PIGMENTATION OF HAIR

Hair Melanins and Hair Color: Ultrastructural

and Biochemical Aspects

Jean-Paul Ortonne and Giuseppe Prota

Department of Dermatology, University of Nice, Sophia Antipolis, France; Istituto di Chimica Organica, University of Naples, Italy

The color variants of mammalian hair, including spotting molecular biology of melanin pigmentation that some pro-
and albinism, are the result of melanocyte activity and have gress is being made in understanding the nature and origin of
been shown to be determined by the action of multiple genes, hair color. It is the purpose of this article to provide an inte-
some of which o erate through the milieu in which the grated overview of the major advances so made and to draw
pigment cell resi tfes; others appear to act intracellularly to attention to certain peculiarities of the melanization proc-
control the type of melanogenesis. Although there has been esses of hair with respect to those underlying skin pigmenta-
much descriptive work on the mode of action of these genes, tion. Key words: melanins, melanocytes, melanogenesis,
it has only been with the recent advances in the chemistry and hair. J Invest Dermatol 101:82S - 89S, 1993

H
air pigmentation in mammals is due to melanins years towards understanding the biochemical and ultrastructural
[ l-31. These igments are produced by specialized bases of hair color as well as its regulation.
dendritic ccl Ps, melanocytes, derived from the
neural crest. Although some of the factors control- THE FOLLICULAR MELANIN UNIT
ling proliferation and differentiation of melano- Distribution of Melanocytes In active hair follicles, melano-
blasts and melanocytes such as basic fibroblast growth factors, mela- cytes characteristically occur in the wall of the pilary canal (infun-
nocyte-stimulating hormone, the c-kit gene, and the ligand for the dibulum) and in the pigmented part of the bulb, close to the upper
c-kit receptor (mast cell-stem cell growth factor [3,4]) have been part of the dermal papilla. Usually, no active melanocytes are ob-
identified, most of them are unknown. Melanin synthesis occurs in served in other locations. However, dopa-positive melanocytes
organelles termed melanosomes. Fully differentiated melanocytes have been observed in the outer root sheaths of hair follicles after
can be found mainly in the hair bulbs of the skin, where they secrete irradiation with X-rays [8], after dermabrasion [8], after exposure to
melanosomes into the surrounding keratinocytes, giving rise to me- ultraviolet rays, and after oral photochemotherapy [o]. Amelanotic
lanized hairs [3]. The pigmentation of hair follicles follows se- melanocytes (dopa-negative) have been observed along the outer
quences of events identical to those seen in epidermis. The color of root sheath of the middle and lower part of the follicle, between the
hair is based on the pigment content of the hair shaft. By analogy to basal portion of the tall epithelial cells that form the outer periph-
the e idermal melanin unit, the pigmentation of hair is regulated by eral layer [8]. Concerning the distribution of these active and inac-
a fol Picular melanin unit. A few distinctive features differentiate tive melanocytes, Staricco [8] divided the hair follicle into four
these follicular melanocytes and their epidermal counterpart. Thus, parts. Portions A and D, melanotic portions, constitute, respec-
the melanocyte population of the skin can be considered as a bicom- tively, the upper part of the follicle (infundibulum) and the upper
partmental system, composed of an epidermal and a follicular com- part of the bulb in contact with the upper papilla. Portion B com-
partment. The melanocytes contain a multifunctional enzyme, ty- prises the middle and lower follicle and possesses amelanotic mela-
rosinase, which catalyzes the initial events of melanogenesis. Other nocytes. Portion C is the generally amelanotic outer root sheath of
regulatory proteins, known as tyrosinase-related protein (T-RP) I the bulb and the hair matrix below Montagna’s critical level.
and II, have also recently been identified [S]. Mammalian follicular
melanocytes produce two types of melanins: the brownish black Source of Bulb Melanocytes for Successive Hair Generation
eumelanin and the reddish yellow pheomelanin that, despite the The source of bulb melanocytes for successive hair generation has
evident differences in molecular size and general properties, are not been fully clarified. During the catagen stage of mouse hair
biogenetically related and arise from a common metabolic pathway follicles, melanocytes are present in the epithelial column [lo].
in which dopa uinone is a key intermediate [6,7]. From these start- They may be the survivors of the preceding bulb population of
ing points, wit x two categories of melanin pigments, how can one melanocytes and persist in an undifferentiated state with few imma-
interpret the biologic complexity illustrated by the wide range of ture melanosomes in their scanty cyto lasm. Pigment cells are
colors of mammalian hair, including human hair, ranging through found in the hair germ in the telogen an dpanagen I stages and in the
shades of yellow, orange, and red as well as black, grey, and white? bulbar region in the anagen II stage [lo]. It may be suggested that
The present review summarizes the progress achieved in recent these melanocytes arise from the transfer of the undifferentiated
melanocytes present in the epithelial column in catagen into the
future resting hair germ. During the catagen and telogen stages, the
Reprint requests to: Dr. Jean-Paul Ortonne, Service de Dermatologie,
melanocytes are scarce, but their number increases in the early ana-
HBpital Pasteur BP 69, 30, Avenue Voie-Romaine, 06002 Nice, Cedex 1,
France. gen stage [lo]. These observations suggest that the bulb population
Abbreviations: AHP, amino-hydroxyphenylalanine; &SH, alpha mela- of melanocytes in mice has a perpetuating system, that is, a melano-
nocyte-stimulating hormone; PTCA, pyrrole-2,3,%tricarboxylic acid; cyte reservoir that contributes to normal hair pigmentation despite
TRP 1, tyrosinase-related protein I; TRP 2, tyrosinase-related protein II. the shedding and replacing of hairs throughout life [lo]. The repop-

0022-202X/93/SO6.00 Copyright 0 1993 by The Society for Investigative Dermatology, Inc.

82s
VOL. 101, NO. 1, SUPPLEMENT, JULY 1993 HAIR MELANINS AND HAIR COLOR 83s

ulation of the new bulb during the early anagen phase involves a the upper part of the outer root sheet resembles that in epidermis. In
proliferation and a dedifferentiation of the surviving melanocytes. the lower art of the sheath the majority of cells contain only a small
Radiation studies on murine follicular melanocytes also suggest the amount 0 P melanin pigment.
existence of this melanocyte reservoir, and it has been estimated that It is likely that these distinctive features between the follicular
there are about four clonogenic melanocytes per hair follicle [ 111. and epidermal melanin units are largely due to environmental in-
Very little is known about the factors controlling the melanocyte fluences. The chemical signals arising from the dermal apilla and
repopulation of hair follicles at each hair cycle. Recent investiga- the follicular keratinocytes are probably quite different Prom those
tions suggest that the c-kit gene encoding a transmembrane tyrosine coming from the epidermal keratinocytes or from the upper dermis.
kinase receptor for the mast cell-stem cell growth factor plays a
The Melanocyte Population of the Skin as a Bicompartmen-
critical role in this process. Injection of an anti-c-kit monoclonal
tal System Several clinical situations suggest that the epidermal
antibody, an antagonist of c-kit function, in the normal or shaved
and follicular compartment of the melanocyte po ulation of the
post-natal mouse skin results in the growth of unpigmental hair [4].
skin are relatively independent. Senile white hair o Pten occurs on a
These results indicate that c-kit is required for melanocyte activa-
scalp epidermis with a normal melanin pigmentation. On the other
tion that occurs concomitantly with the hair cycle and continues
hand, body hairs often keep their normal color in a fully depig-
throughout life after neonatal development of the first hair [4].
mented lesion of vitiligo. It is clear, however, that exchanges may
However, how the activation of hair cycle can trigger c-kit-
occur between these two compartments, which are not closed sys-
dependent proliferation of hair melanocytes remains to be eluci-
tems. This has been demonstrated under certain circumstances in
dated.
which one of the two melanocyte corn artments is altered or de-
stroyed. After dermabrasion (removal o P the epidermis and the in-
Distinctive Features Between the Follicular and Epidermal
fundibulum follicle), amelanotic melanocytes divide in the middle
Melanin Units Hair bulb melanocytes differ from those in the
portion of the hair follicle, become active (dopa-positive), and mi-
epidermis only in some respects. They synthesize larger melano-
grate upward from the outer root sheath to the infundibulum and
somes than the epidermal melanocytes [12]. Follicular melanocytes
later into the basal layer of the healing surrounding epidermis [8]. A
are active only during a specific phase of hair production, namely
similar process occurs during epidermal wound healing after pure
anagen stages III through VI. During the hair growth induced by
epidermal destruction by suction (Ortonne et al, personal observa-
plucking in mice, tyrosinase visualized on polyacrylamide gels by
tion). Evidence for such exchanges has also been obtained from the
Dopa reaction is not demonstrated in skin extracts during telogen or
study of repigmentation of vitiligo skin during oral photochemoth-
during l-3 d after plucking (anagen stages I to III) [I3]. By
erapy [9]. Experiments performed in the white skin of trichrome
radioimmunoprecipitation using specific antityrosinase antibodies,
guinea pigs suggest that melanocytes injected intradermally are in-
it has been demonstrated that tyrosinase synthesis occurs during the
corporated into the hair bulbs and migrate upwards into the outer
early anagen phase of the hair cycle, with reduced levels of the
root sheath.* It is not yet clear, however, if there are other melano-
protein, until late anagen. No synthesis of tyrosinase is detected
cyte precursors distinct from the amelanotic melanocytes present in
during the catagen and telogen stages. During the development of
the outer root sheath of hair follicles. It has been suggested that a
murine anagen hair follicle, the two melanogenic proteins, tyrosin-
reservoir of melanocytes exists in hair follicles of C57 black mice,
ase and Gp75, are regulated in a time-restricted frame [14,15]. The
but its existence has not yet been demonstrated [lo].
signal-transduction mechanisms implied in the control of melano-
Less evidence exists to suggest migration of melanocytes from the
genie activities during the hair cycle are largely unknown. Alpha
epidermis into the hair follicles. In most cases, after destruction of
melanocyte-stimulating hormone @MSH) failed to increase tyro-
hair melanocytes by various physical agents (X-ray, freezing, etc.),
sinase synthesis in human hair follicle. On the other hand, a cyclic
regenerated hair follicles remain depigmented, giving rise to white
AMP analogue stimulated melanogenesis by increasing tyrosinase
hair [I&19]. Few experiments suggest that such exchanges exist. In
synthesis in human dark hair follicles [16]. How melanogenesis is
the guinea pig, an autograft of full-thickness black skin left for 7 d in
linked to the hair cycle is a mystery. When epithelial mesenchymal
white skin, later removed, is followed by the appearance of pigmen-
interactions during mammalian hair follicle development are better
tation due to active melanocytes in the healing wound. Within a
understood, it is likely that many questions related to hair melanin
few months, white hair grows as well as black hair [20]. This may be
pigmentation will be solved [ 171. Ultrastructural changes in melano-
due either to the persistence in the wounds of pigmented hair bulbs
cytes are related to the hair cycle, and in hair follicles of C57 black
from the graft, or to migration of isolated melanocytes from the
mice, early anagen is associated with several modifications [lo]:
pigment grafts that colonize the regrowing white hair bulbs. In
increase in volume of the cytoplasm, increase in dendricity, develop-
humans, after induction of a pure dermal wound, removing the
ment of the Golgi complex and rough endoplasmic reticulum, and,
middle and lowest portion of hair follicles, regrowing hair is still
finally, increase in size and number of melanosomes.
pigmented. It is possible that the melanocytes present in this hair
During catagen and telogen, the melanocytes contain only a few
originate from the overlying e idermis [21]. Repigmentation of
small premelanosomes. They exhibit scanty cytoplasm with less
leukotrichia by epidermal gra P.tmg and systemic psoralen plus
well developed Golgi complex and rough endoplasmic reticulum,
UV-A has been recently reported [22]. In three vitiligo patients,
as well as nuclei with prominent heterochromatin patterns. It has
clinical follow-up suggested a possibility of activation and migra-
also been observed that the shape and internal structure of the mela-
tion of epidermal melanocytes to the hair follicles. Although ele-
nosomes can change during the black hair cycle [lo]. Hair melano-
gant, these experiments do not give conclusive evidence for the
cytes transfer melanosomes to follicular epithelial cells. Medullary
occurrence of movement of melanocytes from the epidermis to-
cells receive their melanin from melanocytes in the upper part of the
wards the hair bulb.
hair bulb, in a manner similar to that described for the epidermis.
Melanosomes are also transferred to immature cortical cells. Due to MELANOGENESIS IN HAIR
their larger size, melanosomes are usually distributed singly, what- Ultrastructural Features Many studies have been carried out on
ever the ethnic background [I2]. The hair melanosomes are 2 to the ultrastructural and biochemical aspects of hair melanin pigmen-
4 X larger than the epidermal melanosomes. As the process of kera- tation. The results will be discussed in relationship to visual hair
tinization continues, melanin granules, along with other cytoplas-
mic remnants, become embedded in keratin. A small number of
melanosomes are found associated with the spongy-appearing kera- Human Red Hair: In human red hair, specified as pheomelanic by
tin of the hair medulla. Melanin granules are mainly in the cortex, chemical analysis, melanocytes contain spherical melanosomes with
their long axis being parallel to the hair surface. The cuticle cells
contain few or no melanosomes. Usually, no melanin is seen in the * Surleve-Bazeille JE, Gauthier Y: Fate of non-tumorous melanocytes
cells of the inner root sheath. The distribution of melanosomes in injected within the dermis (abstr). Pig Cell Res 1:300, 1988.
 84s ORTONNE AND PROTA THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

microvesicular (vesiculoglobular) and proteinaceous matrices on

which melanin deposition is spotty and granular [23,24]. The se-
quences of melanization of these organelles are identical to those of
pheomelanosomes in yellow mice and tortoise-shell guinea pigs
H jJI~$O~HH2N&~$co2H
2 2
(yellow areas) [25]. i OH H OH
In other human red hair specified as “mixed” type melanogenesis
Trichochrome B Trichochrome C
by chemical analysis [23], a majority of melanocytes produce spheri-
cal melanosomes of pheomelanic form. They also contain “mosaic”
Figure 1. Chemical structure of trichochrome B and C.
melanosomes with features of both eumelanosomes (ellipsoidal
shape, regular striation) and heomelanosomes (spotty and micro-
granular melanization, lack o P electron-lucent bodies in mature mel-
anosomes). The nature of these “mosaic” melanosomes, whether zine unit [6]. The same type of unit is also found in the tri-
they are eumelanic, pheomelanic, or mixed, remains to be clarified. chochromes, which are smaller molecules of well defined structure
In addition, there was a second type of melanocyte, producing ellip- and composition [34]. Examples are the isomeric trichochromes B
soidal melanosomes of typical eumelanin form. and C (Fig 1); the latter is the predominant and most representative
member of the series.
Human Blond Hair: Since the identification of pheomelanosomes, From the structural anatomy of these molecules, one can easily
few detailed electronmicroscopic studies of human blond hair folli- recognize their biogenetic origin from dopa and cysteine through
cle have been reported. Melanin granules are smaller and less numer- formation and subsequent oxidation of 5-S-cysteinyldopa and 2-S-
ous in blond than in dark-haired subjects [24 - 261. Melanosomes are cysteinyldopa [35]. Th ere is evidence that the same intermediates
not fully melanized even in the dendritic processes of melanocytes. are involved in the biosynthesis of the polymeric sulfur-containing
This suggests that the light color in blond hair may be due to a pheomelanins, but little is definitely known about the reaction
quantitative decrease in the production and melanization of melano- pathway beyond the benzothiazine stage. Yet, despite this and other
somes. doubts, it is noteworthy that pheomelanins and trichochromes are
Human Black and Brown Hain: Typical ellipsoidal melanosomes, the end products of the same metabolic pathways that diverge after
at various stages of melanization, are observed in follicular melano- the formation of a common intermediate. Such a biogenetic rela-
cytes of black hair. Their ultrastructural characteristics are identical tionship explains why the two types of pigments are often found
to those seen in the epidermis of caucasoids and negroids [26,27]. together in pheomelanic hair.
Melanosomes transferred to neighboring keratinocytes are singly As indicated earlier, in both humans and in some animals yellow
distributed [23]. In brown hair, the follicular melanocytes also con- or reddish pigments exist that are chemically different from those
tain all the developmental stages of eumelanosomes. Lighter brown derived from cysteine and dopa. Currently, little is definitely
hairs have smaller melanosomes [26]. Similar aspects are observed, known about the structure of these varieties of pheomelanins except
whatever the racial background [24]. that they behave like bleached eumelanins. These and other obser-
vations [31] have led to s eculation that these pheomelanin-“look-
Senile Gray and White Hairs: In the melanocytic zone of the senile ing” pigments may in Pact be structural variants of eumelanins,
gray hair bulb, the number of melanocytes appears normal or re- arising from partial peroxidative cleavage of 5,6-dihydroxyindole
duced [28]. These cells show little melanogenic activity and contain units. Such a view is not unlikely, considering the high susceptibil-
very few melanosomes. In senile white hair, there are no dopa-posi- ity of the eumelanin polymer to hydrogen peroxide, which is in-
tive melanocytes. By electronmicroscopy, melanocytes are scarce or volved in the later stages of melanogenesis. Thus, in viva, modula-
entirely absent and there is no melanin in the matrix and cortex. tion of eumelanin color by hydrogen peroxide would provide an
Similar findings have been observed in white hair from vitiligo alternative mechanism to explain the polychromy of epidermal mel-
macules. The senile white hairbulbs do not contain immunoreactive anin pigmentation, which is otherwise difficult to explain on the
tyrosinase antigen [29]. More recently, tyrosinase mRNA or its basis of only two basic types of pigments, i.e., eumelanins and the
protein have been detected in senile white hairs, suggesting the sulfur-containing pheomelanins.
resence of amelanotic melanocytes within the outer root sheat Most natural melanins including hair melanins contain a certain
P301. amount of sulfur. As an example, as high as 3% sulfur has been
found in human black hair [6]. Another study demonstrates rather
Biochemical Aspect
high sulfur content whatever the hair color: 5.3% in Italian brown
Main Types ofMelanins: Hair melanins can be roughly classified hair, 4.9% in Japanese black hair, 8.8% in Irish red hair, and 2.3% in
into those giving dark colors, namely, black and brown and their Scandinavian blond hair [36].
derivatives, and those giving light ones, with a wide range from the Degradation experiments aimed at characterizing the main struc-
bright yellow coat of some mice to the carrot-like red color of tural units and the biosynthetic origins of the pigment are also
certain human hair. The dark colors are usually regarded as deriving useful tools for the study of natural melanins. A simple and rapid
from a fairly homogeneous group of polymeric pigments, the eu- method for the quantitative estimation of eu- and pheomelanins in
melanins, and consisting mainly of 5,6-dihydroxyindole (DHI) tissues based on the analysis of degradation products without isola-
and to a lesser extent of 5,6-dihydroxyindole-2-carboxylic acid tion of melanins made these studies possible [37]. The rationale of
(DHICA). Some of these units appear to be in the oxidized quinone this analytical method is that permanganate oxidation of eumelanin
form, as evidenced by the ability of melanins to undergo reversible gives pyrrole-2,3,5-tricarboxylic acid (PTCA) as a major pyrrolic
reduction. Minor structural contributions include the presence of product, whereas hydriodic acid hydrolysis of pheomelanins yields
5,6_dihydroxyindole semiquinone units and carboxylated pyrrole amino-hydroxyphenylalanine (AHP) as a major phenolic amino-
units; the latter probably arise by the partial fission of the indole acid [37].
units by the hydrogen peroxide formed during melanogenesis In hair of mice and guinea pigs, there is a good correlation be-
[31-331. tween the melanogenesis type defined by ultrastructural analysis
The light colors are usually described under the omnibus term of and by content of melanin [37]. It was demonstrated that black
pheomelanins, but these include pigments with different chemical mouse hairs contain a high level of eumelanin, but a negligible level
and physical properties. The most extensively investigated are on pheomelanins. On the other hand, yellow hair contained the
found in certain types of red hair, as well as in the feathers of lowest level of eumelanins and an intermediate level of pheome-
domestic fowl, e.g., New Hampshire and Rhode Island hens. Ana- lanin, and albino mouse hair contained a medium level of pheome-
lytical and degradative studies have shown that these pheomelanins lanin. As follicular melanocytes in black mice produce ellipsoidal-
are polymers or mixtures of polymers that contain a 1,4-benzothia- lamellar melanosomes, these observations suggest that the pigments
VOL. 101, NO. 1, SUPPLEMENT, JULY 1993 HAIR MELANINS AND HAIR COLOR 85s

synthesized in these organelles are mostly eumelanins. In contrast, recently [16], tyrosinase activity in hair follicles was measured in 23
follicular melanocytes in yellow (Aya) mice that produce spherical- red- and dark-haired individuals. Tyrosinase activity was also found
granular melanosomes are probably mostly associated with pheo- to be greater in the hair follicles of red-haired subjects than in those
melanin production. Similar results were obtained in the black, from dark-haired individuals. Evaluation of tyrosinase synthesis by
yellow, and white hair of tortoise-shell guinea pigs [37]. immunoprecipitation of tyrosinase with a specific antibody after
This technique was applied to the study of human hair [37 - 391. metabolic labeling of the protein suggested that red hair follicles
The main conclusions were that whatever the color, all the human presumably resulted from an increased synthesis of this enzyme.
hair examined contained various amounts of eumelanin and pheo- These three studies have focused on the tyrosine hydroxylase
melanin. This also demonstrated that visual differentiation of hair activity of tyrosinase. A method for measuring the dopa oxidase
color does not always reflect the melanogenesis type in human hair activity of human hairbulb tyrosinase based on the measurement of
follicles. Indeed, in 17 subjects with hair color ranging from white the formation of 5-S-cysteinyldopa has also been developed [45].
to blonde, the hair follicles were engaged in mixed type melanogen- With this technique it has been demonstrated that tyrosine hydrox-
esis. In six subjects with brown and black hair, follicular melano- ylase and dopa oxidase activities are coordinate functions of hair-
cytes synthesize predominantly eumelanins [37]. Finally, in nine bulbs tyrosinase over a broad range of hair color.
red-haired subjects, the follicles of only three were found to be These results from three different groups suggest that in red hair
engaged in typical pheomelanogenesis [23]. From this study, it was bulbs, follicular melanocytes have a high melanogenic activity.
also concluded that chemical analysis, i.e., the ratio of eumelanin to They contrast with the results obtained in mice, in which pheome-
pheomelanin, corresponded well to the fine structural differentia- lanogenesis is clearly associated with lower tyrosinase activity [43],
tion of eumelanogenesis and pheomelanogenesis. and illustrate that striking species differences may exist in the con-
All these results demonstrate that human hair follicles, whatever trol of melanogenesis. It would be of great interest to know if the
the color, contain various proportions of both eumelanins and recently identified regulatory proteins of melanogenesis TRP-1 and
pheomelanins. These observations point towards the key role of the TRP-2 [46] are also increased in these circumstances. The finding of
switching mechanisms that determine whether pheomelanins or a high tyrosinase activity in blond hair follicles suggests that the
eumelanins are synthetized by follicular melanocytes. The availabil- light color is not due to a low melanogenic activity of follicular
ity of sulfhydryls in melanocytes, more specifically within melano- melanocytes, but is rather due to the chemical structure of the mela-
somes, is probably an important factor in this process. Cysteine nins produced or to a post-tyrosinase block in the melanogenesis
and/or glutathione may sidetrack part or all of the generated dopa- pathway.
quinone to form additional intermediates, namely cysdopas and
Regulation of Melanogenesis in Hair Follicles The response
glutathionedopas [6]. It h as b een proposed that the role of cysteine/
of epidermal and follicular melanocytes to exogenous or endoge-
glutathione as a regulatory factor in switching melanogenesis type is
nous stimuli differs slightly. Due to their anatomical localization,
not tied to its absolute presence or absence, but rather to the effective
follicular melanocytes are less exposed to environmental factors
concentration within the melanocyte at a given time [39]. Because
than are epidermal melanocytes. This is the case for ultraviolet (UV)
the addition of cysteine or glutathione to quinones is a rapid reac-
radiation. Although psoralen plus UVA is known to affect hair
tion, the metabolic fate of do aquinone is mainly dependent upon
growth and hair pigmentation, it is likely that in normal conditions,
the activity of the enzymes o P the glutathione system that affect the
UV rays have little or no effect on the determination of constitutive
tissues’ sulfhydryl content. Direct support for this view is provided
hair pigmentation. Intense solar radiation lightens hair. Brown
by comparative analysis of the levels of glutathione and related
hairs lighten more than red hair. This suggests that pheomelanins
enzymatic activities in tortoise-shell guinea pig skin of different
are more resistant to photodegradation than eumelanins [47].
colors (black, yellow, red, and white), as well as in the skin of pure
black (a/a) and yellow (Ay/a) mice [40]. As expected, the lowest Genetic Control: Hair pigmentation is under genetic control. Very
levels of glutathione reductase activity were found to be associated little is known about this control in humans. In the dark races, there
with eumelanin-type pigmentation, whereas the highest ones were is intense selection for dark hair, but in caucasoids there is no strong
found in the skin with light pheomelanin-type pigmentation. More- selection for any particular color [48]. It is generally agreed that the
over, analysis of non-protein thiol pools revealed that GSH levels factors responsible for black hair color and largely also those for
are lower in black skin than in yellow skin of the agouti mice [41]. brown are epistatic to those that determine red hair color [49]. It is
Topical application of catecholic antioxidants, e.g., 4-tertiary butyl also agreed that red hair color is dominant to blond hair [50]. A
catechol, inhibits eumelanogenesis but stimulates pheomelanogen- strong evidence for a major locus concerning brown hair color being
esis in melanocytes of murine and guinea pig skin. This biologic linked to a locus for green eye color and located on chromosome 19
process is associated with elevated activities of glutathione reductase has been obtained. From linkage to the MNS blood group system, a
and gammaglutamyltranspeptidase, further suggesting that sulfhy- major gene for red human hair has been assigned to chromosome 4
dryls and sulfhydryl-related enzymes play a key role in the switch of ]511.
melanogenesis type [42]. Th e melanosomal membrane may play an In mice, more than 50 genetic loci have been identified [1,2].
important regulatory role by controlling the uptake of melanogenic They act either directly on the melanocyte or indirectly through the
substrates and sulfhydryls. follicular environment. These loci can be grouped into four major
classes: those affecting the migration, proliferation, and survival of
Hair Melanin, Hair Color, and Tyrosinase Activity: The possibility melanocytes; those controlling the amount of melanin produced,
that hair color could be related to the melanogenic activity of follicu- those that determine the kind of melanin synthesized, and those
lar melanocytes has been raised. Furthermore, the finding that, in reflected in the sha e and ultrastructure of melanocytes [52]. In
mouse hair follicular melanocytes, higher levels of tyrosinase activ- recent years, the mo Pecular bases of several of these mutations have
ity are associated with eumelanogenesis than with pheomelanogen- been identified. As examples, only a few of them will be discussed in
esis [43] suggested that tyrosinase expression in human hair follicu- detail.
lar melanocytes could depend upon the type of melanin produced. In the first group, the c-kit gene at the W locus affects the prolif-
Using a micromethod allowing the evaluation of tyrosinase activity eration of melanoblasts as well as hemopoietic stem and primordial
(tyrosine hydroxylase) by the Pomerantz’ method in single or germ cells during embryogenesis. Mutations at this locus give rise to
pooled hairbulbs, red and, to a lesser extent, blond hair follicles were white patches or spots in the fur due to a failure of melanoblasts to
shown to have the highest tyrosinase activities compared to black migrate, survive, or proliferate in these regions [8]. Another muta-
and brown hair follicles [44]. A second study using a similar method tion, at the steel locus, which encodes the ligand for the c-kit recep-
[29] also demonstrated that red hairbulbs have significantly higher tor, causes defects in the same three cell lineages as do mutations in
tyrosinase activity than the other colors. Except for red, the melanin the W locus and causes similar phenotypes [51,53]. Mutations of the
hairbulb tyrosinase levels were similar in all other hair colors. More kit protooncogene have been described recently in human piebald-
 3
Table I. Available Data on Tyrosinase Activity, Content of Eumelanin and Pheomelanin and Type of Human Hair Color
S
Hair Color Blonde/ Brown-’ Black/ Reds

Tyrosinase Activity
King et al [44] 2.423 rt 0.525 1.141 f 0.223 1.680 -t 0.482 4.310 f 1.150
(mean +_ SD) Light Medium Dark
Lloyd et a/ [29] 53.5 + 16 49.9 f 12 88.0 +- 18 84.7 f 20 99.0 f 42 201.5 f 45h
(mean + SEM)b (n = 5) (n = 7) (n = 9) (n = 6)
Townsend et al [45] ..:‘,?,=,20.3, From 1.26 f 0.71 to 7.25 f 0.49 5 k”:cz.7 6.17 f 0.45
(mean f SD) DO: 9.9 * 1.2 From 0.5 f 0.1 to 3.5 zk 9 4.6 +_ 1.2 2.6 f 0.9
(n= 1) (n = 1)
Burchill er a/ [ 161 Dark t8; 2 0.06 * 4.!?=0!!8 h
(mean * SEM)d
Ratio of Eumelanin/ Ash-blonde Blonde-brown Medium light brown Medium dark-
Pheomelanitr Light brown brown
Dark brown Light red Fire red Dark red
Jimbow et aP 1.63 + 0.80 3.93 f 1.44 13.5 +_ 9.6 1.7 f 7.5 29.3 f 8.43 0.81 f 0.44 0.72 f 0.55
(mean zk SD) (n = 3) (n = 3) W;) (n = 3) (n = 3) (n = 4) (nyl)
Light (n = 5,
Brown
Thody er al [38] 0.87 3.1 1.96 + 1.3 2.54 + 1.43 0.014
(mean + SD) (n = 1) (n= 1) (n = 3) (n = 6)
Burchill et al [16] Dark 1.36 f 0.46 0.:: 7 $05
(mean * SD) (n = 9j
Type of melanogenesis Mixed Eumelanic EuLlz? Eumelanic Eumelanic Pheomelanic Pheomelanic
Jimbow et a/j’
(;;,‘d (;Y=,‘d (;;,tl (;;,‘d
Mixed
(n = 3) (n = 1) (n = 2) (n = 2)

aTyrosinase activity expressed as pm01 tyrosine oxidized/l20 min.

b Tyrosinase activity expressed as pm01 DOPA formed/3 bulbs/30 min.
‘TH: Tyrosine hydroxylase activity of tyrosinase expressed as pm01 of tyrosine oxidized per h: DO: Dopa ox&se activity of tyrosinase expressed as nmol of 5-S-cysteinyldopa formed per h.
’ Tyrosinare activity expressed as pmol tyrosine converted/h.
*Based on the contents of PTCA (eumelanin indicator multiplied by a factor of 50) and AHP (ph eomelanin indicator multiplied by a factor of 5) in the follicles.
1 Based on the ratio/PTCA/AHP content in the follicles.
8 n within brackets = number of individuals.
h StatisticaIly significant. “z
’From Jimbow K, Ishida 0, Takahashi H, Ito S: Hair color and type of melanogenesis in human hair: characterisation based on chemical analysis of eumelanin and pheomelanin. and ultrastructural analysis of m&nosome structure. h: iz
Structure and Function of Melanin, Vol. 1. Nitto Printing Co. Ltd. Sapporo, Japan, 1984, pp 26-35. Y
?i
VOL. 101, NO. 1, SUPPLEMENT, JULY 1993 HAIR MELANINS AND HAIR COLOR 87s

ism, suggesting that this disorder may be the human homologue of By using an organ-culture technique, it has been demonstrated
dominant white spotting in the mouse [54]. that alpha-MSH and cholera toxin as well as forskolin induce eume-
In the second group, different alleles at the c locus encoding lanin synthesis in explants from lethal yellow mice (Ay/a), whereas
tyrosinase have been analyzed in detail and the molecular basis of these agents did not show a similar effect in the hair follicles of
mutations identified. This is the case for the albino mutant and for recessive yellow (e/e) mice, and eumelanogenesis was not modified
the Himalayan mutant allele, due to a temperature sensitivity of the by these treatments [63]. From these observations, it was suggested
enzyme resulting in a very peculiar phenotype, the mouse having that the product of the “a” locus probably interacts with alpha-
dark ears, feet, and nose against a pale body [52]. Similar mutations MSH at the alpha-MSH receptor. When alpha-MSH binds to its
have also been described in humans with tyrosinase-negative oculo- membrane receptor, the signal is transduced to adenylcyclase,
cutaneous albinism and temperature-sensitive albinism [52]. which results in an increase in cyclic AMP level in the cytoplasm,
In the third group, the agouti and extension loci have been exten- resulting in increased tyrosinase activity leading to eumelanogene-
sively studied, as the corresponding mutations provide a good sys- sis. On the other hand, when Ay/a or A/A follicular cells secrete
tem to study the control of melanogenesis in hair follicles, particu- their product, it competitively interacts with alpha-MSH, blocking
larly the switching mechanisms for pheo- or eumelanogenesis. The the alpha-MSH receptor, resulting in low intracytoplasmic CAMP
agouti locus is an excellent system to study the control of melano- levels, low tyrosinase activity, and, finally, pheomelanogenesis. The
genesis in hair follicles, although it is not present in humans. The action of the “e” locus could be through the control of the function
murine agouti locus in chromosome 2 encodes gene products that of adenylcyclase in the membrane of mouse melanocytes [63].
determine whether eumelanin, pheomelanin, or both of these pig- In the fourth group, the pallid mutation produces defects in at
ments are synthesized in hair bulb melanocytes [2]. The wild-type least three subcellular organelles: platelet-dense granules, kidney
mouse has two types of pigment: eumelanin that colors the tip and lysosomes, and melanosomes. These mice have diluted pigmenta-
base of the dorsal hair, and pheomelanin that colors a stripe across tion due to abnormally small melanosomes. A recent report suggests
the middle of the hair. This is a striking demonstration that individ- that pallid is a mutation in the Epb 4.2 gene, the murine gene for
ual melanocytes can switch between the two types of pigment syn- protein 4.2. This protein interacts with band 3 on the erythrocyte
thesis. Hair follicle melanocytes in transition from black to yellow membrane and ankyrin in solution. By immunofluorescence, pro-
possess both eu- and pheomelanosomes, demonstrating that the tein 4.2 has been localized in melanosomes in human melanoma
shift from eumelanogenesis to pheomelanogenesis or vice-versa cells. Although the function of protein 4.2, renamed pallidin, is
occurs within a single cell [55]. E ven so, melanosomes with features unknown, it may be that it plays a role in membrane stabilization.
of both pheomelanosomes and eumelanosomes have been observed These mouse mutations resemble the human disorders Hermansky-
in such circumstances [56]. Pudlak syndrome and Chediak-Higashi disease. The pallidin gene is
Mutations at the “a” locus disrupt this switching between eu- and certainly a candidate gene for these disorders [64].
pheomelanogenesis. Lethal yellow (Ay) is dominant to all other Many alleles of the pink-eyed dilution on mouse chromosome 7
alleles, resulting in a uniformly yellow coat. At the opposite end of are defined by reduced pigmentation of both coat and eyes. Mutant
the spectrum, extreme non-agouti (ae), recessive to all other alleles, p melanosomes are structurally abnormal and have a reduced ca ac-
produces a totally black coat [2]. Despite considerable effort, the ity to bind or accumule melanin. A recent characterization o P the
specific effect of the agouti locus remains unknown. It is estab- human homologue of the mouse pink-eyed dilution gene revealed
lished, by grafting and cell culture experiments, that the genes at that it may be associated with Prader-Willi and Angerman syn-
this locus produce their effect by altering the follicular milieu (i.e., dromes. From these results, it has been suggested that altered ex-
outside the melanocyte) [2]. S everal ex eriments support the notion pression of this gene may be responsible for the hypopigmentation
that agouti genes either directly or in Birectly modulate the activity phenotype exhibited by certain individuals with these disorders
of tyrosinase. Whether this modulation occurs at the transcrip- b51.
tional, translational, or osttranslational level remains to be estab- The dilute mutation is associated with a reduction or dilution of
lished. Recent data [57 P suggest that the agouti locus modulates igmentation [2]. This is due to a failure of the melanosome transfer
tyrosinase activity at least in part by regulating the total number of f rom melanocytes to keratinocytes due to an inability of the ig-
tyrosinase molecules. This regulation could operate at the level of ment cell to extend dendrites [2]. Recently, the sequence o P the
translation or ost-translational modification of tyrosinase [58]. dilute product has been identified. It is a novel myosin heavy chain
The role of sul R ydryl compounds in the expression of agouti locus that probably plays a major role in the extension of the melanocyte
alleles has also been discussed for a long time 1591. From a large set of dendrites 1661.
- a
experiments, it was concluded that all-melanocytes from “pheome-
Other Factors Normal hair color is altered in many disorders of
lanic” mice of different agouti locus constitutions synthesize eume-
different causes. Among them, hormonal, nutritional, and meta-
lanin under standard in vitro conditions. However, if sulfhydryl
bolic disorders are largely represented (for review see [67]), demon-
compounds such as glutathiones are added to the nutrient medium,
strating the complexity of the regulatory controls of melanogenesis
all melanocytes, regardless of their age or agouti locus genotype,
in hair follicles.
could be induced to nroduce eheomelanin. MSH treatment converts
pheomelanogenesis’ to eum;lanogenesis when the former results Aging of the Follicular Melanin Unit During human life,
from the action of agouti locus alleles [60]. The mouse agouti gene natural hair color changes may be observed. Many fair-haired chil-
has recently been cloned and characterized. It encodes a 131amino- dren gradually become darker and by middle age have brown hair,
acid protein [61]. whereas other children often become brown, sandy, or auburn-
Another locus called extension also controls the type of melanin haired adults [68]. Greying of hair is the most familial manifestation
pigment reduced by follicular melanocytes [2]. The extension of the aging process. The grey ap earance represents a mosaic
series of aPleles is located on chromosome 8 and acts autonomously blending of many white and dark Rairs with a small number of
within the melanocytes. The dominant alleles are black whereas the transitional hairs that are grey. Whitening of hair refers to the end
recessive mutation “err produces yellow mice only when homozy- stage of depigmentation [67].
gous. In contrast, pheomelanogenesis of the coat of recessive yellow This phenomenon is very common and occurs to various extents
mice is not modified by MSH administration [60]. The relation in almost all persons. The onset of physiologic canities seems largely
between these two loci is unknown, but the possibility that it could hereditary, but other factors are probably involved. It is variable but
be a factor/receptor relationship has been proposed [62].* usually occurs in the late fourth or early fifth decade. Neither sex is
spared. Greying appears earlier in dark- than in light-haired sub-
* A recent paper demonstrates that pigmentation phenotypes of various jects, but more fair- than dark-haired subjects become completely
extension locus alleles result from point mutations that alter MSH receptor grey. The possible explanation for this apparent contradiction is that
function (Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss the first signs of greying are seen more directly against a dark back-
L, Brack E, Mountjoy KG, Cone RD: Cell 72:827-834, 1993. ground than against a fair background. Greying is less common in
88s ORTONNE AND PROTA THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

blacks. Greying of the hair is usually irreversible, but return of 10. Sugyiama S, Kukita A: MeIanocyte reservoir in the hair follicles during
pigment has been known to occur [69]. the hair growth cycle: an electron microscopic study. In: Kobori T,
The cellular and subcellular bases of greying and whitening of Montagna T (eds.). Biology and Diseases of the Hair. University
Park Press, Baltimore, 1976, pp 181-200
hair have already been discussed. The precise cause of senile greying
of hair has not been established. Alpha-MSH binding sites, nor- 1 I. Vegesna V, Withers HR, Taylor JM: The effect on depigmentation
mally present in pigmented human scalp hair follicles, are absent in after multifractionated irradiation of mouse resting hair follicles.
Radiat Res 111:464-473, 1987
senile white hair follicles [70]. The role of some disturbance of
immune tolerance has been suggested, but little evidence supports 12. Toda K, Pathak MA, Parrish JA, Fitzpatrick TB: Alteration of racial
differences in melanosome distribution in human epidermis after
this idea 1711. Animal models of senile greying of hair include
exposure to ultraviolet light. Nature 236:143-145, 1972
guinea pigs, rats, rabbits, dogs, sheep, and horses [ 1,2]. A progressive
13. Burnett JB, Holstein TJ, Quevedo WC Jr: Electrophoretic variations
loss of hair nigmentation inherited as an autosomal dominant trait
I ”
of tyrosinase in follicular melanocytes during the hair growth cycle
and influenced by unspecified maternal effects has been described in
in mice. J Exp Zoo1 171:369-376, 1969
wild-type Australian mice [72]. This suggested that heredity is a
14. Burchill SA, Redfern CPF, Thody AJ: Expression of mRNA recog-
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