THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

COPYRIGHT© 1973 by The Williams & Wilkins Co.

Vol. 60, No.4 Printed in U.S.A.

SUBSTRATE IDENTITY IN MAMMALIAN MELANOGENESIS*

RUEY J. YU, PH.D., AND EUGENE J. VAN SCOTT, M.D.

ABSTRACT

A total of 45 chemical substances have been tested as substrates for melanogenesis identified by pigment formation in melanocytes of freshly plucked hair roots incubated in 1 mM solutions of the compounds. Melanocytes of human red and blond hair and of guinea-pig red-blond hair can utilize catechol, tyrosine, dopa, and 14 of their derivatives as substrates for pigment formation. Melanocytes of human red and blond hair, but not guinea-pig red-blond hair, form pigment from dopamine and methyl dopa. Human and guinea-pig hair melanocytes form dark brown pigments from dopa, dopa methyl ester, and dopamine; lighter brown pigments from tyrosine, catechol, dopa ethyl ester, dopa n-propyl ester, and dopa n-butyl ester; and golden yellow pigments from dopa isopropyl and isoamyl esters. Identification of the substrates utilized for melanogenesis may provide new clues to possible means of specifically affecting melanocyte function.

Pigments synthesized by mammalian melano­cytes have been categorized as eumelanin, phaeo­melanin, and neuromelanin depending upon their color characteristics, solubility in dilute alkali, or tissue site of melanogenesis [1]. Eumela­nin, a brown or black pigment characteristically found in dark skin or hair, presumably is formed via a metabolic pathway involving tyrosine, dopa, and 5,6-dihydroxyindole [2]. Phaeomelanin, a yellow or red pigment, has been thought to derive from tyrosine, dopa, dopaquinone, and cysteinyl dopa [3]. A copper-dependent enzyme, tyrosinase, has been found to catalyze the first two steps of the metabolic pathways in the biosynthesis of both eumelanin and phaeomelanin [4, 5]. Very little is known about the nature of neuromelanin found, for example, in the brain stem and in the trigeminal and dorsal root ganglia. It has been suggested that neuromelanin is a polymeric pig­me-nt derived from catecholamines [6] and that a particular enzyme different from tyrosinase is required, namely tyrosine hydroxylase, which cata­lyzes the hydroxylation of tyrosine to dopa [7 ]. In vitro studies have shown that pigments formed from dopa, dopamine, and 5, 6-dihydroxyindole are considerably different in physicochemical properties [8].

Tyrosine and dopa have been the substrates commonly used for investigation of melanogenesis in human and other animal tissues. However, very scarce information exists concerning other natural and synthetic substrates. The present studies were done to determine the range of chemical structures utilizable as substrates for melanin formation by melanocytes in human and guinea­pig hair bulbs. Such information might be useful in designing and synthesizing distinctive sub-

Manuscript received September 29, 1972; in revised form December 21, 1972; accepted for publication De­cember 27, 1972.

*From the Department of Dermatology, Temple University School of Medicine, Skin and Cancer Hos­pital, Philadelphia, Pennsylvania 19140.

strates specifically toxic to melanocytes which could be used for therapy of melanocytic disorders including melanomas.

MATERIALS AND METHODS

Pigment formation in hair-bulb melanocytes oflightly pigmented hairs was used as an index of melanogenesis according to a previously established technique [9, 10]. Hair was obtained from: the scalps of three red-haired men, two red-haired women, and three blond men; the backs of five red-blond-haired guinea pigs and two albino guinea pigs. Hairs were manually epilated with a needle­holding forceps, the jaws of which had been coated with solder to insure uniformly tight closure. The root por­tions of the hairs were cut off with a scissors and immediately immersed in 0.1 M phosphate buffer. At least 10 anagen hair roots, identified through a dissect­ing microscope, were transferred into 0.1 mM, 1 mM, and 10 mM solutions of test substances in 0.1 M phosphate buffer, pH 7.0. Incubation in the solutions and in the control buffer was carried out at 37° C for 18 hr.

Heat inactivation of enzyme systems in guinea-pig hair bulbs was carried out by heating hair roots in the buffer at 65° C for 3 hr prior to incubation at 37° C in test substances.

Melanogenesis was determined qualitatively and semiquantitatively by evidence of pigmentation of cells in the lower matrix of anagen bulbs as visualized through a dissecting microscope. To confirm the cellular site of melanogenesis, representative hair roots were examined histologically by the following techniques: routine for­malin fixation, paraffin embedding, and hematox­ylin-eosin staining of 6-J.L sections; glutaraldehyde fixa­tion, embedding in Araldite, and staining 1-J.t sections with Azure II; and frozen sectioning and staining with hematoxylin-eosin .

RESULTS

Neither human nor guinea-pig hair roots showed any evidence of darkening when incubated in the buffer solutions alone. Albino guinea-pig hair roots showed no evidence of pigment forma­tion in any test substances. Human red hair roots incubated in solutions of catechol, tyrosine, dopa-

234

SUBSTRATE IDENTITY IN MAMMALIAN MELANOGENESIS 235

mine. and dopa showed distinct pigmentation in the matrix portion of the bulb. Incubation in 0. 1 mM, 1 mM, and 10 mM concentrations of sub­strate showed that maximal pigmentation was achieved equally with e ither 1 mM or 10 mM solutions. Thereafter, all experiments were done in 1 mM concentrations of the test substrates. The results are tabulated in the Table.

Specific pigmentation was identifiable in the lower mid-portion of the hair bulb where melano­cytes normally are found (Figs. 1, 2J. The pigment histologically wa~ localized within melanocytes and it outlined the contour of these dendritic cells (Fig. 3). Nonspecific coloration of the whole hair. presumably due to a ir oxidation, occurred with some chroru0 genic substances, e.g., epinephrine and resorcinol. Human red and blond hair and guinea-pig red -blond hair utilized catechol, tyro­sine, dopa, and various dopa esters as substrates for melanogenesis, although d ifferences in degree of pigmentation were found as indicated in the Table. Melanocytes of human red and blond hair. but not those of guinea-pig red-blond hair, uti­lized dopamine and methyl dopa as substrates for pigment formation. Heat-inactivated hair roots

TABLE

Effect on pigmentation' in hair bulbs incubated in I mM substrate solutions

Guinea-Human Human pi~

Substrate red blond red-hair hair blond

hair

Catechol ++++ + + ·t + Catechol Diacetate• + L-Dopa + +-! + + + t + + + Dopamine ++-t-+ + - • DL-Dopa Methyl Ester• ++++ ++ + 1-

DL-Dopa Ethyl Ester' + +++ ..-++ +-I DL-Dopa n-Propyl Ester• ++++ +r DL-Oopa iso-Propyl Ester' ++++ + -'-DL-Oopa n-Butyl Ester' + ++ + .. + DL-Dopa sec-Butyl Ester' + ++ + l

DL-Dopa n-Amyl Ester' + + ++ ·t + + + DL-Dopa iso-Amyl Ester ' -t + + t +· + i + DL-Dopa Cyclohexyl Ester ' ++ -t· + + + Methyl Dopa + + -· L-Tyrosine + t -t· l +' L-Tyrosine iso-Amyl Ester' ++ + L-Tyrosine Amide + + L-Tyrosine Methyl Ester !· t

L-Tyrosyl Glycine + +

' Positive effects relatively expressed as: +, weak; + +, moderate; + t +, intensive; ++++.very intensive.

• Synthesized in our Laboratory of Medicinal Chemis­try.

• No melanogenesis even at 10 mM concentration. • A preferred L-tyrosine concentration was a saturated

solution (2.5 mM ). Addition of catalytic amount of dopa (0.05 mM) did not intensify melanogenesis.

A

FIG. I . Guinea-pig hair roots. x 50. A. From albino guinea pi!(. No pigmentation of matrix melanocytes detectable after incubat ion in anv substrate. Note: keratogenous zone in shaft immediately distal to root is dark due to diffract ion of t ransmitted light , not pigment. B. From red-blond guinea pig. incubated in huller alone. ('. From red-hlond guinea pig, incubated in dnpa. Pigment formation confined to melanocytes in hai r bulb. D. From red-blond guinea pig, incubated in dopa isoamyl ester.

did not form any pigments when incubated in mM dopa solution.

The color of melanins formed from different substrates varied from dark brown to golden yellowish. Whereas the pigments formed from dopa, dopamine. and dopa methyl ester were dark brown. those formed from dopa isopropyl and dopa isoamyl esters were golden yellowish. Tyro­sine, catechol, dopa ethyl ester, dopa n-propyl ester. and dopa n-butyl ester yielded light brown pigments.

The following compounds did not provid e any detectable pigment formation by human red and guinea-pig red-blond hairs: caffeic acid, dopa benzyl ester, :3 , 4-dihydroxybenzoic ac id, epineph ­rine. glycyl tyrosine, hydroquinone. p-hydroxy­ben zoic acid, leucyl tyrosine, methyl dopa ethyl ester. norepinephrine. pyrogal lol. resorcinol, DL-o-tyrosine, DL-m-tyrosine, and ty ramine. No pigmentation was found in guinea-pig red -blond hair when incubated in the following compounds: chlorogenic acid, 5-hydroxyindole. p- hydroxy­phenylacetic acid, p-hydroxyphenylpyruvic ac id, DL-p-hydroxyphenyllactic acid. 0-phenylenedia­mine, m-phenylenediamine, p-phenylenediamine. 0 -aminophenol. m-aminophenol, and p-amino­phenol.

DISCUSSION

The foregoing observations indicate that normal human red and blond hair and guinea-pig red­blond hair utilize to various degrees catechol , tyr osine, dopa, and various esters of dopa as substrates for pigment form ation.

THE .J()L'HNAI. OF l:"VEST!C ... TI\"E DEHMATOL()(;y

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Joo,tr :nn··. l ("•·! t· r . i F+ , :d t·{·h· ; ,Ht •i · FI r t · ~ .• ~r ~ !l\ c· i

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' .,., .. .. ' " ~ "" ,•'

• .... ....

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A 8

Ftc;. :1. His tologic spt'riml'n' <ll human n·d hair roots.- Azurt' II. · :!f>O. A. lnnli.>ated in hufft·r 11lmw. B lnn1hatPd in dopa isnam~·t estt>r. PiKmt>nlatiun uonfinNI l(> d~n.-lnt IC m!•lannc~·tt•s .

It is apparent that pigment;; fornwd from 1 hes<· suhstrates were enzymP-mediiHt•d products ~ince the heat- inactivated hair rnots did not form an~· pigments from dopa. The fact that human red or hlond hair can utilize dopamint> or methyl dopa. whereas guinea -pig red · blond hair cannot. sug · gests that pigmentation from tht>se two suhstrates may rt>quirf> particular E'nz~·matir activitiE's, present in hair roots of ;.orne ~ pecie" but n o t in other~.

While human red hair readily utilized both dopa and 1 yrusinl' for melanogenesi:<. human blond and guinea-pig red-blond hairs did not U!"e

tyrosine as readil~· as dopa even in the presenn• of catalytic amount" of thf> latter. It appf>ars thar different enzyme system~ mav he required to initiate melanogene~is from tyrnsint•. such HS

peroxidase- m erliated pigment at ion Ill 1. l'\"en

though onl~· a single enzyme, l~Tosinase, has been proposE'd to caullyze the synthesis of melanin from both dnpa and tyrosinf' 14. f>l - A purified mam­malian tyrosinase from Harding- Passey mouse melanoma has been reported t o oxidizt' epineph­rine, norepinephrine. and tyramine [1:! 1- However. Wt' f(>und none of these suhstratf•s to he utilized as suhstrate for pigment formation in hair roots.

The pigments formed from CE'rtain dopa estt>rs varied distinctivt>ly in rolor shades from the pigment formed from dopa irseiL Therefore. in hair. melanogerw~i >; an f>Sterase !l('ti,·it~· which hydrolyzes the dopa f>StNs to free dopa may rwt he involved in pigment formation.

Deposition of tht> pigments formed from tht> test su hstratf>s wa~ found to he a~sociat.ed with mela­nocytes histological!~·. Thl' precise cytologic site will ha\'1" to be clarified h~· additional studies.

SUBSTRATE IDENTITY IN MAMMALIAN MELANOGENESIS 237

Use of melanocytes of hair roots incubated in test substrates provides a simple technique for screening large numbers of substances for melano­genic properties. The results we have obtained raise many questions concerning substrate iden­tity in melanogenesis in vivo. Additionally, the fact that melanocytes can incorporate a diversity of chemical structures into pigment may provide new clues for designing drugs to exert special effects on these cells.

REFERENCES

1. Fitzpatrick TB, Quevedo WC Jr. Szabo G, Seiji M: Biology of the melanin pigmentary system, Der­matology in General Medicine. Edited by TB Fitzpatrick, KA Arndt, WH Clark Jr, AZ Eisen, EJ Van Scott, JH Vaughan. New York, McGraw­Hill, 1971, 117-146

2. Nicolaus RA, Piattelli M, Fattorusso E: On some natural melanins. Tetrahedron 20:1163-1172, 1964

3. Prota G, Nicolaus RA: On the biogenesis of phaeo­melanins, Advances in Biology of Skin, vol 8. Edited by W Montagna, F Hu. Oxford, Pergamon Press, 1967, pp 323-328

4. Fitzpatrick TB, Becker SW Jr, Lerner AB, Mont­gomery H: Tyrosinase in human skin: demonstra­tion of its presence and of its role in human melanin formation. Science 112:223-225, 1950

5. Brown FC, Ward DN, Griffin AC: Preparation and

properties of mammalian tyrosinase, Pigment Cell Biology. Edited by M Gordon. New York, Aca­demic Press, 1959, pp 525-535

6. Moses HL, Ganote CE, Beaver DL, Schuffman SS: Light and electron microscopic studies of pig­ment in human and rhesus monkey substantia nigra and locus coerulens. Anat Red 115:167-184, 1966

7. Nagatsu T, Levitt M, Udenfriend S: Tyrosine hy­droxylase: the initial step in norepinephrine bio­synthesis. J Bioi Chern 239:2910-2917, 1964

8. Binns F, King JAG, Mishra SN, Percival A, Robson NC, Swan GA, Waggott A: Studies related to the chemistry of melanins. XIII. Studies on the struc­ture of dopamine-melanin. J Chern Soc [Org] 15:2063-2070, 1970

9. Kugelman TP, Van Scott EJ: Tyrosin·ase activity in melanocytes of human albinos. J Invest Derma to! 37:73-76. 1961

10. Van Scott EJ, Reinertson RP, Steinmiiller R: The growing hair root of the human scalp and mor­phologic changes therein following amethopterin therapy. J Invest Derma to! 29:197-204, 1957

11. Okun MR, Edelstein LM, Or N, Hamada G, Donel­lan B, Lever WH: Histochemical differentiation of peroxidase-mediated from tyrosinase-mediated melanin formation in mammalian tissues. His­tochemie 23:295-309, 1970

12. Lerner AB, Fitzpatrick TB, Calkins E, Sommerson WH: Mammalian tyrosinase: action substances structurally related to tyrosine. J Bioi Chern 191:799-806, 1951