eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes

Pigment Cell Res 1995; 8:202-208 Printed in the United States of America - all rights reserved

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ISSN 0899-5785 PIGMENT CELL RESBnnca

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes GILLIAN HUNT,' SYLVIA KYNE,' SHOSUKE ITO; KAZUMASA WAKARIATSU;

CAROLE TODD,' AND ANTHONY J. THODY' 'Department of DermatoloD, University of Newcastle upon Tyne, Newcastle upon Tyne, UK,

'Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeornelanideumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanideumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanideumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.

Key words: Melanogenesis, Melanins, Skin pigmentation

INTRODUCTION The synthesis of melanin in the epidermal melanocytes

is vital for protection against the damaging effects of ultraviolet radiation. There are two chemically distinct types of melanin: the red-yellow phaeomelanin and the brown- black eumelanin (for review, see Ito, 1993). Both types of melanin are generated from a common precursor, dopaquinone, produced from tyrosine by the action of tyrosinase (EC 1.14.18.1). Dopaquinone undergoes a series of oxidation reactions resulting in the formation of eumelanin or, alternatively, in the presence of sulphydryl com- pounds, acts as a precursor for phaeomelanin.

Both types of melanin have been detected in human hair (Thody et al., 1991), epidermis (Thody et al., 1991; Tobin et al., 1994) and cultured melanocytes (del Marmol et al., 1993a). However, the melanocyte data are complicated by the culture of the cells in medium containing phorbol esters and CAMP enhancers that have been shown to affect eumelanogenesis in murine melanoma cells (Kuwmaki et al., 1993). It is now possible to maintain human melanocytes in vitro without the use of artificial mitogens (Dona- tien et al., 1993; Hunt et al., 1994) and we have recently reported the eumelanin and phaeomelanin contents of hu-

man melanocytes maintained in the absence of artificial mitogens (Hunt et al., 1995).

Although eumelanin and phaeomelanin have been quantified in both human epidermis and melanocytes, the levels of the two melanins have not been examined in epidermis and cultured melanocytes from the same individual. We de- scribe here the eumelanin and phaeomelanin contents of human epidermis and melanocytes from European and non- European individuals.

MATERIALS AND METHODS Epidermis was obtained as suction blisters from the fore-

arms of young adults who had given their informed con-

This work was presented in part at the European Society for Pig- ment Cell Research meeting, Vienna, October 19-22,1994, and at the British Society for Investigative Dermatology meeting, Oxford April 6-7,1995. Abstracts: Melanoma Research, vol. 4, suppl. 2,13-14,1994; Br. J. Dermatol., vol. 132,642,1995.

Received March 22,1995; accepted May 16,1995.

Address reprint requests to Dr. Gillian Hunt, University Department of Dermatology, Royal Victoria Infirmary, Newcastle upon M e . NEl 4LI: UK.

Human Epidermal and Melanocyte Melanins 203

sent. Samples of foreskin and bat ear correction skin from young children and a single sample of breast skin from a young adult were dissected free of subcutaneous fat and cut into 0.5 cm2 pieces. After overnight incubation in 2 mg/ ml dispase in phosphate buffered saline at 4"C, the epidermis was separated from the underlying dermis. Samples of epidermis (> 10 mg wet weight) were freeze-dried, re- weighed and stored at -20°C prior to the analysis of melanin content.

Culture of Human Melanocytes Melanocytes and keratinocytes were isolated by incu-

bating the epidermis obtained as described above in 0.05% trypsin/O.02% EDTA for 5 min at 37°C. The mixed mel- anocytdkeratinocyte population was seeded in MCDB 153 (Sigma) adjusted to 0.3mM tyrosine and 1mM Ca2', supple- mented with various amino acids, growth factors, and 1% bovine hypothalamic extract but lacking cholera toxin and phorbol esters; 1-2% fetal bovine serum was included in the medium for 2-3 days after initial seeding and for 24 hr following subculture to promote cell attachment to the sub- stratum. Melanocytes and keratinocytes were maintained in co-culture for 3-7 days after which the melanocytes were separated from the keratinocytes by differential tryp- sinisation. Pure cultures of melanocytes were maintained in MCDB 153 as before for a further 10-14 days. Aliquots of > 1 x lo6 cells were freeze-dried before melanin levels were quantified.

The eumelanin and phaeomelanin contents of the samples of epidermis and aliquots of melanocytes were quantified as described previously (It0 and Fyjita, 1985; Thody et al., 1991). Eumelanin was oxidized by perman- ganate to pyrrole 2,3-tricarboxylic acid (PTCA), which was then analysed by high-performance liquid chromatography (HPLC) with ultraviolet detection. Phaeo- melanin was hydrolysed with hydriodic acid to aminohydroxyphenylalanine (AHP), which was analysed by HPLC with electrochemical detection. One ng PTCA approximates to 5 ng eumelanin and 1 ng AHP approximates to 5 ng phaeomelanin.

RESULTS Eumelanin and Phaeomelanin in Epidermis

Eumelanin (PTCA) and phaeomelanin (AJ3P) were quantified in 19 samples of epidermis: donor details and results are given in Table 1. Data are single determinations since the skin samples were relatively small and epidermis was also required for the isolation of melanocytes. However, previous data have demonstrated that the PTCA and AHP assays are reproducible, with SEM within 10% of means for four determinations of PTCA and AHP from the same sample of epidermis (Thody et al., 1991). Although there was wide variation in the levels of both melanins, the epidermis from the eight non-European subjects contained significantly more eumelanin and phaeomelanin than the epidermis from the 11 Europeans: P<O.OOl and k O . 0 1 by t-test, respectively. There was excellent correlation between the levels of PTCA and AHP arising from the epidermis: Spearman's rank correlation coefficient (r,) = 0.97,

P<O.Ol for all 19 samples, r, = 0.909, P<O.Ol for the 11 Europeans, and r, = 0.976, P<O.Ol for the eight non-Eu- ropeans (Fig. 1A).

The AHP/PTCA ratios are given in Table 1. While there was less variation in the AHPPTCA ratios than in the levels of AHP and PTCA themselves, the ratios for the non-European subjects were lower than those for the Eu- ropeans: P<0.05 by t-test.

Eumelanin and Phaeomelanin in Human Melanocytes Eumelanin and phaeomelanin were present in all 16 mel-

anocyte cultures although there was wide variation in the levels (lhble 1). Data are single determinations: cultured in the absence of artificial mitogens, limited numbers of melanocytes are obtainable from each skin sample and >2 x lo6 cells are required to assay both PTCA and AHE However, SEM have been shown to be within 20% of means for PTCA and AHP assays on cultured human melanocytes (del Marmol et al., 1993a). In addition, PTCA and AHP arising from a nonmelanocytic cell line were < 1 ng/106 cells and < 10 ng/106 cells respectively (del Marmol et al., 1993a). Similar to the epidermis, the melanocytes from the non-Europeans produced more PTCA and AHP than melanocytes from European subjects: P<O.O1 in each case by t test.

While there was a correlation between the quantities of PTCA and AHP derived from the melanocytes when all 16 cultures were assessed (r, = 0.845, PcO.Ol), there was no correlation between PTCA and AHP when the European and non-European groups were assessed individually (Fig. 1B). This latter observation is in contrast to that for epidermis.

Although the AHPWTCA ratio was relatively high in culture 19, there was no significant difference between the AHPiPTCA ratios in European and non-European cultures. This also contrasts with the data for epidermis.

Comparison of Melanin Profiles in Epidermis and Cultured Melanocytes

Data for both epidermis and melanocytes were available for 14 individuals ( a b l e 1). Because of the differences in the units (ng/mg epidermis vs. ng/106 cells), it was not possible to make direct comparisons between the PTCA or AHP contents of epidermis and melanocytes. However, data were compared by Spearman's rank Correlation test.

PTCA data are shown in Figure 2A. Although there was a correlation between levels in the epidermis and melanocytes when all 14 individuals were assessed (rs = 0.6, P<0.05), the data appeared to segregate according to racial group so that when the European and non-European cultures were assessed separately, there was no correlation between epidermis and melanocyte PTCA levels for either racial group. The segregation according to racial group was less clear for the AHP data (Fig. 2B) and there was a correlation between the AHP levels when all 14 individuals were assessed (rs = 0.7, P<O.Ol) although again there was no correlation when the racial groups were con- sidered separately.

The AHPPTCA ratio in the melanocytes was higher

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Fig. 1. The relationship between PTCA (eumelanin) and AHP ~ i ~ . 2. The relationship between (A) PTCA (B) mp levels in melanocytes from the Same in& (phaeomelanin) contents of A) epidermis (dry weight) and B) mel-

anocytes maintained in vitro for 2-3 weeks. 0 European; .I Eu- ropeadAfrican; A EuropeadMiddle Eastern; + Middle Eastern;

Asian.

(dry weight) and vidud. . ~ Europeadde Eastern;m Asian.

than that in the corresponding epidermis for every subject (Fig. 31, P<O.Ol by paired t-test, although there was no relationship between the two ratios. While there was very little change in the ratio in subject 9 and relatively small increases in subjects 7,8, and 12 (Europeans) and a particularly large increase in Asian subject 19, there was no difference in the percent increase in the AHPPTCA ratio between Europeans and non-Europeans. However, there was a strong inverse relationship between the initial epidermal AHPPTCA ratio and the % increase in this ratio (ra = -0.78, P<O.Ol) (Fig. 4A) and a direct relationship between the subsequent ratio in the melanocytes and the percent increase (rs = 0.89, PeO.01) (Fig. 4B).

Although all samples contained both melanins, the predominant melanin was determined on the basis of the AHPI PTCA ratio as detailed in the footnotes to Table 1. Eumel- anin was the predominant melanin in 16 of the 19 samples of epidermis while neither melanin predominated in the

remaining samples. In contrast, phaeomelanin was the major melanin in l l of the 16 melanocyte cultures with mixed melanins in the remaining three cultures. Only subject nine retained a similar melanin phenotype in the cultured melanocytes as in the epidermis (Table 1).

DISCUSSION We have described the eumelanin and phaeomelanin con-

tents of human epidermis from different races and have provided the most extensive data to date on melanin types in human melanocytes cultured without artificial mitogens. In addition, these are the first data suggesting that the melanin profile of cultured melanocytes differs from that of the epidermis from which they were isolated.

The direct relationship between the eumelanin and phaeomelanin contents of the epidermis confirms previous observations (Thody et al., 1991). A similar relationship between eumelanin and phaeomelanin has been

206 G. Hunt et al.

U

A

epidermis cultured melanocytes

epidermis cultured B melanocytes

Fig. 3. Comparison of AHPIPTCA ratios in epidermis and cultured melanocytes from the same individual. A) 0 Europeans, B) non- Europeans: A CaucasianMddle Eastern; Asian.

described for sheep hair of low melanin content, changing to an inverse relationship at higher melanin contents (Ito, 1993). The present data demonstrate that epidermis from non-European individuals has higher levels of both eumel-

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Fig. 4. The relationship between (A) the initial AHPPTCA ratio in the epidermis or (B) the subsequent AHPPTCA ratio in the cultured melanocytes and the % increase in the ratio for cultured melanocytes relative to that for the epidermis om the same individual. 0 European; A European/Middle Eastern; W Asian.

anin and phaeomelanin but lower AHPPTCA ratios than that from Europeans.

Our preliminary data on the eumelanin and phaeomelanin contents of cultured human melanocytes indicated a direct relationship between the levels of the two melanins in six subjects of differing race (Hunt et al., 1995). How- ever, while the extended data presented here confirm this observation, they demonstrate that, in contrast to epidermis, no such relationship exists when the melanocytes are assessed in their racial groups. In addition, the ratios of the melanins in the cultured melanocytes are unrelated to both the racial origin of the cells and the ratios in the epidermis from which they were isolated.

Tyrosinase activity may be a key factor in regulating melanin type (for review, see Ito, 1993), high activity being

Human Epidermal and Melanocyte Melanins 207

associated with eumelanogenesis, low activity with phaeomelanogenesis and intermediate activity with the formation of mixed melanins (Prota, 1980). Both Black skin and cultured melanocytes contain more tyrosinase activity and melanin than those from Caucasian subjects and melanocytes from individuals with red hair are phenotypically similar to those from other Caucasians (Iozumi et al., 1993; Iwata e t al., 1990; ?\alwar et al., 1993). In general, our melanin data agree with these observations.

The tendency of the isolated melanocytes to become phaeomelanogenic relative to the epidermis, reflected in the increase in the AHPPTCA ratio, and the lack of correlation between the epidermis and melanocyte AHP/ PTCA ratios are, as yet, unexplained. While little. is known of the biological breakdown of melanins (Duchofi 1994), there may be differences in the rates of eumelanogenesis and phaeomelanogenesis within the epidermis and isolated melanocytes and this may account for some of the results described here. In vivo, melanins produced by the melanocytes are transferred to the adjacent keratinocytes, thus assay of melanins in epidermis measures melanins contained within melanocytes and keratinocytes. However, the severity of the conditions under which AHP and PTCA are generated make it unlikely that the differing nature of the samples has influenced the outcome of the assay. Except after initial seeding, the melanocytes are maintained in vitro in the absence of keratinocytes and thus cannot trans- fer melanin. Loss of keratinocyte influence may also have an effect on melanocyte behavior since it has been demonstrated that keratinocytes produce factors that influence melanocyte morphology and stimulate melanogenesis (Gor- don et al., 1989).

In addition to the factors described above and the possible effects of the loss of influence of circulating or epidermis-derived factors, the conditions under which the melanocytes are maintained in vitro may affect melanin type. Phaeomelanogenesis involves the reaction of dopaquinone with cysteine or glutathione and although reduc- ing intracellular glutathione stimulates eumelanogenesis in human melanoma cells (del Marmol e t al., 1993131, the avail- ability of cysteine may be more important in determining phaeomelanogenesis. Most culture media contain relatively high levels of cysteine (MCDB 153, used in this study, contains 240pM cysteine) and this may be the main factor re- sponsible for the high rate of phaeomelanogenesis in cultured human melanocytes. Direct evidence for the effect of cysteine on melanin type is provided by the observations of del Marmol et al. (1993c), who demonstrated that in cysteine-depleted medium, predominantly phaeomelanogenic cells become more eumelanogenic. Although, under the conditions described here, the melanocytes contain predominantly phaeomelanin, eumelanogenesis can be stimulated by the a-melanocyte stimulating hormone analogue N l ~ ~ l F h e ~ t r - M S H (Hunt et al., 1995).

Only one culture maintained an AHPPTCA ratio, and thus a melanin profile, similar to that of the corresponding epidermis and thus these melanocytes may have been rela-

tively insensitive to the factorb) stimulating phaeomelanogenesis.

The shift towards phaeomelanogenesis is greatest in those individuals with a low AHP/PTCA ratio in the epidermis, i.e., containing relatively more eumelanin. As dis- cussed already, melanocytes from these individuals could have relatively high tyrosinase activity that may manifest itself as relatively increased phaeomelanogenesis in the presence of excess cysteine. Indeed, the production of 5- cysteinyldopa, a precursor of phaeomelanin, by human melanoma cells in vitro, has been reported to correlate with their tyrosinase activity (Benethan et al., 1994).

In conclusion, in addition to describing the eumelanin and phaeomelanin contents of both human epidermis and cultured melanocytes, our data suggest that, although epidermis contains predominantly eumelanin, phaeomelanin is the major melanin in melanocytes maintained in vitro. This observation may have important implications for the use of cultured human melanocytes in studies of the control of melanogenesis in man.

ACKNOWLEDGMENTS We acknowledge the assistance of Dr. A.G. Quinn in ob-

taining suction blisters from volunteers and thank the Medical Research Council (UK) for financial support.

REFERENCES Benathan, M., C. Scaletta, and E. Frenk (1994) Demonstration of ty-

rosinasedependent and tyrosinase-independent 5-S-cysteinyldopa genesis in vitro. Melanoma Res., 4 (supp 2):14-15.

del Marmol. V, S. Ito, I. Jackson, J. Vachtenheim, E Bern, G. Ghanem, R. Morandini, K. Wakamatsu, and G. Hem (1993a) TRP-1 expression correlates with eumelanogenesis in human pigment cells in culture. FEBS Lett., 327:307310.

del Marmol, V, E Solano, A. Sels, G. Huez, A. Libert, E Lejeune, and G. Ghanem (199313) Glutathione depletion increases tyrosinase activity in human melanoma cells. J. Invest. Dermatol., 101:871-874.

del Marmol, V, B. Bouchard, R. Mosselmans, A. Libert, S. Ito, G. Ghanem, and F. Solana (1993~) TRP-1, cysteine and eumelanogenesis. Pigment Cell Res., 6:293.

Donatien, ED., J.E. Surlhe-Bazeille, AJ. Thody, and A. W e b (1993) Growth and differentiation of normal human melanocytes in a TPA- free, cholera toxin-free, low-serum medium and influence of keratinocytes. Arch Dermatol Res 285:385-392.

Duchoil, J. (1994) Some unsolved problems in the biochemistry of melanogenesis. Melanoma Res., 4, supp 2:39.

Gordon, ER., C.E Mansur, and B.A. Gilchrest (1989) Regulation of human melanocyte growth, dendricity and melanization by keratinocyte derived factors. J. Invest. Dermatol., 92:565-572.

Hunt, G., C. Todd, J.E. Cresswell, and A.J. Thody (1994) a-Melano- cyte stimulating hormone and its analogue Nle4DPhe7a-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes. J. Cell Sci., 107205-211.

Hunt, G., S. Kyne, K. Wakamatsu, S. Ito, and A.J. Thody (1995) Nle4DPhe7a-melanocyte stimulating hormone increases the eumelanin : phaeomelanin ratio in cultured human melanocytes. J. In- vest. Dermatol., 104:83-85.

Iozumi, K., G.E. Hoganson, R. Pennella, M A Everett, and B.B. Fuller (1993) Role of tyrosinase as the determinant of pigmentation in cultured human melanocytes. J. Invest. Dermatol., 100:806-811.

Ito, S. (1993) High-performance liquid chromatography (HPLC) analy- eie of ou- and phaoomolanin in melanogoneeh control. J. Inveat. Dermal., 100:166S-l71S.

Ito, S. and K. Fhjita (1985) Microanalysis of eumelanin and phaeomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal. Biochem. 144:527-536.

208 G. Hunt et al. Iwata, M., T. Corn, S. Iwata, M.A. Everett, and B.B. Fuller (1990)

The relationship between tyrosinase activity and skin colour in human foreskins. J. Invest. Dermatol., 959-15.

Kuzumaki, 'I!, A. Matsuda, K. Wakamatsu, S. Ito, and K. Ishikawa (1993) Eumelanin synthesis is regulated by co-ordinate expression of tyrosinase and tyrosinase-related proteins. Expl. Cell Res., 20733-40.

Prota, G. (1980) Recent advances in the chemistry of melanogenesis in mammals. J. Invest. Dermatol., 75:122-127.

Talwar, H.S., C.E.M. Griffiths, G.J. Fisher, A. Russman, K. Krach, S.

Benrazavi, and J.J. Voorhees (1993) Differential regulation of tyrosinase activity in skin of white and black individuals in vivo by topi- cal retinoic acid. J. Invest. Dermatol., 100:800-805.

Thody, A.J., E.M. Higgins, K. Wakamatsu, S. Ito, S.A. Burchill, and J.M. Marks (1991) Phaeomelanin as well as eumelanin is present in human epidermis. J. Invest. Dermatol., 9P340-344.

Tobin, D., A.G. Quinn, S. Ito, and A.J. Thody (1994) The presence of tyrosinase and related proteins in human epidermis and their relationship to melanin type. Pigment Cell Res., 7:204-209.

eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes

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