could be more photolabile, leading to little photoprotection but rather to photocarcinogenic events. This possibility was suggested by Haywood et al. (2006, 2008) showing that synthetic and hair melanins at low concentrations undergo photo-oxidation (producing superoxide radical) upon UVA irradiation. We wish to predict that since the UVA irradiation eventually reduces the photoprotection by melanins, this would make UVA-tanned skin even more susceptible to subsequent DNA damage by UVR (Miyamura et al., 2011). Finally, the UVA-induced degradation of eumelanin and pheomelanin should be confirmed on cultured human melanocytes and human skin in future studies using biomarkers developed in this study.

Methods

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

Melanins and hair samples

Synthetic eumelanin (DHICA-melanin) was prepared from DHICA (10 mM) and synthetic pheomelanin (Dopa + Cys-melanin) was from an equimolar mixture of dopa (10 mM) and cysteine (10 mM) under vigorous mixing (Wakamatsu et al., 2009). 7-Alanyl-5-hydroxy-3-oxo-3,4-dihydro-2H-1,4-benzothiazine (ODHBT), 6-alanyl-4-hydroxybenzothiazole (BZ), and decarboxytrichochrome C were prepared as described previously (Wakamatsu et al., 2009; Ye et al., 2003).

A mixture of DHICA dimers, trimers, and tetramers was prepared by oxidation of 1 mmol/l DHICA in 0.05 mol/l sodium phosphate buffer, pH 6.8, with 100 U/ml tyrosinase at 25°C for 30 min, the oxidation being stopped with 1 mM phenylthiourea plus 1 mM ascorbic acid (Ozeki et al., 1997). Identification of dimers and trimers were confirmed on the isolated compounds prepared according to the reported method (Pezzella et al., 1996). A mixture of tetramers and hexamers was prepared according to Pezzella et al. (2003).

Mouse black (a/a) and recessive yellow (e/e) hair samples were obtained from dorsal area by Dr. Tomohisa Hirobe. Human black hair samples (n = 11) were obtained from Japanese females and red hair samples (n = 6) were from German females. Hair samples were cut 1 cm from the base. Those subjects gave informed consents.

UV irradiation

For UVA or UVB irradiation we used an Oriel 300W Solar UV light simulator (Oriel Instruments, now the Newport Corporation, Stratford, CT, USA) and the doses used were measured using a Photo-Radiometer [Delta Ohm srl, HD2302.0; Casella di Selvazzano (Pd), Italy] prior to each exposure. For UVA (315–400 nm), UVB wavelengths were removed using a combination of cold filter, longpass filter/325 nm, and red rejection UV filter/340 nm (Asahi Spectra Co., Tokyo, Japan). For UVB (280–315 nm), UVA was removed using a combination of cold filter and bandpass filter/310 nm (Asahi Spectra Co.). Wavelengths above 400 nm were minimized using a dichromic mirror.

Hair samples were homogenized with a Ten-Broeck glass homogenizer at a concentration of 10 mg/ml and then ultrasonicated. Water suspensions of hair sample were irradiated with UVA by the Oriel Instrument. Solutions of DHICA-melanin or Dopa + Cys-melanin (0.2 and 0.02 mg/ml) were also irradiated. The dose of UVA was 3.97 mW/cm2 (UVB 0.04 mW/cm2), which is similar to the irradiance in Greece during midday in June (Haywood et al., 2011). The dose of UVB was 0.79 mW/cm2 (UVA 0.06 mW/cm2).

Chemical analyses

Alkaline H2O2 oxidation to measure PTCA (Total PTCA) and TTCA was performed as described in Ito et al. (2011). In brief, 100 μl of water suspensions of samples (0.1 mg synthetic melanin or 1.0 mg hair) were placed in 10-ml screw-capped conical test tubes, to which 375 μl 1 M K2CO3 and 25 μl 30% H2O2 were added. The tubes were mixed vigorously at 25 ± 1°C for 20 h. The residual H2O2 was decomposed by adding 50 μl 10% Na2SO3 and the mixture was then acidified with 140 μl 6 M HCl. Each reaction mixture was centrifuged at 4000 g for 1 min, and an aliquot (80 μl) of each supernatant was injected directly into the HPLC system (Ito et al., 2011). Free PTCA was extracted under essentially the same conditions as for the above-mentioned H2O2 oxidation except that H2O2 was omitted. Water suspensions of 100-μl aliquots of samples were placed in 10-ml screw-capped conical test tubes, to which 25 μl H2O, 50 μl 10% Na2SO3, and 375 μl 1 M K2CO3 were added. The tubes were mixed vigorously at 25 ± 1°C for 20 h. The mixture was then acidified with 140 μl 6 M HCl.

HI reductive hydrolysis to measure pheomelanin (as 4-AHP and 3-AHP) was performed as described in Wakamatsu et al. (2002).

Soluene-350 solubilization to measure total melanin (TM) was performed as described in Ozeki et al. (1996) with a minor modification (Ito et al., 2011). Data were corrected against the absorbance of 0.020/mg hair for the background (Ozeki et al., 1996).

Size exclusion HPLC analyses

Solutions of DHICA-melanin and Dopa + Cys-melanin were analyzed on a column (8 × 300 mm, 6 μm particle size) of Shodex OHpak SB-802.5 HQ (Showa Denko, Kawasaki, Kanagawa, Japan). The stationary phase is made of polyhydroxymethacrylate gel, and molecules are separated based on their molecular size (gel filtration or size exclusion). The mobile phase was 0.05 M sodium phosphate buffer, pH 6.8: methanol, 60:40 (v/v). HPLC was run at 35°C at a rate of 0.7 ml/min. The exclusion limit, according to a Shodex brochure, is 10 000 Da using polysaccharide molecular weight markers. The void volume of this column using human serum albumin (66 kDa) was 4.99 ml (retention time 6.975 min).

Statistical analyses

Students’t-test and multivariate statistics were employed with jmp 7.01 software (SAS Institute Inc., Cary, NC, USA).

Acknowledgements

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

This work was supported, in part, by a Japan Society for the Promotion of Science (JSPS) grant (No. 23591659) given to S.I. and K.W. We wish to thank Dr. Tomohisa Hirobe of the National Institute of Radiological Sciences (Chiba, Japan) for kindly providing us with hair from black and recessive yellow mice and Dr. Vincent J. Hearing of the Laboratory of Cell Biology, National Cancer Institute (Bethesda, MD, USA) for his helpful discussion.

References

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Conclusions
  8. Methods
  9. Acknowledgements
  10. References
  11. Supporting Information

Figure S1. (Photo)degradation of eumelanin in black hair from 11 individual subjects.

Figure S2. UVA-induced changes in UV-visible absorption spectra of (A) DHICA-melanin (0.2 mg/ml( and (B) Dopa + Cys-melanin (0.2 mg/ml).

Figure S3. UVA-induced changes in fluorescent spectra of (A) DHICA-melanin (0.2 mg/ml) and (B) Dopa+Cys-melanin (0.2 mg/ml).

Figure S4. Size exclusion HPLC analysis of synthetic melanins and DHICA oligomers.

Figure S5. UVB-induced changes in UV-visible absorption spectra of (A) DHICA-melanin (0.2 mg/ml) and (B) Dopa + Cys-melanin (0.2 mg/ml).

Figure S6. UVB-induced degradation of synthetic eumelanin and pheomelanin.

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