J. Soc. CosmeticChemists,19, 565-580 (Aug. 19, 1968)

Melanocytesand Melanin Pigmentation* FUNAN

HU,

M.D.

PresentedDecember6, 1567, New York 6¾ty

Synopsis During their normal development,melanocytesundergochangesin size and shapeas well as in enzymaticactivitiy. Their developmentalphasescan be studiedin vitro. Young, mature, and old melanocytesare defined accordingto their morphology,enzyme activity, and ultrastructure. The enzyme tyrosinasethat catalyzes the hydroxylation of the melaninprecursortyrosineto dihydroxyphenylalanine and to dopa quinoneresidesin a minute cytoplasmicstructure in melanocytesknown as the premelanosome.The biosynthesisof melaninis regulatedby the availability of free tyrosineas a substrate.the presence of factorsthat activate tyrosinase,and the presenceor absenceof inhibitorsof tyrosinase. The colorof skin dependsnot somuch on the numberof mdanocytesin it but on the amount of melaningranulesthat the melanocytes cansynthesize and distributeeitherin the epidermis or in the dermis. Deviation from normalfunctioningresultsin abnormalpigmentation. INTRODUCTION

The colorof the skinis oneof man'smajor concerns. This area naturally is of great interest to the cosmeticchemists.

Skin colorvarieswith the over-allthicknessof the integument,the stateof vascularityandthe amountof the pigmentin the skin. Accordingto EdwardsandDuntleythe fiveprimarypigmentswhichcontribute to the colorof humanskinarecarotene(yellow),oxyhemoglobin (red), reduced hemoglobin (bluish),melanin,andmelanoid(1). Amongthese, * Publication No. 293 from the OregonRegionalPrimateResearch Center,supported by Grant FR 00163 of the National Institutes of Health and Grant CA 08499 froin the National

Cancer Institute.

} OregonRegionalPrimary ResearchCenter,505 N.W. 185th Ave., Beaverton,Ore. 97005.

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melanin and melanoid are the most important since these are the pig-

mentswhich distinguishthe dark-skinnedfrom the light-skinnedindividuals.

It is well establishedthat melanocytes,located in the basal layer of the epidermis,are the only cellscapableof melaninproduction. Therefore, normal or abnormal melanin pigmentation is directly related to anatomical,physiologicaland biochemicalvariations of the melanocyte. Hence,when onespeaksof melaninpigmentation,onehas to speakof the melanocyte. A thorough knowledge of the biological properties of melanocytesis an important prerequisite for the understandingof normal and abnormal pigmentation. Terminology

For convenientdiscussionit is important to defineclearly the various terms used in the text.

The terminology used here follows that proposedby Fitzpatrick, et al. (2), which is a modificationof the one adoptedby the Third Conferenceon the Biology of the Normal and Atypical Pigment Cell in 1931. This new version has the approval of the Sixth International Pigment Cell Conference (3).

Melanocyte.* A cell which synthesizesa specializedmelanin-containing organelle,the melanosome. Melanophore. A type of melanocyte that participates with other chromatophoresin the rapid colorchangesof certain animalsby the intracellular displacement(aggregationand dispersion)of melanosomes.

g/Ielanoblast. An undifferentiated precursor of the melanoeyte (and melanophore). g/[elanosome. t A discrete melanin-containing organelle in which melanization is complete; shownby electronmicroscopyto be more or lessuniformly "electron dense"; tyrosinase activity not usually demonstrable.

?remelanosome. All distinctive particulate stagesin the maturation of melanosomes,with variable electron density; possesses an active tyrosinasesystemafter the onsetof melanin synthesis. * Included here are differentiated cells which synthesize nonmelanized or partly melanized premelanosomes as terminal products. It is suggested that in albinism the •nelanoeytes containing nonmelanized premelanosomesbe called albino rnelanocytes.

• Multiple melanosomesimbedded in supportingmatrices (for example, as in the maerophagesand malpighian cellsof mammals) may be designatedrnelanosorne complexes.

MELANOCYTES

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367

CYTOLOGY OF THE MELANOCYTE

The cytology of the melanocyte varies remarkably. Description of developmentalstagesof melanocyteshas largely been basedon observations of strainsof mousepigmentcell grownin vitro (4). Figures1 and 2 demonstrate the morphological variations of these cells in culture. They vary from small to large; although the majority of the cells are bipolar or spindle in shape they may be round, oval, bipolar, spindle, epithelial-like or polydendritic. The amount of the intracytoplasmic melanin pigment granules that they contain is variable. In actively growing cultures the number of nonpigmentedcells often exceedsthat of pigmented ones. Whether or not they contain pigment these cells are all potentially capable of synthesizingmelanin. There

are also differences

in the fine structure

of these cells.

The

small round or bipolar nonpigmented cells contain only nonmelanized premelanosomes,while the large epithelial or polydendritic pigmented cells contain premelanosomes,melanosomesand melanosomecomplexes (3, (3)(Fig. 3). The activity of the enzyme, tyrosinase, is also not uniform. With the use of autoradiography, the incorporation of Dopa-2-C TMwas not usually observed in those cells that contain large amounts of melanin pigment. The small round, ovoid, or spindle-shapedcellswere not regularly labeled. The small bipolar or dendritic cells, on the other hand, often showedthe uptake of the radioactive substances(7). Similar variationshave beenrecordedin human melanoeytes(8-11). By correlating the morphology, enzyme activity and ultrastructures of these cells, one can divide the pigment cells into young, active, and old. These different forms are included in Table I.

The melanoblasts,propigment cells, or immature pigment cells are small, may be round, ovoid, or somewhattriangular in shape,are usually enzymatically inactive, contain no visible melanin pigment, but have premelanosomes. Table

I

Cytological and Enzymatic Variations in the Melanocytes Propigment Cell Tyrosinase

Premelanosome Melanosome Melanin complex

Active Melanocyte

Old Melanocyte

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....... . .:•.. :,• . :•: ;•.•.• .'•••

FiguresI and •. Pigmentcell strain. 10-daymonolayerculture. May-Oruenwald-Oiemsa. 160X. Note the great variation in size and shape of both pigmented and nonpigmented cells

The youngand physiologicallyactivemelanocytesare usuallylarger than the propigmentcells and their shapemay be stellate, bipolar or dendritic. They are usuallytyrosinaseor dopapositive,and their cytoplasmusuallycontainspremelanosomes andmelanosomes. The old melanocytesusually are the largest of the three; they have

MELANOCYTES

AND

MELANIN

PIGMENTATION

.:.

569

.

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Figure 3. Pigment cells in monolayer culture. In situ fixation glutaraldehydc and osmium. 21760X. Part of a pigmented cell showing melanosomes and prcmelanosomes with varying degree of melanization. p--prcmelanosome, m--melanosome, and N--nucleus

much larger cell bodies and shorter processes,in contrast to the small cell body and long and slender processesof the younger melanocytes. They are enzymatically inactive, contain intracytoplasmic melanin granules and usually do not kave premdanosomes.

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BIOSYNTHESIS

OF COSMETIC

CHEMISTS

OF MELANIN

The Metabolic Unit of Melanin Formation

Seiji et al. (12) hypothesized that the tyrosinaseis synthesizedin ribosomesand transferredvia the endoplasmicreticulum to the Golgi area, where tyrosinase is separated into small units, each o[ which be-

comessurroundedby a membranousenvelope. Within eachenvelope, tyrosinasemoleculesbecomealignedin the orderedpattern, after which melaninsynthesisbeginsand the particleis known as a premelanosome.

As melaningraduallyaccumulates, the premelanosome is eventually transformedinto a uniformly dense and structurelessparticle called melanosome. Seiji and Iwashita (13) found that an incorporationof Dopa-CTMoccurredonly in premelanosomes and concludedthat premelansomes are the specificsite of melaninformation. Sincetyrosinase activity wasfoundin premelanosomes, smoothsurfacemembranes,rough surfacemembranesand ribosomes,the presenceof tyrosinaseactivity is not necessarilycorrelated with melanin formation.

Nakai and Shubik

(14), usingelectronmicroscopic radioautography,observedthat an appreciableamount of Dopa-CTMwas incorporatedwith premelanosomes.

On the other hand, Zelicksonet al. (15), using similar techniques, demonstratedin S-91 mousemelanoma that initial melanin synthesis occurred predominantly in the endoplasmicreticulum and associated RNP particles. The label was located to a large extent at the level

of ribosomesand ER.

It wasthe authors'opinionthat this label prob-

ably representsan insolublemonomerbeforepolymerizationrather than

melaninitself,with movementtoward and5nal polymerizationoccurring within

the melanosome. MELANIN

PIGMENTATION

The capacity of a melanocyte to synthesizemelanin is determined by its geneticmake-up, which limits its range of pigmentarychanges. Within this limit melanin pigmentation of skin varies with:

1. The number of activemelanocytes. Either the changein the rate of tyrosinasesynthesisor in the rate of melanosomeproduction in the melanocytecan affectthe degreeof pigmentation. 2. The amount of melaningranulesin epidermalcells(melanocytes and epithelialcells). 3.

The number of melanin-containing cells in the dermis--Dermal

melanocytesand melanophages(macrophages with phagocytosed melanin).

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/[ TYROSINASE:

Hl

H•,

TYROSINE:

H2

DOPA

Dopoquifiofie

(max 280 re.u)

HO

, z•' HO

0•

5,6 Dlhydraxylfidale (mox 275; 298 mr)

COOH

HO

DOPACH ROME.

COOH

Lmacodopachrome

Red

i

(max. 305• 475 m•a)

Ifidole-5,60ulfiofie Yellow

ITYRO$1NE:-ME:LANIN I Browfi { Eumelanlfi)

(max 300; 450mu)

Yellow(Pheomelofilfi) (Generol Absorption)

Metabolicpathwayof tyrosincto melanin.

Figure {.

Mctabolic pathway of tyrosineto melanin. From Fitzpatrick el •l. {49)

FACTORS THAT INFLUENCE

MELANIN

PIGMENTATION

2Ej•ect on theEnzyme Melanin formation involves the conversion of the colorlessamino acid,

tyrosine,to an insoluble brown polymer (Fig. 4).

This process can

be carried out in mammalian tissue only by the catalytic action of the

enzyme,tyrosinase. In the presenceof tyrosinaseandmolecularoxygen, tyrosine is invariably oxidized to dopa. Dopa formed in the first reaction is oxidized enzymatieally by a reversiblereaction to dopa-quinone (dopa-quinonethus formed may be reducedback to dopa when a reducing agent such as aseorbieacid is present in the reaction system in vitro). Further stages of the reaction can proceed in the absence of enzymes(16). Tyrosinase is a copper protein complex. Melanin formation is inhibited when tyrosinase activity is inhibited. General factors such as temperature, pH, concentrationsof the substrate, and the presenceor absenceof inhibitors which normally affect any enzymatic reaction will naturally affect tyrosinasereaction. Other factors which specifically affecttyrosinaseactivity may be groupedasfollows: CopperBinding Agents: Copper is an essentialpart of tyrosinase moleculefor the enzyme activity; any agent that binds coppermakesthe

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enzymeinactive. Amongtheseagents,substances possessing a reactive sulfhydrylgroupwill inhibit the enzymereaction. Early in the 1940's Rothman and co-workersreportedthe presenceof a dialyzable,watersoluble-sulfhydryl-containing componentin human epidermiswhich inhibitedthe formationof melaninfrom tyrosineand dopa (17). Hal-

prin and Ohkawara(18) have sincedemonstrated that reducedglutathione is the sulfhydryl compoundpresentwithin epidermalextracts that inhibitsmelaninformation. They alsocomparedNegroand Caucasianskinsandfoundthat Negroskincontainslessreducedglutathione and glutathionereduetasethan the Caucasianskin. Adaehi in our Department comparedthe enzymeactivitiesof the mela•_otic and amelanotie mouse melan.omas and found that the amela-

notietumorshaveno demonstrable tyrosinaseactivity but haveapproximately twice the activity of glutathione reduetase as the melanotic tumors (19).

Hu, by incorporatingagentsthat have reactivesulfhydrylgroups, such as ergothionine and Cleland's reagent, in culture media failed to elicit any inhibiting effectson melanogenesis. At high concentrations these substancesinhibit cell growth completely, while lower concentrationshave 1•oeffectwhatsoever(7). ReducingAgents: Since the primary reaction involved in the con-

versionof tyrosineto dopaquinoneis an oxidativeprocess, the presence of strong reducing agents will affect this reaction. Ascorbic acid is one

example of what is consideredto be a depigmentingagent in vitro. When incorporated in the culturemediumin whichthe mousepigment cellsgrow,it showsno evidenceof inhibitingpigmentformation(20). Unknown TyrosinaseInhibitors: Satoh and Mishima (21) and Chian and Wilgram (22) havedemonstrated the presenceof tyrosinase inhibitorsin Forther'shamstermelanomaand $91, B16 and HardingPassey mouse melanomas. These inhibitors were found in both the pigmentedand nonpigmentedtumorswith higheractivity in the latter. Ultraviolet light inaetivates the inhibitor isolatedby Chian and Wilgram. The inhibitorin the Forther'smelanomais not changedby the addition of Cu++ and thereforedoesnot appearto be a sulfhydryl compound.

Effectson theActivity of Melanocytes The effects of hormones, chemicals and radiations have been demon-

stratedon melanoeytes.Snell(23) reportedthat a-MSH (melanocytestimulatinghormone) causedan increasein size and complexityof

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573

dendritic processesof the melanocytesas well as in the amount of free melanin in the guinea pig skin. Similarly, Lerner and McGuire (24) observeddarkening of human skin by a and/•-MSH. Other hormones such as estroõen and progesteronehave also been reported to affect variously both the melanocytes and melanin pigmentation in the animals. Single ultraviolet irradiation has been reported to stimulate melanin pigmentation with or without concomitant increase in melanocytepopulation (25-7). X-irradiation and local applicationof thorium X to the skin also led to increasedactivity of the melanocytesin the form of hypertrophic changesof the melanocytes(28, 29). All the above changesin pigmentation and in melanocyteswere observed only in the intact skin of living animals, but the mechanism of their action is not known. These agents are not effective when tested on isolated mammalian pigment cells grown in vitro. Hu (20) studied the action of ACTH, MSH, tyrosine, copper sulfate, dopa, melatonin, and ascorbicacid on the pigment cellsin culture and found no significant change in either the number or the morphology of these cells. Klaus and Snell (30) who employeda similar in vitro systemfound no changes in either the cell morphology or melanin granule activity when a-MSH, acetylcholine, norepinephrine,and melatonin were incorporated in the media in which guinea pig epidermal melanocyteswere cultured. On the other hand, colchicine, a mitotic poison, is not known to stimulate melanocytes. But Hu (20) has shown that, when these cultured pigment cellswere treated with colcemidfor a prolongedperiod of time (24 to 48 hours), the number of the melanin-containing cells increased. In addition, the cells enlarged to several times their usual size (Fig. 5, 6). This latter effect was evident in both pigmented and nonpigmentedcells. High concentrationsof ACTH, i.e., 0.5 or 5 t•g/ml seemed to produce hypertrophic changes of both the melanotic and amelanotic cells (7) (Fig. 7, 8) similar to those produced by colcemid. a- and/•-MSH at the same concentrationsdid not produce an effect on the pigment cells as significant as that of ACTH. Hydroquinone in certain concentrations, however, had a selective toxic effect on the

pigmentedcells (20). This effect appearsto parallel its action in vivo. Chavin reported similar changes in pigment cells when he injected hydroquinoneinto goldfish(31). In human beings,local applicationsof hydroquinoneproduce depigmentationof the skin (32). Silver and Hu failed to see stimulation of melanogenesiswhen they

irradiated pigment cellsin culture with ultraviolet light and x-ray (33). The difference between in vivo and in vitro effects indicates that,

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Figures 5 and 6. Nine-day monolaycr culture. Phase contrast. 140X. Figure 5. Control. Melanin-coutaining cells mingled with nonpigmented cells. These are predominantly small cells. Figure 6. Colcemid 0.3 vg/ml for 48 hours. Note the size of both pigmented and nonpigmented cells, especially the giant epithelial-like cells, which are many times larger than

the cells in control

cultures

with the exception of hydroquinone, the action of these substancesis not

a simple,direct one. This differenceperhapsmay be explainedin part by the concept of the epidermal melanin unit proposedby Fitzpatrick

MELANOCYTES

Figures 7 and 8. trol. Figure 8.

AND

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PIGMENTATION

575

Twelve-day monolayer culture. Phase contrast. 256 X. Figure 7. ConACTH 5 t•g/xnl. Compare the cell sizesof both pigmented and nonpigmented

cells in control

and treated

cultures

and Breathnach (34). The epidermal melanin unit has been defined as a melanocytewith an associatedpool of malpighian cells (Fig. 9, 10). In this view, factors influencingany componentin the epidermal melanin unit can be expectedto modify the function of the entire system. Perhaps ultraviolet stimulates melanogenesis,not as a direct action on the

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Figure 9.

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Epidermal melanin unit.

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CHEMISTS

Modified aftcr Fitzpatrick et al. (49)

melanocytes,but indirectly, by inducing proliferation of malpighian cellsand thereby providingmore vehiclesfor the transportationand/or storage of melanin.

In discussingpigmentary disturbancesin vitiligo, Hu et al. (35) suggestedthat the defect of vitiligo may lie not only in melanoctyesbut also in the basal and malpighian cells of the epidermis. A block of transfer of the pigment between the melanocytesand the epithelial cells may account partially for the lack of pigment in the diseasedareas. The melanocytesin the hyperpigmentedbordersof the vitiliginous skin are often hypertrophic and engorgedwith melanin granules while the surroundingepithelial cells are completely devoid of pigment. Hypertrophic melanocytesalsowere seenin inflammatory conditions of the skin.

Pinkus et al. (36) observed that inflamed skin exhibited

large melanocyteswith widespreadpigmented processesin the intercellular spaces,while the edematousmalpighiancells,on the other hand, contained little or no pigment. Rappaport (37), who studied this phenomenonin atopic dermatitis, has suggestedthat the malpighian

MELANOCYTES

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577

i::' 'a o• •' .'?• ,•' .•

:

•C

• • '

'•

.::.•,• •---.•..

•.

.

-

•

....

Figure 10. 21-day human skin culture. cytcs (arrows) iu sheets of epithelial cclls. Note the dcndritcs of these •nclanocytcs reach out to the neighboring epithelial cells

cellsbecameunable to acceptpigment. Papa and Kligman (38) showed that the characteristic responseof the epidermal melanocytes in inflammatory skin conditionswas both a hyperplasia and a hypertrophy of the pigment cells. •,lJelaninTransfer BetweentheMelanocytesand Epithelial Cellsof theSkin Masson (39) believed that melanocytes transfer melanin to mal-

pighiancellsby an "inoculation-like process"and proposedthe "cytocrine theory" of melanin transfer, i.e., that an active part is taken mostly

by the melanocytes. Cruickshankand Harcourt (40), who basedtheir theorieson observationsby time-lapse cinemicrographyof cultured cells of human and guinea pig skin, speak of the melanin transfer processas "pigment donation." They believe that this is accomplishedboth by actual phagocytosisof a portion of the dendrite and by direct passage of organellesinto the malpighian cell not unlike the processdescribedby Masson. Early in 1936,the author demonstratedthe closerelationship between the melanocytesand the epithelial cells basing her conclusions on observations of the dynamics of human epidermal cells grown in vitro as recorded by time-lapse cinemicrographictechnic (41). The extreme activity of these two cell types at their contact surfacesis clear evidence of the dual participation in the pigment transfer process. With the use of electron microscopy, Barnicot and Birbeck (42) and

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Melanin

OF COSMETIC II

Transfer and Block in Normal

and Abnormal

Transfer

Normal

CHEMISTS

Skins

Block

skin

Pigmented basal cell epithelioma Pigmented seborrheic keratosis (46) Ephelis

Vitiligo (30) Precancerous keratosis (45) Melanoacanthoma (46) Inflammatory dermatoses (atopic dermatitis, chronic eczematous dermatitis, Lichen planus) (36, 37) Thorium X (29) Eccrine poroma (47) Epithelium of the intraepider-

Lentigo

mal part of sweat duct (47) Outer root sheath epithelium of fetal hair (48)

Drochmans (43, 44) showed that segments of melanocytic dendrites containing the pigmented organellesactually penetrate into the malpighian cells and are there nipped off. Melanin transfer and block between the melanocytesand malpighian cells occur both in normal and abnormal

conditions.

Table II lists some

examples of these conditions. Hypopigmentation results from the destruction of melanocytes, as in inflammatory conditions of the skin, or from the block of melanin transfer to the epithelial cells in spite of the presence of hypertrophic and hyperplastic melanocytes in the neighboringareas. Conversely,hyperpigmentationresultsfrom an increasedactivity of the melanocytesand from increasedpigment transfer from the active melanocytes to the epithelial cells. Since in human

epidermisthe number of the epithelial cells far exceedsthat of the melanocytes,and the degreeof melanin pigmentationis directly correlated to the amount of melanin in the epidermis, any change in the amount of melanin in the epithelial cellswill influenceskin pigmentation more effectively than the changein the melanocytes. (Received December 8, 1967)

REFERENCES

(1) Edwards, E. A., and Duntley, S. W., Pigment and color of living human hair, Am. J. Anat., 65, 1-33 (1939). (2) Fitzpatrick, T. B., Quevedo,W. C., Levene, A. L., McGovern, V. J., Mishima, ¾., and Oettle, A. G., Terminology of vertebrate melanin-containingcalls, Science,152, 88-89 (1966).

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(3) Fitzpatrick, T. B., Quevedo, W. C., Levene, A. L., McGovern, V. J., Mishima, V., and Oettle, A. G., Terminology of vertebrate melanin-containing cells, their precursors, and related cells:

a report of the Nomenclature Committee of the Sixth International

Pigment Cell Conference, in Structure and Conlrol of the Melanocyte. Della Porta, G., and Mfihlbock, O., eds. Springer-Verlag, New Vork, N.Y., 1966, pp. 1-5.

(4) Hu, F., The developmentalcycleof B16 melanornacell in culture, TexasRept. Biol. Med., 23, suppl. 1,308-20 (1965). (5) Hu, F., and Cardell, R. R., Jr., The ultrastructure of pigmented melanoma cells in continuous culture, J. Invest. Dermatol., 42, 67-79 (1964). (6) Hu, F., Swedo, J. L., and Watson, J. H. L., Cytological variations of B16 •nelanoma cells, in Advancesin the Biology of Skin, Montagna, W., and Hu, F., eds., Perga•non Press,Oxford, Vol. 8, The Pigmentary System, pp. 549-79, in press. (7) Hu, F., Unpublished data. (8) Hu, F., Cytological studies of human pigment cells in tissue culture, in Pigment Cell Biology, Gordon, M., ed., Academic Press, Inc., New York, New York, 1953, pp. 147-58. (9) Hu, F., and Lesney, P. F., Comparative studiesof human melanocytesin skinsof varying degreeof pigmentation, Henry FordHosp. Med. Bull., 8, 52-4 (1960). (10) Hu, F., Melanocytes and nevus cells, J. Am. Med. Women'sAssoc.,22,257-60 (1967). (11) Mishima, ¾., Macromolecular changesin pigmentary disorders, A .M.A. Arch. Dermatol., 91,519-57 (1965). (12) Seiji, M., Fitzpatrick, T. B., and Birbeck, M. S.C., The melanosome; a distinctive subcellular particle of mammalian melanocytes and the site of melanogenesis, J. Invest. Dermatol., 36,243-52 (1961). (13) Seiji, M., and Iwashita, S., Intracellular localization of tyrosinase and site of melanin formation in melanocyte, Ibid., 45,305-14, (1965). (14) Nakai, T., and Shubik, P., Electronmicroscopicradioautography: the melanosomeas a site of melanogenesisin neoplasticmelanocytes,Ibid., 4:t, 267-9 (1964). (15) Zelickson, A. S., Hirsch, H. M., and Hartmann, J. F., Melanogenesis: an autoradiographic study at the ultrastructural level, Ibid., 4:t, 327-32 (1964). (16) Seiji, M., Melanogenesis, in Ultrastructure of Normal and Abnormal Skin, Zelickson, A. S., ed., Lea and Febiger, Philadelphia, Pa., 1967, pp. 183-201. (17) Rothman, S., Krysa, H. F., and Smiljanic, A.M., Inhibitory action of human epidermis on melanin formation, Proc. Soc.Exp. Biol. Med., 62,208-9 (1946). (18) Halprin, K. M., and Ohkawara, A., Glutathione and human pigmentation, Arch. Dermatol., 94,355-7 (1966). (19) Adachi, K., Unpublished data. (20) Hu, F., The influence of certain hormones and chemicals in mammalian pigment cells, J. Invest. Dermatol., 46,117-24 (1966). (21) Satoh, G. J. Z., and Mishima, Y., Subcellular enzyme-substrate relationships between amelanotic and melanotic malignant m elanoma, FederationProc., 25,294 (1966). (22) Chian, L. T. Y., and Wilgram, G. F., Tyrosinase inhibition: its role in suntanning and in albinism, Science,155,198-200 (1967). (23) Snell, R. S., Hormonal control of pigmentation in man and other mammals, In Advances in the Biology of Skin, Montagna, W., and Hu, F., eds., Pergamon Press, Oxford, Vol. 8, The Pigmentary System, pp. 447-66, in press. (24) Lerner, A. B., and McGuire, J. S., Effect of alpha and beta melanocyte stimulating hormoneson the skin color of man, Nature, 189,176-9 (1961). (25) Snell, R. S., The effect of ultraviolet irradiation on mclanogenesis, J. Invest. Dermatol., 40,127-32 (1965). (26) Quevedo, W. C., Jr., Szabo, G., Virks, J., and Sinesi, S. J., Melanocyte populations in U. V.-irradiated human skin, Ibid., 45,295--8 (1965). (27) Pathak, M. A., Sinesi, S. J., and Szabo, G., The effect of a single dose of ultraviolet radiation on epidermal melanocytes,Ibid., 45,520-8 (1965).

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(28) Staricco, R. J., and Pinkus, H., Quantitative and qualitative data on the pigment cells of adult human epidermis, Ibid., 28, 33-45 (1957). (29) Staricco, R. J., Qualitative and quantitative data on melanocytes in human epidermis treated with thorium X, Ibid., 29,185-96 (1957). (3o) Klaus, S. N., and Snell, R. S., The response of mammalian epidermal melanocytes in

culture to hormones,Ibid., 48,352-8 (1967). (31) Chavin, W., Effects of hydroquinone and hypophysectomy upon the pigment cells of black goldfish, J. Pharmacol. Exptl. Therap., 142,275-90 (1963). (32) Arndt, K. S., and Fitzpatrick, T. B., Topical hydroquinone as a depigmenting agent. Y. Am. Med. Assoc.,194,965-7 (1965). (33) Silver, S., and Hu, F., The effects of ultraviolet light and x-irradiation on mammalian pigment cellsin vitro, in preparation. (34) Fitzpatrick, T. B., and Breathnach, A. S., Das epidermale Melanin-einheit System, Dermatol. Wochschr.,147,481-9 (1963). (35) Hu, F., Fosnaugh,R. P., and Lemey, P. F., In vilro studieson vitiligo, Y. Invest.Dermalol 33,267-80 (1959). (36) Pinkus, H., Staricco, R. J., Kropp, P. J., and Fan, J., The symbiosisof melanocytesand

human epidermis under normal and abnormal conditions, in Pigment Cell Biology, Gordon, M., ed., AcademicPressInc., New York, N.Y., 1959, pp. 127-38. (37) Rappaport, B. Z., Studies on atopic dermatitis, II. Melanin distribution and dopa oxidasereaction, A.M.A. Arch. Pathol., 61,318-32 (1956). (38) Papa. C. M., and Kligman, A.M., The behaviour of melanocytes in inflammation, Y. Invest. Dermatol., 45,465-74 (1965). on the (39) Masson, P., Pigment cells in man, in The biologyof melanomas,in Conference

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