tolleson jeshc 2005 melanocyte review.pdf

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Journal of Environmental Science and Health, 23:105161, 2005

Copyright C Taylor & Francis Inc.

ISSN: 1059-0501 (Print); 1532-4095 (Online)

DOI: 10.1080/10590500500234970

Human Melanocyte Biology,Toxicology, and Pathology

William H. Tolleson

National Center for Toxicological Research, U.S. Food and Drug Administration,Jefferson, AR, USA

The human melanocytes of the skin, hair, eyes, inner ears, and covering of the

brain provide physiologic functions important in organ development and maintenance.

Melanocytes develop from embryonic neural crest progenitors and share certain traits

with other neural crest derivatives found in the adrenal medulla and peripheral nervous

system. The distinctive metabolic feature of melanocytes is the synthesis of melanin pig-

ments from tyrosine and cysteine precursors involving over 100 gene products. These

complex biochemical mechanisms create inherent liabilities for melanocytic cells if in-

tracellular systems necessary for compartmentalization, detoxification, or repair are

compromised. Melanocyte disorders may involve pigmentation, sensory functions, au-

toimmunity, or malignancy. Environmental factors such as ultraviolet radiation and

chemical exposures, combined with heritable traits, represent the principal hazards

associated with melanocyte disorders.

Key Words:Melanocytes; Melanin; Melanoma; Ocular toxicity; Ototoxicity; Oxidative

stress

MELANOCYTE PHYSIOLOGY

Melanocytes are melanin-producing somatic cells that provide important phys-

iological functions in the skin, eyes, inner ear, and meninges (Table 1). Rec-ognized functions associated with melanin production in humans include pho-

toprotection, trapping reactive oxygen species, sequestering metal ions, and

binding certain drugs and organic chemicals (1, 2). Moreover, exfolliation pro-

vides a minor route for the elimination of toxic materials bound to melanin

embedded in the hair and outer skin layers. Human melanogenesis also

Address correspondence to William Tolleson, National Center for ToxicologicalResearch, U.S. Food and Drug Administration, Jefferson, AR 72079, USA; E-mail:[email protected]

This article is not an official guidance or policy statement of U.S. Food and DrugAdministration (FDA). No official support or endorsement by the U.S. FDA is intendedor should be inferred.

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106 W. H. Tolleson

Table 1: Typical melanocyte functions.

Function Example Relevant melanocyte properties

Photoprotection Intrafollicular cutaneousmelanocytes

Melanin biosynthesis anddistribution to neighboringkeratinocytes

Scavenge organic andinorganic cations

Leptomeningealmelanocytes

Affinity of melanin fordrugs/chemicals

Elimination of excessmetal ions

Cutaneousmelanocytes

Shedding melanized hair shaftsbearing sequestered metals

Defense againstphotooxidative stress

Cutaneous and uvealmelanocytes

Consumption by melanin ofsinglet oxygen, hydroxylradical, and superoxideanion

Vision Uveal melanocytes;also retinal pigmentepithelium

Pigmented cells absorb straylight preventing internalreflections

Organogenesis Choroidal melanocytes Development of the iris andciliary

Organ maintenance Otic melanocytes Maintenance of the scalamedia of the cochlea

Antigen presentation Cutaneousmelanocytes

Phagocytosis; expression ofMHC II and ICAM-1;stimulate proliferation ofcytotoxic T-lymphocytes

influences interactions between individuals and cultures; the moral, social, andpsychological implications of this function have been addressed by others (35).

Additional functions of melanin are apparent in other species, such as creating

an opaque barrier that helps marine cephalopods evade potential predators,

imparting structural strength to seed pods in plants, and providing antibiotic

properties for insect immune systems (1). The relevance of these properties to

human melanocytes is unclear.

Unlike epithelial and mesenchymal constituents of the skin and eyes,

melanocytes are not responsible for the principal functions of these tissues.

Instead, melanocytes take up residence in between other cell types to provide

accessory functions, typically involving pigmentation. Melanoblasts, the embry-

onic precursors of melanocytes, migrate to different sites during development

to establish the melanocyte populations from which various melanocytic lesions

may arise (for review, see Piris and Rosai [6]). This review will emphasize four

populations of melanocytes located in the skin, eyes, inner ears, and covering

of the brain (Figure 1).

Cutaneous Melanocytes

The largest numbers of melanocytes are found in the skin and hair follicles.

The majority of proliferating cutaneous melanocytes in normal, out-bred, adult,

furred mammals are present in the papillary bulbs of hair follicles in an ap-

proximate one-to-five ratio with keratinocytes (Figure 2). Human interfollicular


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Human Melanocyte Biology, Toxicology, and Pathology 107

Figure 1: Distribution of human melanocytes. Human melanocytes populate theintrafollicular epidermis, hair follicles, uveal tract of the eye, stria vascularis of the cochlea,and meninges covering the brain and spinal cord.

Figure 2: Hair follicle structure. Melanocytes are concentrated around the papillary bulb ofthe hair shaft. Additional melanocytes are present in the inner and outer root sheaths.


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108 W. H. Tolleson

melanocytes are particularly abundant in the face, scalp, and the genital

regions, but are also found throughout the epidermis, including mucosal epithe-

lia such as the oral cavity and the anus, but are less abundant in the thicker

afollicular epidermis of palms and foot soles. Interfollicular melanocytes are

less common in the keratinized epidermis of nonhuman species. In human skin,

interfollicular melanocytes are found within the basal layer of the epidermis,

attached to up to 36 neighboring keratinocytes that comprise the epidermal

pigmentation unit (Figure 3) (7). Melanin is packaged within specialized vesi-

cles known as melanosomes for distribution to the surrounding keratinocytes

of the epidermal melanin unit. This occurs via a network of dendritic processes

originating from a melanocyte located within the basal layer of the epidermalstratified epithelium.

Figure 3: Human epidermal pigmentation unit. Melanosomes are distributed toneighboring keratinocytes via dendritic processes originating from a melanocyte residing in

the basal layer of the stratified epithelium. Viable pigment-carrying keratinocytes of thebasal layer undergoing differentiation are pushed upwards through the skin to form thestratum spinosum. Nuclear degeneration, crosslinking of the cell envelope, andkeratinization occur in the stratum granulosum. Fully keratinized, enucleated squams areexfollilated from the stratum corneum.


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In contrast to the continually renewing epidermal pigmentation unit of

the intrafollicular epidermis, a different pigmentation mechanism is apparent

within the hair follicle (reviewed in ref. [8]). The follicular melanin unit un-

dergoes a cyclic pattern of melanogenesis synchronized with the hair cycle.

The human scalp hair cycle is comprised of a 35 year growth period (anagen

stages IIII), a three week regression phase (catagen), and a three month rest-

ing phase (telogen). Undifferentiated amelanotic melanocytes or melanoblasts

are recruited to the dermal papilla at beginning of anagen from a supply of

immature melanocytes located in the upper, permanent portion of the outer

root sheath. The immature melanocytes in the anagen hair bulb differentiate

and form dendritic connections with keratinocytes of the hair shaft cortex dur-ing anagen I and II. Melanogenesis and transfer of melanosomes to precortical

keratinocytes occurs primarily during anagen III and continues through cata-

gen, when melanogenesis decreases and the dendrites of bulbar melanocytes

retract. Extensive apoptosis occurs within the hair bulb during late catagen,

but the preexisting fully pigmented club hair shaft is usually retained in the

follicle through the end of telogen. After telogen it is often displaced by a newly

formed hair shaft produced in the subsequent anagen cycle.

The proliferation of cutaneous melanocytes is normally suppressed by close

physical association with epithelial cells. The co-expression of the E-cadherin

cell adhesion molecule by melanocytes and keratinocytes facilitates gap junc-tion formation and contributes to suppressing melanocyte proliferation (9).

Some cutaneous melanocytes switch from E-cadherin to N-cadherin expression;

melanocytes expressing N-cadherin are released from growth suppression via

attachment to epithelial cells, allowing them to proliferate and self-aggregate

to form moles or nevi, latin for nests. Switching from E- to N-cadherin expres-

sion confers increased resistance to apoptosis and also permits the migration of

melanocytes among N-cadherin-expressing dermal fibroblasts (10). The more

rapidly proliferating keratinocytes of the epidermal pigmentation unit retain

melanin granules donated to them by melanocytes as they continue to progress

through their characteristic program of terminal differentiation to establish

the keratinized epithelium of the skin. This process creates a sheltered envi-

ronment for epidermal melanocytes, protecting them under a stratified canopy

of melanized keratinocytes that provides a tough, waterproof, and pigmented

physical barrier. Also, because intrafollicular cutaneous melanocytes are lo-

cated primarily within the basal layer of the epidermis, they experience dimin-

ished oxygen tension and have restricted access to other factors transported via

the capillary blood supply of the dermis.

Ocular Melanocytes

Another large population of melanocytes resides within the uveal tract of

the eye, comprised of the choroid, ciliary body, and iris (Figure 4). The choroid,


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110 W. H. Tolleson

Figure 4: Ocular anatomy.

located in the posterior portion of the eye, is separated from the retinal pigment

epithelium and neural retina by Bruchs membrane and is surrounded on its

outer boundary by the densely fibrous sclera. The cillary body extends from

the choroid in the anterior direction and forms the site of attachment for the

lens. The pigmentation of the iris provides photoprotection for the system of

capillaries, muscles, and motor nerves that regulate light entering the pupil.

Cells of the iris, ciliary, and retinal pigment epithelium are also pigmented, but

they are not true melanocytes, and the adenomas and adenocarcinomas that

arise from pigment epithelium cells are rare in humans (1114).

The microenvironment of uveal melanocytes differs significantly from that

of cutaneous melanocytes. The uveal tract is very highly vascularized, which

creates a higher oxygen tension than that of the central nervous system.

Melanocytes of the choroid have ready access to blood-borne factors and are

in physical contact with fibroblasts that form the sclera and with vascular en-

dothelial cells that form the choriocapillaris. Uveal melanocytes are typically

attached to other melanocytes, unlike cutaneous intrafollicular melanocytes

that are distributed singly among clusters of keratinocytes. The anterior

structures of the eye that overlay choroidal melanocytes (e.g., Bruchs mem-

brane, retinal pigment epithelium, photoreceptor layer, neural retina, vitre-

ous humor, lens, aqueous humor, and cornea) absorb or scatter the major-

ity of UV, visible, and infrared radiation found in sunlight (1517). Thus,

the potential for direct photodamage is greatest for interfollicular cutaneous


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melanocytes, followed in decreasing order by follicular cutaneous melanocytes,

conjunctival melanocytes, and iris melanocytes. Melanocytes of the choroid and

cililary body receive virtually no exposure to UV or visible light. Unlike cuta-

neous melanocytes, which distribute pigment to adjacent keratinocytes, uveal

melanocytes retain the melanosomes they produce (18). Until recently it was

believed that melanin synthesis occurred continuously in interfollicular cuta-

neous melanocytes, cyclically during the anagen phase of the hair cycle in fol-

licular cutaneous melanocytes, but only during prenatal development by uveal

melanocytes (19). However, because optic treatments with the prostaglandin

analog latanoprost result in heterochromia and increased pigmentation of the

iris, it is apparent that melanin synthesis can be induced in adult eyes (20, 21).Further evidence that uveal melanocytes retain the potential for melanin syn-

thesis in adults is apparent in the observations that: (1) induction of tyrosinase

activity by latanoprost in cultured adult human uveal melanocytes is blocked

by the tyrosinase inhibitor 1-methyltyrosine, (2) the artificial melanin precur-

sor [3H]-methimazole labels the uveal tract of pigmented adult DBA mice, and

(3) tyrosinase activity is evident in the uveal tract of bovine eyes (2224). Thus,

the potential for melanin synthesis is present in adult ocular melanocytes.

Otic MelanocytesMelanocytes are also found in the stria vascularis of the cochlea. The stria

vascularis forms the lateral wall of the scala media, the endolymph-filled cham-

ber that houses the organ of Corti, which is the principal sound-detecting com-

ponent of the inner ear (Figure 5). The melanocytes of the stria vascularis are

termed intermediate cells and provide functions that are necessary for nor-

mal hearing (Figure 6). Deafness results from loss of these cells via trauma,

infection, or perhaps noise (25).

Ototoxic agents, such as ticarcillin, may damage intermediate cells, result-

ing in hearing loss (26). The range of functions provided by intermediate cells

has not been characterized extensively but it is known that the stria vascularis

is responsible for endolymph maintenance (27). Unlike most other bodily fluids,

the major cation of endolymph is not sodium, but potassium supplied by the

stria vascularis. Potassium transport is regulated to maintain the endocochear

potential at +80 mV via ion-selective channels expressed by cells of the scala

media (28). Expression of the Kir4.1 potassium channel is restricted to interme-

diate cells, suggesting that this melanocyte function is essential for endolymph

maintenance and hearing (29, 30).

Cephalic Melanocytes

Melanocytes are distributed through the meninges covering the central ner-

vous system, particularly within the ventrolateral leptomenigenes overlying


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112 W. H. Tolleson

Figure 5: Structure of the inner ear. Otic melanocytes are found within the stria vascularis(SV) which covers the spiral ligament and bony labyrinth of the cochlea. The scala mediais the endolymph-filled chamber that houses the organ of Corti (OC). The scala media isseparated from the scala vestibuli by Reissers membrane (RM) and from the scalatympanium by the basilar membrane. (Guinea pig, second turn of cochlea, H&E stain, bypermission A.N. Salt, Ph.D., Washington Univ., St. Louis, MO, http://oto.wustl.edu/cochlea/accessed Feb. 2005).

the pons and medulla oblongata (31) (Figure 7). In addition to their well known

role as light-absorbing sunscreens for tissues exposed to the environment,melanosomes can scavenge potentially toxic organic and inorganic cations and

free radical species (32, 33). The physiological functions of leptomeningeal

melanocytes have not been established, but the tendency of melanotic cells

to cluster around blood vessels within the pia mater of other species (cats (34)

and Zucker rats (35)) suggests that they have a protective role, probably by

sequestering toxic materials from the circulation.

MELANOCYTIC CELLS DURING EMBRYOGENESIS

Melanocytes are derived from neural crest cells that migrate after neural tube

closure. The initial set of migratory neural crest cells follow a ventral pathway

and ultimately give rise to the peripheral nervous system, craniofacial bones


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Figure 6: Melanocytes of the stria vascularis. Melanocytes of the stria vascularis are termedintermediate cells (IC). Dendritic intermediate cells are found between the pigmentedmarginal cells (MC) and the basal cells (BC), which are attached to the spiral ligament

(SL). A capillary (C) is also shown.

Figure 7: Diagram of meninges covering the medulla. Melanocytes are more prevalent inthe leptomeninges covering the pons and medulla in humans.


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114 W. H. Tolleson

Figure 8: Embryonic origins of human melanocyte populations. Neural ectodermdifferentiates to form the neural plate and neural fold. Upon closure of the neural fold,neural crest cells migrate through dorsolateral pathways to form the peripheral nervoussystem, adrenal medulla, craniofacial structures, cardiac structures, and variousmelanoblast subpopulations. Melanocyte progenitor cell populations are shown in boldtype.

and cartilage, cardiac structures, and adrenal medulla. Later migrating neural

crest cells include melanoblasts, which follow a dorsolateral migratory path-

way between the mesodermal and ectodermal layers (Figure 8). Melanoblast

migration is directed vectorially by chemotactic signal gradients and patterns

of cell surface molecules and receptors expressed by the mobilized melanoblasts

or present within the extracellular matrix through which they move. Migrating

melanoblasts are responsive to various signals including: endothelin, stem cell


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factor (c-kit ligand), ephrin, fibronectin, Wnt3a, bone morphogenic protein-4

(BMP4), transforming growth factor- (TGF), retinoic acid, and hepatocyte

growth factor/scatter factor (HGF/SF) (3642). Melanoblast migration is termi-

nated and mesenchymal-epithelial transition occurs as melanoblasts encounter

the proper signals, such as those expressed by epithelial cells of the embry-

onic periderm or mesenchymal cells of the periocular mesoderm and the otic

vesicle.

Neural crest cells arise from the primitive neuroectoderm. Later, in prepa-

ration for migration, neural crest cells undergo a characteristic epithelial-to-

mesenchymal transition. Migratory cells arriving at their destinations undergo

the reverse process, mesenchymal-to-epithelial transformation, and initiatetheir specific differentiation programs. Neural crest derivatives may acquire

enhanced motility via a second epithelial-to-mesenchymal transition cycle

instituted in the process of carcinogenic transformation. The tendency of

some melanomas to develop an aggressive metastatic phenotype recapitulates

aspects of embryonic melanoblast programming, including responsiveness to

chemotactic signals, particularly those mediated by the c-met: HGF/SF and

Eph : ephrin receptor systems (42, 43)

Melanocytes share similarities with some neural crest derivatives, partic-

ularly the adrenal medulla and dopaminergic neurons, but are clearly dis-

tinct from others, such as those that give rise to craniofacial bones and car-tilage. For example, migrating dendritic neural crest cells communicate with

neighboring cells via gap junctions involving connexin-43 (44). Many neu-

ral crest derivatives function as secretory cells to produce and release neu-

rotransmitters or pigment. A significant biochemical feature that these cells

share is the utilization of tyrosine to generate the catechol intermediate L-

3,4-dihydrophenylalanine (DOPA), a precursor in the synthesis of dopamine,

epinephrine, norepinephrine, melanin, pheomelanin, and neuromelanin. Cate-

chol biosynthesis is compartmentalized within specialized organelles in these

cell types, limiting exposure to dangerous catechol, quinone, and reactive oxy-

gen species formed as intermediates or by-products. It has been suggested

that inherited mutations or chemical treatments may compromise the contain-

ment or scavenging of toxic derivatives generated during catechol biosynthe-

sis, contributing to disorders such as contact/occupational vitiligo or melanoma

progression (45, 46).

MELANOSOME ASSEMBLY AND MATURATION

Melanin biosynthesis in mammals has been the subject of numerous chemical,

biochemical, and molecular biological studies. At least 127 genetic loci involved

in pigmentation have been identified in mice and other species (47). Genes in-

volved in human pigmentation were recently reviewed by Sturm et al. (48), and


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116 W. H. Tolleson

Spritzet al. (49) reviewed disorders of human and mouse pigmentation. Slo-

miniskyet al. (8) provided a comprehensive review of hormonal regulation of

melanogenesis. The roles of many genes in the expression of a fully pigmented

phenotype have been elucidated or implicated using biochemical and molecu-

lar biological techniques. Inherited pigmentation patterns often follow simple

Mendelian genetics. The genes associated with human oculocutaneous albinism

types 14 (tyrosinase, pink-eyed dilution, TRP-1, and MATP) are representative

of autosomal recessive traits. Piebaldism (c-kit), Waardenburg syndrome types-

1 (PAX3), -2 (MITF), -3 (PAX3), and -4 (SOX10, endothelin 3, and endothelin-B

receptor) exhibit autosomal dominance.

Melanogenesis is tightly coordinated with genesis of melanosomes in hu-man melanocytes. The process of melanosome assembly in relation to melanin

biosynthesis was recently reviewed by Hearing (50). Melanosome develop-

ment begins when spherical stage I melanosomes, or premelanosomes, bud

from the endoplasmic reticulum (ER). The major scaffold protein Pmel 17, also

known as gp 100 (encoded by the silver locus in mice), is transferred to Stage

I melanosomes. Proteolytic cleavage of Pmel 17 activates its condensation to

form the characteristic internal fibers of melanosomes and marks the transi-

tion to ellipsoidal Stage II melanosomes. Tyrosinase, essential for melanogen-

esis, is synthesized within the ER, and abnormal transport of tyrosinase from

the ER has been established as a mechanism for some forms of oculocutaneousalbinism. Inactivation of the tyrosinase gene is responsible for oculocutaneous

albinism type-1 (OCA-1). The genetic basis for oculocutaneous albinism type

2 (OCA-2), the most common form of albinism globally, is associated with mu-

tations of the pink-eyed dilution gene, the p locus in mice (51); yet, until re-

cently, the intracellular localization and functions of the OCA-2 protein product

(P protein) were uncertain. Current evidence establishes that the P protein is

required for proper transit of tyrosinase from the ER and that the majority

of P protein is located within the ER (52). Other studies show that increased

expression of the wild type P protein is associated with decreased intracellu-

lar glutathione levels and decreased resistance to arsenic (53). Earlier studies

detected P protein associated with melanosomes (54). Peptide sequence homolo-

gies between the P protein and bacterial Na+/H+ transporters suggested that it

might function in tyrosine transport into melanosomes (55), but tyrosine trans-

port is evident within Pnull melanocytes, arguing against involvement of the P

protein (56).

While within the lumen of the ER, tyrosinase is folded properly via

chaperone-type interactions with TRP-1 (tyrosinase-related protein 1, also

called catalase B) and calnexin (57, 58). Oculocutaneous albinism type 3 (OCA-

3) is associated with TRP-1 mutations that prevent tyrosinase export from the

ER (59, 60). Watabe and coworkers (61) showed that abnormal acidification of

the ER also prevents tyrosinase export from the ER, resulting in albinism via

proteasome-dependent digestion of tyrosinase but that raising the pH of the ER


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reversed this effect. Thus, the pH of the ER exerts a regulatory effect on tyrosi-

nase expression. Following its initial glycosylation step in the ER, tyrosinase is

processed through the cis-Golgi and trans-Golgi where it is glycosylated further.

Tyrosinase acquires its two essential copper ions by the point it reaches the

trans-Golgi, based on DOPA-positive staining of this organelle (62). Mature ty-

rosinase is first transported via AP3 (adapter protein 3) sorting vesicles to early

or late endosomes, and finally delivered to Stage I and Stage II melanosomes.

Oculocutaneous albinism type 4 is associated with inactivating mutations of

MATP (membrane associated transporter protein), resulting in abnormal extra-

cellular delivery of melanosome-specific proteins after normal transit through

the ER, Golgi, and endosomal compartments (63, 64). Other melanosome-specific components, such as TRP-1, TRP-2 (tyrosinase-related protein-2, also

called dopachrome tautomerase), MART-1 (also called melan A), and OA-1 (ocu-

lar albinism-1) proteins, are delivered to maturing melanosomes via alternative

sorting pathways that have not been characterized completely (57, 6567).

Pigment synthesis begins within Stage III melanosomes. Stage IV melanosomes

are mature, fully pigmented, and ready for transfer to neighboring cells.

BIOSYNTHESIS OF MELANINS

Tyrosinase, a mono-oxygenase Type-3 copper protein with o-phenol oxidase and

catechol oxidase activities, catalyzes the synthesis of dopaquinone via hydrox-

ylation and oxidation of L-tyrosine or L-DOPA (Figure 9). Inactive tyrosinase,

which cannot bind molecular oxygen, is termed deoxy-met-tyrosinase. The two

active site copper centers of deoxy-met-tyrosinase are both in the Cu(II) oxi-

dation state and are liganded by two clusters of three histidine residues (68)

(Figure 10). Reduction of the two active site Cu(II) centers to the Cu(I) state is

typically accomplished via the sacrificial oxidation of L-DOPA to dopaquinone

yielding deoxy-tyrosinase. Deoxy-tyrosinase readily binds molecular oxygen

as a bridged 2 2 peroxy complex, forming active oxy-tyrosinase prior

to binding either tyrosine or DOPA substrates (69). It was previously ac-

cepted that dopaquinone was formed from tyrosine sequentially in two steps:

(1) hydroxylation at C-3 followed by (2) two-electron oxidation to convert the

3,4-catechol to the o-quinone final product (70). Recent studies showed that

oxy-tyrosinase converts tyrosine to dopaquinone directly (Figure 9, step 1), per-

haps via a bidentate copper/catechol complex that decomposes with oxidation

of the catechol to the o-quinone (71, 72). Fenoll et al. (73) have challenged

this view and, among other considerations, proposed that formation of a dead-

end complex between tyrosine and met-tyrosinase alters the flux of interme-

diates formed in tyrosinase-mediated reactions. Computer simulations using

rate constants for intermediate steps in dopaquinone synthesis indicated that

separate hydroxylation and oxidation reactions of tyrosine provided the best fit


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118 W. H. Tolleson

Figure 9: Biosynthesis of melanin pigments from L-tyrosine and L-cysteine.


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Figure 10: Catalytic cycle of the two copper centers in the active site of humantyrosinase. Each copper is liganded by histidinyl residues (his). The enzyme is representedby the plane passing through the two copper centers.


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120 W. H. Tolleson

to their experimental data. Regardless of whether tyrosine is converted in one

or two steps, formation of dopaquinone is the common end result. Alternatively

acting as a catechol oxidase, oxy-tyrosinase binds L-DOPA and catalyzes its

two-electron oxidation to form the o-quinone product (Figure 9, step 2). The

catechol oxidase activity of tyrosinase is also required to recycle L-DOPA gen-

erated non-enzymatically as a by-product in later steps of melanogenesis.

The rate constants for spontaneous chemical reactions of dopaquinone in-

volved in melanin synthesis have been determined using spectrophotometry

and pulse radiolysis techniques (7476), providing important mechanistic ra-

tionales for inter-individual differences in the type and quantities of melanin

produced (for review, see [7]). Dopaquinone has three potential chemical fatesthat alternatively generate black, brown, or reddish-yellow pigment forms. The

synthetic pathway favored and the type of pigment produced depends primarily

on the relative influx of L-tyrosine and L-cysteine into the melanosome, the ac-

tivities of the three melanogenic enzymes (tyrosinase, TRP-1, and TRP-2), and

the pH of the melanosome. Formation of 5-S-cysteinyldopa from dopaquinone

via a Michael addition is favored in the presence of excess L-cysteine (Figure 9,

step 3). Pheomelanin, a reddish-yellow polymeric pigment ultimately formed

from 5-S-cysteinyldopa, is predicted to be the predominant product when the

intramelanosomal cysteine concentration is >107 M (77). In the absence of

thiols, dopaquinone cyclizes (Figure 9, step 4) to form leukodopachrome (cy-clodopa), which is readily oxidized nonenzymatically by excess dopaquinone to

form dopachrome and L-DOPA (Figure 9, step 5). Tyrosinase catalyzes the ox-

idative recycling of L-DOPA to dopaquinone (Figure 9, step 6). Dopachrome

tautomerizes spontaneously at a moderate rate to form dihydroxyindole-2-

carboxylic acid (DHICA) (Figure 9, step 7) or partially decarboxylates to form

dihydroxyindole (DHI) (Figure 9, step 8). Alternatively, in the presence of

dopachrome tautomerase (TRP-2/DCT), dopachrome derived from dopaquinone

is converted to DHICA more rapidly, and without decarboxylation. Polymeriza-

tion of DHI generates a dark colored, high molecular weight, insoluble pig-

ment, termed DHI-melanin (Figure 9, step 9). Polymerization of DHICA yields

a lighter colored, lower molecular weight pigment, called DHICA-melanin

(Figure 9, step 10), which is slightly more soluble than DHI-melanin. DHI-

melanin and DHICA-melanin are collectively termed eumelanins. In mice, an-

other enzyme, TRP-1, catalyzes the further oxidation of DHI and DHICA cate-

chols to their respective o-quinone forms (Figure 9, steps 1112). The absence

of this activity confers a brown color to eumelanin pigments. This function of

TRP-1 is absent in humans, where DHI and DHICA oxidation can be catalyzed

by human tyrosinase instead (78).

In summary, essentially no melanin pigment is formed within human

melanosomes in the absence of active tyrosinase (oculocutaneous albinism

types 14). Pheomelanin synthesis, conferring red or yellow color, is favored

when cysteine is readily available. Active TRP-2 accelerates dopachrome


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tautomerization, thereby increasing the efficiency of eumelanin production and

the generation of darker pigments. Tyrosinase (or TRP-1 in mice) catalyzes fur-

ther oxidation of eumelanin precursors to indolequinones, yielding the darkest

black pigments. Although a mixture of pigments ranging from yellow (blonde)

to jet black can be produced by human cutaneous melanocytes, the presence of

pheomelanin may be visually masked by the darker eumelanins. Pheomelanin

pigments are visually dominant when TRP-2 levels are lower, due to diminished

flow of dopaquinone through dopachrome to eumelanins.

Other factors are required for proper melanogenesis and/or melanosome

development in addition to expression of the three melanogenic enzymes

tyrosinase, TRP-1, and TRP-2 and the intracellular vesicular traffickingsystems described above. Basrur et al. (79) used a combination of SDS gel

electrophoresis, trypsin digestion, and tandem mass spectrometric analysis of

peptides separated by liquid chromatography to identify 68 proteins associated

with Stage III melanosomes obtained from cultured melanocytes. These pro-

teins included 6 novel melanosomal proteins, 56 proteins shared with other

organelles, and the 6 melanosomal proteins known previously. The majority

of proteins identified possess functions consistent with the overall nature of

melanosomes and melanin synthesis. On the other hand, some factors known

to play critical roles in melanogenesis, such as the OCA-2 protein, were not

detected in melanosomes, supporting the view that the majority of P-proteinis located within the ER where it functions in tyrosinase export. Both tyro-

sine and cysteine are necessary substrates for melanogenesis and saturable

melanosomal transporter systems for both amino acids have been demonstrated

(56, 8083). The identities of the protein components of the melanosomal tyro-

sine and cysteine import systems have not yet been determined genetically or

biochemically. The nature of these amino acid transport systems, particularly

with regards to their selectivity for substrates and inhibitors, could be relevant

to mechanisms of chemically-induced depigmenting disorders.

Contact between cutaneous keratinocytes and melanocytes affects the ratio

of pheomelanin to eumelanin. Using reconstructed human epidermis systems

and melanocyte/keratinocyte co-culture experiments in vitro, Duvalet al.(84)

showed that synthesis of eumelanin increases concomitantly with decreased

pheomelanogenesis under conditions favoring keratinocyte/melanocyte contact.

The majority of Caucasians with red hair are hetero- or homozygous for variant

alleles of the melanocortin-1 receptor (MC1-R), the receptor for -melanocyte

stimulating hormone (-MSH) (48). Normally,-MSH binding by MC1-R stim-

ulates adenylate cyclase which increases cAMP levels, and among its other

physiological effects, stimulates the expression of TRP-2 expression and eume-

lanin synthesis. Common MC1-R variations occurring in humans account for

either decreased affinity for -MSH or diminished coupling to adenylate cy-

clase (85). Interestingly, the anesthetic requirement is increased in redheads

homozygous for variant MC1-R alleles and compound heterozygotes, perhaps


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122 W. H. Tolleson

due to depressed cAMP levels (85, 86). The human agouti signaling protein

acts as an endogenous MC1-R antagonist that blocks MC1-R dependent signal-

ing and suppresses eumelanin synthesis (87). A genetic polymorphism for the

agouti signaling protein was found to be more common among West Africans,

African-Americans, and East Asians than among American Caucasians, and

was associated with dark hair and brown eyes, but not melanoma (88, 89).

PROPERTIES OF MELANINS AND MELANOSOMES

Human melanocytes from very darkly pigmented tissues, such as the choroid,

are enriched with ellipsoidal melanosomes, approximately 0.9 0.3m, con-taining eumelanins as the predominant pigment. Melanosomes of this type,

sometimes called eumelanosomes, exhibit the classic stages IIV of melanosome

development and the characteristic well-ordered structural features described

in the previous section. Pheomelanosomes are spherical melanosomes (0.7m

diameter) and exhibit pheomelanin as their primary pigment; these organelles

are more abundant within the melanocytes of red or blonde hair (48). The fin-

gerprint pattern of regularly spaced internal fibers found in Stage II eume-

lanosomes is replaced with an irregular vesiculoglobular matrix in developing

pheomelanosomes (18).

A variety of model systems have been utilized to study the fine structureof melanosomes: (a) native melanosomes examined in situ within fixed human

tissue specimens, (b) intact melanosomes isolated from marine invertebrates

or human melanocytic tissues, and (c) synthetic melanin polymers formed in

vitro. Particular advantages and limitations are associated with these model

systems so a complete view of melanosome ultrastructure is gained by consid-

ering studies of each of them.

Human melanosomes embedded in melanocytic tissues studied using elec-

tron microscopy provide standard reference information regarding their na-

tive structure. Electron microscopes adapted to perform electron energy loss

spectroscopy (EELS) allow elemental analyses of ultrastructural features.

Muller and Bereiter-Hahn (90) demonstrated calcium accumulation within

melanosomes of dermal melanocytes in Xenopus laevis using EELS. Nagai

and coworkers (91) applied EELS to oral melanosis and melanoma to com-

pare relative sulfur levels within eumelanosomes and pheomelanosomes in

situ. Secondary ion mass spectrometry (SIMS) has been used to image sulfur

distribution in hair shaft cross sections (0.20.5 m probe beams) (92) and to

detect iodobenzamide, a melanoma tracer compound, within the melanosomes

(0.05m resolution) of metastatic B16 melanomas colonizing lung tissue (93).

Hallegot and coworkers (94) applied in-situ imaging mass spectrometry, a

multi-ion technique related to SIMS, to study cryosections of human hair. By

imaging the distribution of 16O, 12C 14N, 32S, and 34S ions simultaneously across

the hair shaft, the chemical composition of individual melanin granules could


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be determined. Melanin composition contrasted with that of other architec-

tural features of the hair shaft, including the medulla, cortex, endocuticle, and

exocuticle. Analytical techniques such as these should continue to provide in-

formation regarding the chemical environment of melanocyte substructures

within melanocytic tissues.

Eumelanosomes can be isolated from cultured melanocytic cells or the ink

sacs of cuttlefish (Sepia officinalis) using relatively mild techniques, minimizing

the potential for damage to delicate ultrastructural features. Although eume-

lanin isolated from Sepia ink sacs is chemically similar to that produced by

human melanocytes, Sepia eumelanosomes are spherical and smaller (0.2 m

diameter) than ellipsoidal human eumelanosomes (0.9 0.3 m). The basisfor the morphological differences in human and Sepia eumelanosomes is not

known, although differences in metal ion and protein composition are appar-

ent. However, both human and Sepia mature eumelanosomes contain similar

internal ultrastructural features to be described later.

Pigmented hair contains abundant melanin granules, and human eume-

lanosomes have been extracted from hair using acid, base, and/or peroxide

treatments. The chemical composition of human melanin has been studied

using these preparations, but recent studies showed that bound metal ions

were altered by the aggressive chemical treatments necessary to disrupt the

hair matrix. Novellino and coworkers (95) discovered that appropriate deter-gents and exhaustive proteolysis allowed the recovery of undamaged eume-

lanosomes from bovine irides and human hair. Adapting this approach allowed

intact eumelanosomes and pheomelanosomes to be isolated from black or red

human hair samples, respectively, facilitating chemical analyses and detailed

ultrastructural analyses using electron microscopy and atomic force microscopy

(Figure 11, Table 2) (96).

Figure 11: Atomic force microscopic images of melanosomes isolated by proteolyticmethod from human hair. (A) Eumelanosomes isolated from dark hair, (B)pheomelanosomes isolated from red hair. Atomic force microscopy, tapping mode, bars200 nm. (by permission, Liu et al. Photochem Photobiol. 2005;17(3):262269 [96])


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124 W. H. Tolleson

Table 2: Composition of purified melanosomesa.

Chemical analyses

Eumelanosomes Pheomelanosomes Eumelanosomes(dark human hair)b,c (red human hair)b (Sepia ink sac)

Amino acid (w/w%)d 14.115.3 43.8 5.1Total melanin/mge 58006240 1430 10810PTCA (ng/mg) f 27002900 750 166004-AHP (ng/mg)g


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Atomic force microscopy of eumelanosomes isolated from Sepia and darkly

pigmented human hair reveal three categories of fine structural features: (a) fil-

aments approximately 4.5 nm diameter and variable lengths, (b) smaller par-

ticles 415 nm high with diameters 20 nm, and (c) larger roughly spherical

particles approximately 150 nm in diameter (Figure 12) (96, 105). The melanin

filament category was detected in native melanosome preparations but could

also be recovered by ultrafiltration from the 10003000 Da fraction of son-

icated Sepia eumelanosomes. The 10003000 Da fraction correlates with the

estimated size of melanin protomolecule aggregates (2250 Da). Thus, the 4.5 nm

diameter filaments detected by atomic force microscopy, which are somewhat

larger than the pores of the ultrafiltration membranes used (13 nm), likelyrepresent reassembled structures, reminiscent of 6.6 nm diameter filaments

formed from synthetic melanin in the presence of copper ions (104). The physi-

cal integrity of the larger 150 nm melanosome particles was probed using atomic

force microscopy. These particles appeared to be composed of smaller substruc-

tures that correlated roughly with the dimensions of the 20 nm particles and fil-

aments. Thus, the current model of eumelanin structure proposes that melanin

protomolecule aggregates are arranged to form the ultrastructural features de-

tected in intact eumelanosomes, and that the smaller ultrastructural features

may represent components of larger 150 nm melanin particles.

The biophysical mechanisms controlling the deposition and organization ofnascent melanin polymers to form the distinctive features of human pheo- and

eumelanosomes are not completely understood. It is clear that melanosomal

proteins and metal ions influence melanin polymerization and organization.

The spectrum of functional groups provided by melanin monomers includes

potential hydrogen bond donors and acceptors, hard and soft ligands for

metal binding, aromatic groups capable of -stacking, and reactive centers

for Michael-type addition reactions. Sharma and coworkers (106) showed that

melanosomal proteins affect melanogenesis by facilitating or inhibiting poly-

merization of melanin precursors and the binding of melanin polymers to

melanosomal proteins. Chakraborty et al. (107) and Lee et al. (108) showed

that polymerization of 5,6-dihydroxyindole-2-carboxylic acid was facilitated by

the Pmel 17 melanosomal scaffolding protein in a superoxide-dependent man-

ner. Amino acid analysis of human eu- and pheomelanosomes isolated from

hair revealed that arginine residues were the most abundant, suggesting that

this residue could play a special role in melanogenesis. It has already been

mentioned that copper ion concentration influences the assembly of synthetic

melanin into sheet-like or rod-like structures (104), and it is well known that

bound metals and protein contribute considerably to the mass of human eu-

and pheomelanosomes (Table 2).

A recent comparison was performed of metals bound to eumelanosomes

obtained from Sepia ink sacs or human eu- and pheomelanosomes isolated

from hair samples using proteolytic enzymes (96). Ca(II) and Mg(II) were the


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126 W. H. Tolleson

Figure 12: Hypothetical model of human melanosome fine structure. Melanin precursors(bottom right) are polymerized to form melanin protomolecules, which associate via-stacking to form 1.5 nm 1.2 nm aggregates. Melanin aggregrates organize to formrods (4.5 nm diameter), 20 nm particles, and 150 nm particles which comprise substructuresdetected in human eumelanosomes using atomic force microscopy. (by permission,Clancy and Simon, Biochemistry, 2001;40(44):1335313360).


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most abundant bound ions in each case. Melanosomes from human hair con-

tained higher levels of the transition metals Fe(III), Cu(II), and Zn(II), with

particularly higher levels of bound Fe(III) present in pheomelanosomes. The

ion exchange properties and metal binding capacity of eumelanin was studied

using Sepia melanosomes washed extensively with EDTA (109). Cu(II) and

Fe(III) ions were able to bind EDTA-stripped melanosomes under acidic condi-

tions, presumably to hydroxyl or amine functional groups. Mg(II), Ca(II), and

Zn(II) binding occurred above the pKa for carboxyl groups, implicating DHICA

as the primary ligand for these metals. Fe(III) and Cu(II) binding to EDTA-

washed melanosomes reached saturation at 1.2 and 1.1 mmol/g, representing

maximum metal binding capacities of 6.7 and 7.0% by mass, respectively. As-suming a DHICA:DHI ratio of 1:1.12 for Sepia melanosomes and combining the

parameters from Table 2, yields the rough estimation that up to 20% of avail-

able melanin monomers can be recruited for metal binding, as described by

Potts and coworkers (110). This deduction reveals that the structure of Sepia

melanosomes must efficiently allow for metal ion access to potential binding

sites.

Melanins readily bind inorganic metal cations, neutral organic compounds,

and organic cations (111117). Borel et al. (118) showed that line width mea-

surements from high resolution magic angle spinning proton NMR could be

used to determine weak binding constants (Kd 1.818.0 mM) of compoundsto synthetic melanin. The ability of melanin to sequester toxic metal cations

and hazardous organic compounds is believed to provide a protective effect for

melanocytic tissues and impart other biochemical properties. It has been argued

by various investigators that drug binding to melanin is, or is not, relevant to

ocular phototoxicity and to ototoxicity (113, 116, 119121). UV-induced lipid

peroxidation is inhibited by eumelanin (122). However, superoxide-mediated

lipid peroxidation is enhanced when Fe(III)-adenosine diphosphate complexes

are combined with synthetic melanin and incubated with microsomal phospho-

lipids (123). The presence of melanin-bound Cu(II) and Fe(III) ions increases

single stranded breaks in supercoiled plasmid DNA incubated aerobically

either in the dark or with UV-irradiation (109).

MELANOCYTE PHOTOBIOLOGY

Photoprotection via absorption of biologically dangerous solar UV radiation is

the primary function for melanin in the skin and eyes. Solar radiation pen-

etrates exposed tissues in a wavelength-dependent fashion (16, 17, 124, 125)

(Figure 13). The very small amount of UVC (100280 nm) in sunlight that is not

filtered by atmospheric ozone is typically absorbed by the outermost cornefied

layers of the epidermis or cornea. UVB (280320 nm) is far more abundant in

terrestrial sunlight than UVC. UVB is able to reach viable keratinocytes of the

stratum spinosum and penetrates the anterior chamber of the eye where it is


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128 W. H. Tolleson

Figure 13: Tissue penetrating properties of sunlight. (A) skin, (B) eye. (by permission, Tollesonet al. Int. J. Env. Science Pub. Health, 2005;2(1):147155).


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absorbed by the outer layer of the lens. UVA (320400 nm) is also more abundant

in sunlight, penetrates more deeply into the lens, and penetrates the full depth

of the epidermis to the level of the papillary dermis. Visible light (400760 nm)

and IR (76010,000 nm) constitute the majority of solar energy at sea level.

Visible light reaches the ascending loops of the capillary bed in the dermis and

penetrates the eye to be absorbed completely by the retina and retinal pigment

epithelium. IRA (7601400 nm) penetrates the skin completely and reaches the

subcutis. IRA can also penetrate the eye deeply but IRB (1,4003,000 nm) and

IRC (3,00010,000 nm) are absorbed by water and do not penetrate beyond the

superficial layers of the cornea. Acute overexposure of unprotected skin or eyes

to visible or IR radiation via lasers is typically associated with thermal damage,although photodynamic or photo-thermal effects can result in the presence of

photosensitizing agents (126, 127).

The absorption spectrum of eumelanin isolated from human hair reveals

that it absorbs the most hazardous UV components of solar radiation very

efficiently. Furthermore, melanin is recognized as an efficient scavenger of su-

peroxide radical anions, hydroxyl radicals, and singlet oxygen generated un-

der conditions of photooxidative stress (32). However, reactive oxygen species

are released when synthetic melanin or melanosomes isolated from Sepia

or human hair containing normal levels of bound transition metals are ex-

posed to UV in the presence of oxygen (128, 129). Dontsov and coworkers(130) detected increased lipid peroxidation when melanosomes isolated from

the retinal pigment epithelium of human eyes were irradiated with an argon

laser. Addition of synthetic melanin increased the UVA-induced formation of

8-hydroxy-2-deoxyguanosine in calf thymus DNA (131). Interestingly, the ab-

sorption spectrum of the lower molecular weight fraction (1,000 Da), matches the action spectrum

for this effect (129, 132). Lower molecular weight melanin fractions isolated

from melanosomes have a tendency to reassemble spontaneously over time.

Incubation of the low molecular weight melanin fraction (


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130 W. H. Tolleson

for blonde hair lightening that implicates UV-induced melanosome structural

damage, perhaps via attack of reactive oxygen species on melanosomal mem-

brane components. Landet al. (135) and Pilaset al. (136) reported that UV irra-

diation of DOPA generated dopa semiquinone, dopaquinone, and dopachrome,

but UV irradiation of 5-cysteinyldopa led to S-CH2 photohomolysis in which

carbon centered alanyl radicals and aryl thiyl radicals were formed. Koch and

Chedekel (137) showed that the pheomelanogenic precursor 5-S-cysteinyldopa

was approximately 10-fold more susceptible to UV photolysis than DOPA and

that it promoted more photoinitiated DNA single strand breaks and photomod-

ification of calf thymus DNA. These results show that the pheomelanogenic

precursor 5-S-cysteinyldopa is particularly labile to UV and is more effectivein promoting UV-induced DNA damage than the corresponding eumelanogenic

precursor, DOPA.

UV radiation participates in a mixture of beneficial and deleterious

photoreactions in the skin. UV is required for synthesis of cholecalciferol, a

precursor for the active form of vitamin D3,1,25-dihydrocholecalciferol. Pho-

tooxidation by UVA or visible light converts the catechol melanin constituents

DHI and DHICA into darker colored 5,6-indole quinone and 5,6-indole quinone-

2-carboxylic acid components. This constitutes the mechanism for the imme-

diate tanning response, a transient increase in pigmentation that occurs im-

mediately in people who tan normally when reexposed to sunlight. Unlike thedelayed tanning response, the effects of the immediate tanning response begin

to fade when photoexposure stops. UVB is more effective than UVA in stimu-

lating the delayed tanning response, in which synthesis of additional melanin

and melanosomes provides a longer lasting pigmented phenotype.

The UV component of sunlight exerts a suppressive effect on the im-

mune system. Suppression of immune survellience is believed to be relevant to

development of melanoma and nonmelanoma skin cancers in humans (138).

UV-treated humans (139) and laboratory animals (140, 141) exhibit local and

systemic immune suppression, which can be quantified using contact hy-

persensitivity and delayed hypersensitivity assays. Chemical mediators for

photoimmune suppression include: photoinduced formation of pyrimidine

dimers in DNA, photoisomerization oftrans- to cis-urocanic acid (UCA), and

photogeneration of free radicals and lipid peroxidation products (138). Strate-

gies that scavenge these mediators also block UV-induced suppression of the

immune system. Damian et al. (142) showed that broad-spectrum sunscreens

blocking both UVA and UVB were required to prevent photoimmune suppres-

sion in human volunteers.

Immune survellience of the epidermis begins with dendritic Langerhans

cells that patrol the epidermis, phagocytizing antigenic materials, and migrat-

ing to local draining lymph nodes to present antigens to T-cells (143). UV-

treatment blocks the ability of Langerhans cells to present antigens to the

immune system, thus thwarting its ability to mount a contact hypersensitivity

reaction (144, 145). Murine Langerhans cells previously exposed to antigens


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can be isolated from lymph nodes and injected into recipient mice to provide

passive immunity (dendritic cell vaccine). UV-treatment of isolated Langerhans

cells prior to injection destroys the transferred passive immunity they would

have provided otherwise (146). However, if UV-induced pyrimidine dimers in

the isolated Langerhans cells are first repaired using the T4N5 bacteriophage

excision repair enzyme delivered via artificial liposomes, the effectiveness of

the transplanted cells to provide passive immunity is restored (147).

Histidase (histidine ammonia lyase) activity in the stratum corneum of the

skin converts L-histidine to trans-urocanic acid that can accumulate in large

amounts due to the lack of urocanase (148). UV stimulates isomerization of

trans-UCA to cis-UCA, which inhibits contact hypersensitivity and delayed typehypersensitivity (149, 150). Sleijffers et al. (151) correlated decreased specific

cellular immunity against hepatitis B surface antigen with increased cutaneous

cis-UCA levels in human volunteers exposed to five daily UVB treatments fol-

lowed by a standard two-dose hepatitis B vaccination protocol.

The hypothesis that photogeneration of radicals and lipid peroxides

contributes to immune suppression stemmed from the observation that antiox-

idants blocked UV-induced suppression of contact hypersensitivity and immune

tolerance (152, 153). Ullrich (138) proposes that cutaneous UV treatments

initiate a signaling cascade that begins with generation of platelet activating

factor signaling (PAF) and PAF-like molecules derived from lipid peroxidationof membrane lipids (154). PAF and PAF-like molecules stimulate PAF receptors

on adjacent cells, which then amplify the signal by activating phospholipase

A2 to generate additional PAF. Activation of the PAF receptor also stimulates

transcription of the COX-2 gene (155). Induction of COX-2 leads to increased

prostaglandin E2levels and increased transcription of IL-10, which suppresses

the immune system (156). In fact, injecting mice with PAF-like molecules

generated by UV-treating phosphatidylcholine induced immune suppression

that could be blocked by prior injections with PAF receptor antagonists (157).

The melaninization of skin blocks UV-dependent accumulation of pyrimi-

dine dimers,cis-UCA, and PAF-like molecules that mediate photoimmunosup-

pression. Melanin granules transferred from epidermal melanocytes to neigh-

boring keratinocytes should inhibit generation of PAF-like molecules primarily

via absorption of UV and secondarily via scavenging reactive oxygen species.

Conversely, UV exposure of unpigmented skin impairs the ability of the immune

system to mount cell-mediated defenses against potential tumor antigens such

as those related to melanoma.

MELANOCYTE PATHOLOGIESGenetic factors play a major role in many melanocyte pathologies, but pho-

toexposures, hazardous chemicals, and drugs also contribute. Melanocyte-

specific traits, such as the development of melanosomes and melanogenesis,


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132 W. H. Tolleson

Table 3: Representative melanocyte disorders.

MelanocyteRelevant functions

Disease Characteristics melanocytes affected

Vitiligo andalbinism

Depigmentingdisease; variouscauses

All melanocytes All functions

Vogt-Koyanagi-Haradadisease

Inflammatory diseaseinvolvingmelanocytes of themeninges, eyes,inner ear, and skin

All melanocytes Vision, hearing,photoprotection

Drug-induced

ototoxicity

Tinnitus, hearing loss Intermediate

cells of thestria vascularis

Hearing

Microphthalmia Inactivation ofmicrophthalmiagene; absence ofuveal tract; failure ofthe eye to develop

Uvealmelanocytes

Vision,organogenesis

Tietz albinism-deafnesssyndrome

Mutation ofmicrophthalmiagene; congenitaldeafness, mildalbinism

Intermediatecells of thestria vascularis

Hearing,photoprotection

Uvealmelanoma

Intraocular cancer ofthe eye

Uvealmelanocytes

Vision

Cutaneousmelanoma

Melanoma of the skin Cutaneousmelanocytes

Congenitalnevussyndrome

Abnormally largecongenital moles

Cutaneousmelanocytes

provide unique vulnerabilities for pigment-producing cells. In the cases of con-

tact/occupational vitiligo and drug-induced uveitis, exposure to chemicals or

drugs can induce syndromes similar to those typically associated with genetic

or autoimmune dysfunctions (45, 158). Representative pathologies involving

human melanocytic tissues include heritable or induced defects of pigmenta-tion, developmental abnormalities, melanocyte-specific immune disorders, sen-

sitivities to chemicals or drugs, impaired vision or hearing, and melanomas

(Table 3). Although the biochemical mechanisms have not been well character-

ized in some circumstances, melanocytes can provide organ-specific functions

unrelated to their obvious role in melanin biosynthesis; impaired organ func-

tion is associated with abnormal or absent melanocytes in these cases.

Melanocytosis

Disorders that perturb melanocyte functions not only lead to primary syn-

dromes or diseases but may induce additional sequelae in some cases. For ex-

ample, oculodermal melanocytosis, also called nevus of Ota, is a proliferation


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of melanocytes within the facial dermis and mucosa innervated by the first and

second branches of the trigeminal nerve. Nevus of Ota occurs more frequently in

women, who represent 80% of cases, and is more common among Asians, where

it accounts for 0.20.8% of dermatology visits (159161). Nevus of Ota and ne-

vus of Ito, a related syndrome that affects parts of the body other than the face,

are characterized by a blue-grey or blue-black pigmentation and melanocyte

proliferation deep within the dermis. The unusual persistence of melanocytes

within the dermis and exclusion from their normal destinations within the

intrafollicular epidermis of the face is suggestive that failure of melanoblast

precursors to complete the final phase their migratory journey from the neu-

ral crest during embryogenesis may be part of the etiology of oculodermalmelanocytosis. Approximately 10% of patients with nevus of Ota develop glau-

coma due to proliferation of melanocytes within the anterior chamber of the

eye (162). Moreover, nevus of Ota is also associated with an increased risk

for melanoma of the skin, eye, and brain, particularly when it occurs in Cau-

casians (163, 164), or it may lead to tinnitus or deafness in a few individuals

(164, 165).

Immunity, Autoimmunity, and Melanocytes

A recent review by Spritz and coauthors (49) noted that human disor-ders of depigmentation can be grouped into diseases of melanocyte develop-

ment (piebaldism and Waardenburg syndrome), function (oculocutaneous al-

binism types 14, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome,

and Griscelli syndrome), or survival (vitiligo). The absence of melanocytes may

impact adversely the neighboring cells within their microenvironment through

diminished availability of factors required for tissue growth, maturation, or

maintenance. For example, melanocytes are required during embryonic organo-

genesis of the eyes and inner ears and are important postnatally for organ

function and maintenance. The relationship between melanocyte survival and

organ function is illustrated in the case of autoimmune disorders targeting

melanocytes of the inner ear in which tinnitus and/or loss of hearing can

result. In particular, Vogt-Koyanagi-Harada syndrome and sympathetic oph-

thalmia, a closely related syndrome, are inflammatory disorders that target

the four melanocytic organs described in this review (skin, eyes, inner ears,

and meninges). Specific autoimmunity against melanocytes and melanocyte

antigens is associated with Vogt-Koyanagi-Harada syndrome (166169), sym-

pathetic ophthalmia, (170) and vitiligo (171). Vogt-Koyanagi-Harada syndrome

and sympathetic ophthalmia are grouped among other uveo-meningeal syn-

dromes including Behcet disease, Wegeners granulomatosis, sarcoidosis, and

acute posterior multifocal placoid pigment epithliopathy (167, 172); the latter of

these are inflammatory autoimmune disorders for which melanocyte specificity

has not been associated, although clinical effects are evident within melanocytic


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134 W. H. Tolleson

organs. Likewise, Cogan syndrome and autoimmune inner ear disease,

disorders that cause sensorineural hearing loss, have not been associated with

melanocyte immunospecificity; however, no serologic or immunologic tests pro-

vide sufficient diagnostic accuracy for these disorders and antigen specificity

is unclear (173). Although the etiologies of these syndromes have not been es-

tablished definitively, the effects produced in at least some cases of vitiligo,

Vogt-Koyanagi-Harada syndrome, and sympathetic ophthalmia are consistent

with systemic, depigmenting immune reactions attacking melanocytes in the

skin (vitilgo, poliosis, alopecia), eyes (photophobia, uveitis, retinal detachment),

inner ear (tinnitus, dysacusis, vertigo), and meninges (headache, stiff neck,

nausea) (167).Interestingly, an accessory role for melanocytes in immune defense has been

established. This raises the question: do normal or transformed melanocytes

participate in presentation of melanoma tumor-specific antigens to the immune

system? Cultured mouse cutaneous melanocytes exhibit traits normally asso-

ciated with components of the immune system, including: phagocytic capac-

ity (174), expression of MHC I and II and intracellular adhesion molecule-1

(175, 176), antigen-presentation to immune cells (177), and stimulation of T-

lymphocyte proliferation (178). The ability of melanocytes to act as professional

antigen-presenting cells provides another paradigm relevant to both autoim-

munity and cancer. Auto-antibodies reactive against the melanocyte-specificantigens tyrosinase, Pmel 17, TRP-1, and TRP-2 have been detected in the

serum of some vitiligo patients, and cytotoxic T-lymphocytes reactive against

the melanocyte antigen melan A are present in peripheral blood samples from

some vitiligo patients (171). Peptide-based and dendritic cell-based immuniza-

tion strategies have been developed for melanoma therapy exploiting these

melanocyte specific traits (179, 180). Krone and coworkers (181) present an

intriguing hypothesis that immunization with Bacille Calmette-Guerin or vac-

cinia vaccines, or prior infection by certain pathogens, provides cross-reacting

immune protection against melanoma due to peptide sequence homologies with

the melanoma antigen HERV-K-MEL, derived from theenvgene of the HERV-

K human endogeneous retrovirus. The HERV-K-MEL antigen is not detectable

in normal skin or eyes but is expressed by cutaneous and ocular melanomas,

presented by HLA-2 molecules, and recognized by cytotoxic T-lymphocytes

from melanoma patients (182). A similar phenomenon is evident in murine

melanomas which, unlike normal mouse melanocytes, express the p15e env

antigen encoded by an endogenous retrovirus (183) recognized by mouse cyto-

toxic lymphocytes (184). Immunization against this antigen provides protection

against melanoma (185). Thus, the expression of antigens such as HERV-K-

MEL by transformed or damaged human melanocytes marks these cells for

elimination by the immune system, potentially providing a survival advantage

for the organism.


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Vitiligo

Disorders of pigmented cells are prevalent in humans. Cutaneous and oc-

ular melanocytes are more numerous in the body and, due to their anatomic

placement, experience greater exposure to chemical and physical hazards than

the more highly-shielded melanocytes located within the inner ear or the

meninges. Thus, disorders of cutaneous and ocular melanocytes are more com-

mon and are more easily detected. Nevertheless, rare disorders derived from

extracutaneous and extraocular melanocytes also occur in humans, including

neurocutaneous melanosis (186) and melanomas of the cerebellopontine angle

(187).

Approximately 32 million individuals globally are affected by vitiligo, com-prising 0.5% of the world population (188). Vitiligo, a depigmentation disorder

that is particularly noticeable in the skin and eyes, can lead to increased photo-

sensitivity and night blindness due to depletion of ocular melanocytes. Active

cases of vitiligo may progress beyond the skin and eyes and affect melanocytes

in other sites, such as the inner ear (189191) and leptomeninges (31). High

frequency hearing defects are more common among cases of active vitiligo than

among healthy subjects, particularly among males (192). In a review of vitiligo,

Njoo and Westerfof (188) compared six hypotheses of vitiligo pathogenesis:

(1) genetic, (2) autoimmunity, (3) neural, (4) self-destruction, (5) growth fac-

tor defect, and (6) convergence; the latter presumes that combinations of theformer five mechanisms are required for acquisition of vitiligo. Compelling ev-

idence is available in support of each of these theories. Perhaps, as in the case

of carcinogenesis for which a diversity of targets are related to the disease,

no single risk factor accounts for all cases of vitiligo (Table 4). Boissy and

Manga (45) proposed that unspecified genetic factors render melanocytes fragile

and thus susceptible to contact/occupational vitiligo. This model speculates that

vitiligo is induced by chemical agents activated by melanocyte-specific enzymes;

particularly tyrosinase or tyrosinase-related protein-1, to form toxic products

that attack melanocytic cells. In fact, the majority of proven chemical induc-

ers of vitiligo are aromatic or aliphatic phenols and catechols with structural

similarities to melanin precursors or intermediates. Thiol compounds analo-

gous to cysteine, thiol-reactive substances, certain drugs, and other miscella-

neous compounds have been associated with occupational vitiligo (Table 5). In

most cases of chemically-induced depigmentation, a direct association of the

depigmenting agent with melanin or melanin synthesis is apparent or can be

inferred.

Albinism

Oculocutaneous albinism types 14, ocular albinism, Hermansky-Pudlak

syndrome, and Chediak-Higashi syndrome are heritable pigmentation


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136 W. H. Tolleson

Table 4: Vitiligo pathogenesisa.

Proposedmechanism Evidence

Genetic family history for vitiligo in 6.2538% cases vitiligo in monozygotic twins correlations with VIT1, catalase, tenascin, and FOXD mutations various alleles for HLA antigens associated with vitiligo among

different study populationsAutoimmune coincidence with other autoimmune diseases

presence of anti-organ or melanocyte specific antibodies(tyrosinase, TRP-1, or TRP-2)

infiltrating mononuclear cells and T-cells in vitiligo lesions response to corticosteroid treatments

Neural segmental vitiligo confined to body zones definedneurologically

onset of vitiligo associated with periods of emotional stress association with neurologic disorders (viral encephalitis,

multiple sclerosis with Horners syndrome, and peripheralnerve injury)

Self-destruct metabolic precursors of melanin are cytotoxic organic chemicals activated by melanogenic enzymes

induce contact/occupational vitiligo (e.g.,4-(phenylmethoxy)phenol, 4-tert-butylphenol,4-tert-butylcatechol)

association with hyperpigmented skin and sun-exposure association with defective pterin homeostasis, MAO activity,

redox cycling, increased H2O2, and cytotoxic pterinby-products response to UVB+pseudocatalase therapy (pilot study)

Growth factordefect

cultured melanocytes from vitiligo lesions respond to growthfactors derived from fetal lung fibroblasts

melanocytes from repigmenting lesions proliferate in culture response to melagenine, an extract from human placenta

Convergence different clinical forms of vitiligo and different histories vitiligo cases respond to various, unrelated therapeutic modes

aCompiled from reviews by Njoo and Westerhof (Am. J. Clin. Dermatol. 2001;2:167181) andBoissy and Manga (Pigm. Cell Res. 2004;17:208214).

disorders with no apparent association with environmental factors. However,

albinism caused by these genetic traits is associated with additional squelae

relevant to depigmentation disorders, such as vitiligo, for which environmen-

tal causes are sometimes involved. Ophthalmic disorders common among al-

binos include photophobia, reduced visual acuity, astigmatism, nystagmus,

strabismus, misrouting of axons at the optic chiasm, and, occasionally, ambly-

opia (193). Furthermore, the incidence of albinism affects significant number of

individuals globally (Table 6). The incidence of oculocutaneous albinism among

South African Bantu is particularly high, 1:3,800, and is associated with a very

high rate of squamous cell cancer, 504 per 100,000, a rate 15-fold higher than

that estimated for the US, 34 per 100,000 (194, 195).

Ocular albinism type 1 (OA1) is an X-linked recessive trait (Xp22.3) that af-

fects human males in which there is relatively normal pigmentation of the skin


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Table 5: Chemicals associated with contact/occupational vitiligoa.

Phenols:

p-tert-butylphenolp-tert-amylphenolp-octylphenolp-nonylphenolp-phenylphenolhydroquinone (1,4-dihydroxybenzene; 1,4-benzenediol; quinol; p-hydroxyphenol)p-cresol (1,3-methylphenol)monomethyl ether of hydroquinone (p-methoxyphenol, p-hydroxyanisole)monoethyl ether of hydroquinone (p-ethoxyphenol)monobenzyl ether of hydroquinone (4-(phenylmethoxy)phenol,

p-(benzyloxy)phenol)butylated hydroxytoluene (BHT)butylated hydroxyanisole (BHA)Catechols:p-tert-butylcatecholp-isopropylcatecholp-methylcatecholpyrocatechol (1,2-benzenediol)Sulfhydryls:-mercaptoethylamine hydrochloride (cysteamine)N-(2-mercaptoethyl)-dimethylamine hydrochloridesulfanolic acidcystamine dihydrochloride3-mercaptopropylamine hydrochloride

Miscellaneous:MercurialsArsenicCinnamic aldehydeP-PhenylenediamineBenzyl alcoholAzaleic acidCorticosteroidsOptic preparations:Eserine (physostigmine)Diisopropyl fluorophosphate(N,N,N-triethylene-thiophosphoramide)GuanonitrofuracinSystemic medications:ChloroquineFluphenazine (prolixin)aCompiled from Boissy and Manga (Pigm. Cell Res. 2004, 17: 208214).

and hair but reduced or absent fundus pigmentation in the eyes, photophobia,

nystagmus, strabismus, reduced visual acuity, iridal translucency, decussing of

axons at the optic chiasm, and lack of foveal reflex (193, 196, 197). The periph-

eral regions of the retinas often exhibit a characteristic mottled or mud spat-

tered pigmentation pattern in female carriers of the trait (198). The normal

OA1 gene product is a seven helix transmembrane G-protein coupled receptor

melanosomal protein (199). The absence of function OA1 protein inhibits proper

G-protein dependent budding of stage I melanosomes from the ER, resulting in


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138 W. H. Tolleson

Table 6: Albinism.

Human GlobalDisease chromosome Gene product incidence

Oculocutaneous albinism type 1 11q14q21 tyrosinase 1:40,000Oculocutaneous albinism type 2 15q11.2q12 P locus,

pink-eyeddilution

1:15,000

Oculocutaneous albinism type 3 9q23 TRP-1 unknownOculocutaneous albinism type 4 5p MATP,

underwhiteunknown

Ocular albinism 1 Xp22.3 OA1 1:50,000Chediak-Higaski Syndrome 1q42.1q42.2 Lysosomal

protein, LystExtremely

rareHermansky-Pudlak Syndrome type 1 10q23.1 HSP1,

lysosomalprotein

Hermansky-Pudlak Syndrome type 2 5q14.1 HSP2, AP3B1,pearllocus

Hermansky-Pudlak Syndrome type 3 3q24 HSP3,cocoalocus

Rare

Hermansky-Pudlak Syndrome type 4 22q11.2q12.2 HSP4, lightear locus

Opthalmic complications associated with human albinismreduced visual acuity

photophobianystagmusstrabismusdecussing axons of the optic chiasmincreased risk for skin cancers

the formation of the macromelanosomes that are diagnostic for this condition

(197, 200, 201). Cutaneous pigmentation is clinically normal, but reduced com-

pared to unaffected first degree relatives and macromelanosomes are apparent

within cutaneous melanocytes of OA1 patients (198).

Melanoma

The age-adjusted incidence rate for melanoma of the skin is 2.7 per 100,000

globally, accounting for 160,000 cases, but the age-adjusted incidence is much

higher in the Australia and the US (68.0 and 29.3 per 100,000, respectively)

(202). Melanoma of the skin accounts for approximately 4% of new cancers

detected in the US, where the incidence rate increased by 6% per year from

19751981 and continued to increase at 3% per year until the present (195).

Increased risk for cutaneous melanoma does not exhibit a direct relationship

with chronic photoexposure in the same manner as nonmelanoma skin cancers

(Table 7). Epidemiological studies of cutaneous melanomas in humans reveal

that increased risk is related to intermittent over-exposure of unprotected and

unconditioned skin to solar radiation, particularly during adolescence (OR 1.5,


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Table 7: Risk factors for cutaneous melanoma.

Risk factors OR Cl Ref.

Intermittent sun exposure, meta-analysis 1.5 1.21.8 (205)Chronic sun exposure, meta-analysis 1.1 0.91.4 (205)Exposure to arsenic 2.1 1.43.3 (206)Regular swimmers in chlorinated pools 2.2 1.14.6 (211)Exposure to 50 Hz magnetic fields, 0.2 Tesla 2.7 1.45.0 (209)Occupation in metal industry 2.6 1.07.1 (212)Occupation in electronics 2.0 0.66.6 (212)Common nevi (101120), meta-analysis 6.9 4.610.3 (215)Atypical nevi, meta-analysis 6.4 3.810.3 (215)Red hair, data pooled from 8 studies 2.4 1.93.0 (250)

Blonde hair, data pooled from 8 studies 1.8 1.52.2 (250)Light brown hair, data pooled from 8 studies 1.5 1.31.7 (250)Freckling, data pooled from 6 studies 2.3 2.02.5 (250)Family history of melanoma, data pooled from 8 studies 2.2 1.82.9 (251)Blue eyes, adolescents 4.5 1.513.6 (252)Inability to tan, adolescents 4.7 0.924.6 (252)

95% Cl 1.21.8; OR 1.1, 95% Cl 0.91.4 for intermittent or chronic sun exposure,

respectively, from meta-analysis of 19 case control studies) (203205).

Exposure to industrial and environmental carcinogens have not been identi-

fied as important risk factors for cutaneous melanoma, with the exception of tworeports associating exposure to arsenic with cutaneous melanoma (206, 207) A

study by Guoet al.(208) evaluated skin cancer cases recorded in the National

Cancer Registry (Taiwan, ROC) reported an association between exposure to

arsenic and nonmelanoma skin cancers, but failed to detect an association with

cutaneous melanoma. A recent study by Beane-Freeman et al.(206) found ele-

vated arsenic levels detected in toenail clippings (0.084g/g) from cutaneous

melanoma cases reported to the lowa Cancer Registry compared with those from

matched control colorectal cancer patients (OR 2.1, 95% Cl 1.43.3). Kennedy

et al. (207) also detected an association between arsenic exposure (ever vs.

never) among a Dutch population and increased risk for cutaneous melanoma

(OR 7.1), although the relatively wide confidence interval renders that conclu-

sion more equivocal (95% Cl 1.145.5). An association between exposure to elec-

tromagnetic fields and cutaneous melanoma is more controversial. Although

residential exposure to 0.2 Tesla was found to increase risk for cutaneous

melanoma in a Norwegian population study (OR 2.7, 95% Cl 1.45.0), a lack of

association was found in an earlier Finnish cohort study (OR 1.1, 95% Cl 0.9

1.2) (209, 210). Inconclusive or equivocal associations with risk for cutaneous

melanoma have also been proposed for exposure to chlorinated swimming pools

and occupations in electronics and the metal industry (211, 212)

Phenotypic risk factors contributing to cutaneous melanoma include pres-

ence of dysplastic or abundant nevi and freckling (213215). It has been es-

timated that an affected first or second degree relative can be identified for


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140 W. H. Tolleson

approximately 10% of melanoma cases. This observation suggests a role for her-

itable risk factors in melanoma. Hayward (216) identified two high penetrance

genes associated with increased risk for cutaneous melanoma: CDKN2A tumor

suppressor locus (9p21) and CDK4 (12q14) and described four other low pene-

trance melanoma susceptibility genes: MC1R (16q24.3), EGF (4q25), GST M1

(1p13.3), and CYP2D6 (22q13.1). The CDKN2A tumor suppressor gene encodes

the overlapping p16ink4a and p14Arf genes, which share exons 2 and 3 but uti-

lize separate promoters and first exons. The p16ink4a tumor suppressor protein

functions in the retinoblastoma signaling pathway through inhibition of cyclin

dependent kinases 4 and 6 (CDK4 and CDK6). CDK4 is activated during G1

phase of the cell cycle through binding cyclin D where it phosphorylates the Rbtumor suppressor protein to allow transcription of S-phase genes. The p14Arf

tumor suppressor influences the p53 signaling pathway by inhibiting MDM2-

dependent ubiquitinylation and degradation of p53. The MC1R protein is the

receptor for-MSH and regulates the expression of melanogenic traits. Numer-

ous MC1R polymorphisms have been identified and the majority of Caucasians

with red hair are homo- or heterozygous for variant alleles (48). Melanocyte

proliferation is stimulated by epidermal growth factor (EGF) and a variant

EGF allele is more common in melanoma cases (P < 0.0001) (216). Many Cau-

casians do not possess a functional glutathione S-transferase GSTM1 gene, but

Kanetskyet al. (217) found that individuals with red or blonde hair were 2.2-fold(95% Cl 1.24.2) more likely than controls to be GSTM1 null and 9.5 fold (95%

Cl 1.273.0) more likely than controls to be null for both GSTM1 and GSTT1.

These authors speculate that these GST polymorphisms could contribute to

increased risk for melanoma among blondes and red heads. The association

between cytochrome P450 2D6 (CYP2D6) polymorphisms and melanoma is not

well supported, with one study of Northern European Caucasians detecting an

association (P = 0.039) and two others failing to detect a clear association with

melanoma (218220).

Ocular melanomas represent two types of tumors, those that arise within

the uveal tract (iris, ciliary body, and choroid) or others that arise within the

conjunctiva, the thin layer of mucous epithelium overlying the surface of the eye

and the inner (posterior) surface of the eyelid (11). The incidence of conjuncti-

val melanomas in the US increased by 5.5% from 1973 through 1999, especially

among men, and currently accounts for 0.046 cases per 100,000 (221). Thus, the

increase in conjunctival melanoma incidence parallels the increased incidence

of cutaneous melanoma; this correlation is presumed by some to reflect com-

monality in etiology related to changing sun exposure behavior over time (11,

221). On the other hand, the incidence rate for uveal melanomas has remained

stable in the US from 1973 to 1997 at 0.430.46 cases per 100,000 (222). The

role of UV and sun exposure in uveal melanoma has been debated, with some

epidemiologic studies demonstrating increased risk with increased photoexpo-

sure, but others disputing those results (Table 8) (11, 222228). However, it is


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Table 8: Risk factors for uveal melanoma.

Risk factors OR Cl Ref.

Atypical cutaneous nevi 7.3 2.719.4 (253)Occupational exposure to uv (artificial sources - welders) 7.3 2.620.1 (223)Snow blindness, welders burn or sunburn of the eye 7.2 2.520.6 (225)Family history of ocular melanoma 6.9 6.767.4 (224)Chemists, chemical technicians, or chemical engineers 5.9 1.622.7 (254)Nevus of the iris 3.2 1.85.4 (253)Exposure to formaldehyde 2.9 1.27.0 (254)Exposure to asbestos 2.4 1.53.9 (254)Exposure to antifreeze, 2.5 1.06.5 (254)Iris color: green/gray/hazel iris 2.5 1.83.5 (255)

Exposure to carbon tetrachloride 2.3 1.14.7 (254)Exposure to pesticides 2.3 1.34.1 (254)Personal history of cutaneous melanoma 2.4 0.96.6 (224)Iris color: blue/gray iris 1.9 1.32.9 (256)Tendency to sunburn easily 1.8 1.32.5 (225)Personal history of nonmelanoma skin cancer 1.5 1.02.3 (224)

clear that virtually all of the UV and visible light present in sunlight is scat-

tered or absorbed by the anterior structures of the eye (cornea, aqueous humor,

lens, vitreous humor, neural retina, retinal pigment epithelium, and Bruchs

membrane) overlying the posterior choroid, where the majority of uveal

melanomas arise. Thus, if an association between photoexposures and uveal

melanoma is valid, it is highly unlikely to be due to direct absorption of UV

by melanocytes in the posterior choroid. Nevertheless, it can be presumed that

UV photoexposures could facilitate uveal melanoma by an indirect mecha-

nism, such as photoimmunosuppression, which does not require the absorp-

tion of light by the target cells. Hypothetically, longer wavelengths of sunlight,

which have the potential for deeper tissue penetration, could elicit a photo-

dynamic effect within the choroid through interaction with photosensitizing

chromophores. The very high optical density of the choroid would make this

hypothesis untenable except in cases of very lightly pigmented retinas. Inter-estingly, only 26 cases of cutaneous melanomas and no cases of uvea

tolleson jeshc 2005 melanocyte review.pdf

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