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.
566
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
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
AND
MELANIN
PIGMENTATION
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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:-;..•*•
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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).
MELANOCYTES
AND
MELANIN
PIGMENTATION
571
/[ 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
MELANOCYTES
AND
MELANIN
PIGMENTATION
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
MELANIN
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.
OF THE
SOCIETY
Epidermal melanin unit.
OF COSMETIC
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
AND
MELANIN
PIGMENTATION
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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Table
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).
MELANOCYTES
AND
MELANIN
PIGMENTATION
579
(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).
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in the Biology of Skin, Montagna, W., and Hu, F., eds., Pergamon Press, Oxford, Vol.
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580
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P., ]•tudeau microscope
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