Zinc in pigmented cells and structures, interactions and possible roles

Jan Borovanský

2nd Department of Biochemistry, 1st Faculty of Medicine,

Charles University, 128 53 Prague 2, Czech Republic

Reprinted with permission from Dr Milan Spala, Editor-in-Chief of Sbornik lékasky Journal

Zinek v pigmentovych bukách a strukturách-jeho interakce a moné role. Sborn. lék. Vol. 95 (1994) No. 4, p. 309-320.

SUMMARY: Zinc is a feature trace element of pigment cells and tissues. Organelles, in which melanin is synthesized and stored, i.e. melanosomes, represent a zinc reservoir at the subcellular level. In order to understand function of metals in tissues, cells and their constituents, knowledge is needed on metal interactions with intracellular targets. The possible zinc ligands in pigment cells include melanin, metallothionein, melanotransferrin, B700 and related proteins, ferritin, zinc enzymes and low molecular weight ligands. Areas of a special interest in relation of pigment cells and structures to zinc - such as zinc effect on melanogenesis, zinc excretion and buffering by melanosomes, zinc function in free radical processes as well as zinc role in melanomas - have been reviewed. High level of zinc in pigment cells may indicate a physiological defense against the potential danger of oxidative stress.


A large number of natural pigments is associated with metals, namely with iron, copper, manganese or vanadium [7]. Feature trace element of melanoprotein pigments is zinc.


The strikingly high zinc level in pigment tissues was first noticed in pigment structures of eye [17,19,36,58,59,85] and later demonstrated in pigmented normal [45] and tumour tissues [46,58,65]; high level of zinc was demonstrated also in pigmented regions of human brains [29,48].

Experiments with radioactive 65Zn revealed high uptake of zinc into murine tumours - Cloudman S91 melanoma [65], B16 melanoma [10,75] and Harding-Passey melanoma [10]. Newsome and Rothman [63] described the ability of human retinal pigment epithelial cells in vitro to accumulate and retain zinc, later study of the same group verified in vivo that pigment eye tissues of humans and primates took up and retained zinc [62]. Dencker and Tjälve [28] mentioned retention of 65Zn in hair of pigmented C57BL6 mice.


With the development of cell fractination techniques it became obvious that at the subcellular level zinc was deposited especially in melanosomes [41,86,90]. Our comparative studies demonstrated that melanosomes represent unique subcellular storehouses of zinc because the Zn concentration in the isolated organelles exceeded that in the whole original pigment tissue 3-5fold [12,46] - Tab. 1.

Table 1 - Zinc concentration in pigment tissues and in melanosomes isolated from them

bovine uvea

human hair

Harding-Passey mouse melanoma

horse melanoma

human melanoma

Bomirski hamster melanoma (line Ma)

138.4 ± 2.3

158.0 ± 23.2

75.5 ± 1.8

112.0 ± 1.9

181.1 ± 7.5


598.0 ± 4.2

664.0 ± 376.6

383.3 ± 2.2

544.3 ± 4.1

612.1 ± 5.2


The result s are expressed in µ Zn/g dry sample (x ± SD). Compiled from [12, 45, 46]

The initial data derived from colorimetric measurements were later confirmed by modern techniques such as neutron activation analysis [78] or mass spectrometry [92] but there still has persisted a question if the zinc was not absorbed artificially by melanosomes during isolation procedure. Only X-ray microanalysis of melanosomes in situ brought a conclusive evidence for the presence of zinc in trout skin melanosomes [72], in melanosomes of inner ear and uveal tract [60], in retinal and choroidal pig melanosomes [82] and in melanosomes of human retinal pigment epithelium [94]. Only Takaya [91] using X-ray microanalysis found neither zinc nor copper in hair melanosomes.

The presence of zinc was demonstrated also in the pigment extracted by a mild procedure from substantia nigra of human brains [101]. If zinc is the abundant trace element of melanosomes (e.g. its concentration in human hair melanosomes is the highest Zn concentration attained in a structural element of human body), the next question striking mind is where and why it is localized in these organelles.

Zinc-melanin and zinc-protein interactions can be expected to occur in melanosomes. What is the distribution of zinc between melanin and protein moieties of melanosomes has not been clearly defined because only a few studies have addressed the cardinal question of zinc distribution within melanosomes.

Procházková et al. [77] having digested the isolated melanosomes of Harding-Passey mouse melanoma with chymotrypsin separated the proteins electrophoretically on agar and studied by neutron activation analysis the Zn distribution among protein fractions. All the protein fractions displayed the presence of zinc, but a colourless protein band with the highest anodic mobility contained more than a half of the zinc associated with melanosomal proteins.

Zinc pool of melanosomes seems to be quite labile: It was possible to remove all hot Zn by 5 day exchange diffusion against 1mmol/l ZnCl2 from B16 mouse melanoma melanosomes labelled with 65Zn in vivo [10,11]. Treatment with 0.5 mmol/l acetic acid released 100% of radioactive zinc from the melanosomes as well. If the B16 melanosome acetic acid supernatant was passed over a Biogel P-2 column, 55% of 65Zn was eluted in the void volume indicating a bound form of 65Zn, less than 50% of 65Zn was eluted in the salt volume (= free 65Zn) [11]. When the supernatant of SDS-treated B16 mouse melanoma labelled melanosomes was passed over an Ultrogel AcA54 column, 65Zn was eluted in a fraction of a molecular weight in the region 15,00 - 18,00 [11].

There have been also observations suggesting indirectly the importance of non-pigment moieties of melanosomes for zinc binding. To this category falls e.g. a report of Shibata et al.[87] showing that Zn level was higher in premelanosomes than in melanosomes of Green's hamster melanoma.


In order to understand the function of metals in living systems, knowledge is needed on the biochemical basis of metal interactions with intracellular targets. The balance between essentiality and toxicity of metals can be regulated by specific binding sites for metals and hence knowledge concerning intracellular biochemical speciation is of importance.

3.1. Melanin

Melanin behaves as a natural cation exchange material [97] and is therefore able to incorporate various ions both in vitro and in vivo [23]. The analysis of the affinity of synthetic and natural melanins for inorganic ions showed interestingly that zinc was on the lower scale of ionic affinity [74]. Detailed study on binding capacity of metal ions to synthetic dopa melanins demonstrated that two classes of independent binding sites participated in the interactions of cations with dopa-melanin, with association constants for Zn K1=5.87 x 105mol-1, K2=4.85 x 103mol-1 [25].

Situation in vivo is expected to be more complicated: 1) Competition between various metal ions for binding sites on melanin can influence the binding parameters as evidenced by model experiments in vitro [9]. 2) Melanin pigments in melanosomes in vivo are always associated with a protein moiety which can also influence metal ion - melanin interactions. Among various metals only zinc was found in a higher amount in the melanin-human albumin-Zn complexes, unlike Mn, Cu and Fe binding of which decreased in the presence of albumin [3]; recently the binding capacity of melanoprotein isolated from bovine eyes for Zn2+ was found to be by 10 - 20 % lower compared with that of protein-free melanins [2]. The importance of protein in melanin-protein complexes for zinc binding was emphasized already by Bowness and Morton [18] but their results are difficult to interpret due to the usage of phosphate buffers in their experiments.

3.2. Metallothionein

Metallothionein is an important intracellular ligand for zinc and copper as well as for some other transition metals [70].It is believed to be involved in the homeostatic control of Zn absorption, in cellular detoxification, in the control of differentiation and in direct activation of Zn-dependent enzymes [21,31,79].

The metabolic and growth demands of neoplastic tissue may make tumours the predominant site of Zn uptake [10,70,96] which is accompanied by hypozincemia [26,31,70,89]. This is a result of a number of factors, some unrelated to tumour. Hypozincemia has been also recorded in melanoma patients [47]. Further zinc redistribution during tumour-related stress can be induced by a rise in the amount of hepatic metallothionein [70,93]. Some authors suppose [70] that release of Zn2+ from lysing tumour cells may subsequently enable hepatic metallothionen synthesis to proceed.

Quantification of the copper-binding compounds in equine melanoma tumours revealed that as much as 50 - 60 % of total tissue copper was associated with metallothionein whereas tyrosinase and Cu2Zn2-superoxide dismutase accounted for appr. 2% of total copper [56]. The same situation is assumed for human melanoma tissue. Zn binds less strongly than Cu to metallothionein and can, therefore, be readily displaced by Cu [21] but Krauter et al. [56] found equimolar concentrations of zinc and copper in their samples which suggested that metallothionein might be the major protein ligand for zinc in pigment cells.

This would be in accord with the generally accepted concept of metallothionein as an autoregulated intracellular zinc (and copper) buffer [79] establishing intracellular steady state kinetics for Zn and Cu levels. As for pigment cells there have been only rare reports dealing with a specific role of metallothionein in these types of cells: Koropatnick and Pearson [55] studied B16 melanoma cells with low and high metallothionein constitutive expression and concluded that metallothionein was associated with cisplatin resistance. Oliver et al. [66] demonstrated that induction of metallothinein synthesis in human retinal pigment epithelial cells was correlated with an increased capacity for 65Zn uptake into cultured cells.

Zinc bound to metallothionein is released after degradation of the metallothionein protein in lysosomes (unlike the fate of Cu-metallothionein which is different) [79], hence lysosomes may be involved in the accumulation of zinc [84]. If we accept the more and more common opinion that melanosomes are related to lysosomes [88,102], this mechanism would offer an explanation for high Zn level in melanosomes.

3.3. Melanotransferrin

Melanotransferrin, also known as the tumour-associated antigen p97, is a monomeric glycoprotein expressed at high levels in most human melanomas but present in only trace amounts in normal adult tissues [22]. The comparison of the primary structure of p97 with that of other members of the transferrin superfamily revealed a Zn-binding concensus sequence found in metallopeptidases within the N-terminal lobe and in the C-terminal lobe a glutamic acid residue capable of completing a potential thermolysin-like Zn binding site [37]. Thus p97 may have a Zn-binding potential, unique amongst the transferrin superfamily. In contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule [5]. Functional consequences for melanoma cells with high p97 expression in melanoma cells have not so far been investigated.

3.4. B700 and related proteins

B700 protein is the major protein of the murine melanoma cell's melanosomal membrane; it is also present in the membrane of other cytoplasmic organelles as well as in the plasma membrane [44]. There are related proteins in melanomas of other species [39]. It has become obvious that the B700 protein is part of the serum albumin family of proteins [38]. A number of studies underscored the importance of controlling the relative concentrations of Zn and its ligands in Zn transport kinetic research and suggested that varying their concentrations might be a method of regulating the distribution of Zn into specific cells and tissues [8]. Albumin belongs to Zn ligands with physiologically high Zn affinity (circa 107) [1,40]. There has been no information on the B700 affinity for zinc. However, if it maintained the Zn-ligand affinity typical of serum albumin, it would become another hot candidate to explain Zn presence both in melanosomes and pigment cells.

3.5. Ferritin

Ferritin is a "fashionable" molecule because it can be engaged in the deactivation of increased iron load. In the substantia nigra the disbalance between iron and transferrin levels has been suspected from triggering free radical damage in Parkinson's disease [29].

It is less known that ferritin may fulfill also zinc-sequestering and -dispensing tasks. It has been postulated that ferritin may serve as the initial chelator for Zn2+ (and other metal ions) prior to the synthesis of metallothionein is initiated as the second line of defence [76]. No data on the concentration of ferritin in pigment cells have been available, though.

3.6. Zn-enzymes

The magnitude of the stability constants of metal binding proteins varies quite widely and has served to differentiate operationally between two classes, metalloproteins and metal-protein complexes [95] with firm and loose metal binding, respectively. Zinc containing enzymes fall in both groups.

There has been no Zn enzyme described the concentration of which in pigment tissues would be profoundly different from other tissues. It is only possible to mention high a-D-mannosidase expression in melanomas [32], (this enzyme has been suggested as a possible general indicator of Zn status [34]), and early papers emphasizing the importance of carbonic anhydrase to explain high Zn level in eye pigment tissues [36,59].

The marker enzyme of melanogenesis - tyrosinase - belongs to copper-containing proteins. It would be interesting to ascertain whether the recently discovered tyrosinase-related proteins are metalloenzymes and if so, what is their metal dependence.

3.7. Binding of zinc to low-molecular-weight ligands

Metal ion interactions with low-molecular-weight ligands in vivo are extraordinarily difficult to study due to the very low concentrations which are involved and due to the labile nature of most such associations. Our present knowledge about the chemical binding which may, or may not, take place between zinc and low-molecular-weight agents has had to be inferred largely from computer simulations of the equilibria which are thought to dominate the low-molecular-weight fraction of the metal ion [21]. These studies have demonstrated that e.g. in blood binding is clearly dominated by cysteinate with histidine acting as the other important coordinating partner [21,40]. Reduced glutathionate seems likely to supersede cysteinate inside most, if not all cells [21]. The presence of Zn cysteinate was cytochemically confirmed in cat tapetum lucidum rod-shaped paraplasmic inclusions considered by some authors as melanosomes [53]. 1H and 13C NMR studies revealed that Zn2+ binds also with oxidized glutathione in aqueous medium with 1:1 stoichiometry [73]. Taking into account a significant role of glutathione for pigment cell metabolism [6], Zn-glutathione complexes may make the metabolic relations still more complex.

In pigment cells zinc - dopa interactions are also to be expected since L-dopa can bind zinc using its orthophenolic groups [51].

According to the prevailing opinion the small Zn2+-species are involved in processes which exploit their kinetic advantages over the complex formed by proteins. For the most part, these involve transport to or through membranes and exchange between high-molecular-weight species [21] (Fig.1).


25.jpg (6960 bytes)

Fig. 1 - Points of special interest in zinc relation to pigment cells and structures.
B700 = B700 and related proteins, E = zinc enzymes, MF = melanotransferrin,
MT = metallothionein, LML = low-molecular weight-Zn ligands
ZG = zinc gene regulatory proteins.


Physical and chemical properties of zinc, including its coordination flexibility, make it highly adaptable to meeting the needs of proteins and enzymes that carry diverse biological functions and are involved in the metabolism of proteins, nucleic acids, carbohydrates and lipids as well as in the control of gene transcription and other fundamental biological processes such as cell division, differentiation, development, immune phenomena and receptor activity. The advance in knowledge of zinc chemistry and biochemistry in the past two decades has been striking and reached a level that provides predictive capacity for both the physiology and pathology of zinc metabolism. The astoudingly large body of observations and an encyclopedic analysis of the data have been subject of numerous reviews [e.g.4,27,95,98], but surprisingly no attempt to discuss the roles of zinc in melanin-containing structures has been made.

4.1. Participation of Zn2+in melanogenesis

Catalytic function of Zn2+ in the synthesis of 5,6-dihydroxindole derivatives was noticed as early as 1950 [43] and included as a fact in the Raper-Mason scheme of melanogenesis. Observations of Prota and his associates have recently revived attention to the role of zinc in biosynthesis of melanins. They observed that various transition metals including Zn2+ affected markedly the chemical properties of melanin formed by the tyrosinase-catalyzed oxidation of L-dopa by increasing the incorporation of 5,6-dihydroxyindole-2-carboxylic acid into the pigment polymer [67,68]. Zn2+ can thus imitate function of dopachrome oxidoreductase. When acting together the inhibition of 5,6-dihydroxyindole-2-carboxylic acid decarboxylation was greater than that produced by Zn2+ or dopachrome oxidoreductase separately [50]. The suggestion that the presence of carboxylated indole units in natural melanins is due to the intervention in the melanogenesis of metal ions can be accepted. However, the role of Zn2+ namely in this respect appears to be uncertain because the free Zn2+ cation is damaging to biological systems and thus is associated with other molecules as Zn-ligand complex (see the section 3) resulting in a actual free Zn2+ ion concentration that is 10-3 - 10-6 that of the total zinc concentration [8,98]. Whether Zn2+-ligand complexes can influence melanogenesis it has not been tested. Zn2+ ions were shown to inhibit the initial rate-limiting reaction of melanogenesis - tyrosine hydroxylation and thus to have a role in the regulation of melanogenesis [50].

4.2. Excretory function of melanosomes and pigment tissues

Melanin can participate in excretion of some substances under physiological conditions [81]. As hair melanosomes represent rich tissue reservoirs of zinc lost during removal of keratin structures, we tried to quantitate the Zn excretion via hair [12]. The daily Zn loss in man by this way varies around 20 µg which compared to the major Zn2+ portion excreted via pancreatic juice (10 mg/day) and to the output via urine (0.5mg/day) is low. However, if we add also Zn2+ loss by means of epidermal melanosomes, the value will increase.

4.3. Zinc and free radical processes

Since the 1970s it has been anticipated that an essential biochemical function of zinc is to serve as a natural antioxidant [20,99,100]. Two mechanisms of zinc action have been elucidated - the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive oxygen species catalyzed by some transition metals, especially by displaced iron [20,42,100].

On this basis it was predicted that relatively high concentration of zinc might be present in those tissues vulnerable to oxidation such as the hair, skin, eye and spermatozoa. When this was shown to be the case, Willson [100] proposed the following corrolaries: 1 -"in healthy cells, vital molecules are protected from the action of decompartmentalized iron by the presence of zinc"; 2 -"normal cells are designed in such a way that division is not initiated until the zinc concentration at critical sites within the cell is sufficient to protect them from decompartmentalized iron that might normally be present. Zinc thus plays protective and stimulatory role".

The frequent occurence of necroses in melanoma tissue [13] and the presence of H2O2 [24] make the metal driven free radical processes in pigmented tumours probable. Moreover, increased malondialdehyde levels found in the livers of B16 and S91 melanoma-bearing mice [13,71] suggest that the tumours alter host antioxidant defenses. Alteration of iron metabolism and increased levels of lipid peroxidation are characteristic of substantia nigra in Parkinson's disease [30] and the fact that also zinc levels in substantia nigra are markedly increased under these circumstances may indicate a physiological response to oxidative stress [29].

Melanin in melanosomes in pigment cells and tissues represents another source of free radical activity. The melanin polymer has long been known to exhibit stable free radical properties, because of semiquinones, which appear to have a protective action in cells probably by acting as a sink for diffusible free radical species [80]. Data derived from in vitro experiments have indicated that melanins can function as a scavenger of the superoxide anion radical and can protect cellular structures against photochemically induced lipid peroxidation also due to the absorption of light energy [35]. Zn2+ ions were shown to stabilize semiquinone anion radicals in melanin and to increase free radical activity in melanosomes [2,83]. Melanin polymerization is thought to occur by a free radical process in which semiquinones are formed by redox equilibration interactions between melanin precursors which as reactive species are strictly compartmentalized [13,80], and if leaked metabolically detoxified [13].

Evidence documenting that a number of catecholic melanin precursors, including cysteinyldopas and dihydroxyindoles, are photochemically unstable in vitro in the presence of biologically relevant ultraviolet radiation was presented by Koch and Chedekel [52]. Definitive evidence of occurrence of these reactions in vivo is currently unavailable, nevertheless these photochemical processes are expected to have a role in the pathogenesis of various pathological processes. The high level of zinc in epidermal and eye pigment cells may again indicate a physiological defense against the potential danger of oxidative stress.

4.4. Metal ion "buffering" by melanosomes - mobile pool of Zn2+

Melanosomes have been proposed to represent a physiologically important "reservoir" for essential trace elements, a short term storage deposits, which by binding or releasing the metal ions may play a key role in the control of various processes, e.g. in the action of ionic pumps. Such mechanism is believed to be involved in the secretion of endolymphatic fluid in inner ear [60].

According to Pffeifer and Mailloux [69] melanin should be investigated as a storage bank for useful cations such as calcium, potassium, sodium and zinc. The binding of these ions would prevent a disruption in the body's osmotic balance. If the mineral balance was disrupted by dietary or physiological causes, the increased concentration of copper and lead with their greater affinity for melanin would lead to the displacement of more favourable cations - Zn2+ and Ca2+ which may have implication for hypertension and its therapy [69].

Scavenging role in the elimination of metals, when they reach too high levels in the cell, was ascribed to neuromelanin granules [101].

The complexity of zinc intercellular transport can be illustrated by earlier work of O'Rourke et al [64] demonstrating that zinc secreted by the ciliary body is made bioavailable and absorbed by the chorioretinal complex.

However interesting these theories sound, until zinc melanosomal binding sites and their binding parameters are clearly defined, we can hardly ponder upon the importance of these proposals. All we can say is that the melanosome pool of zinc is mobile as evidenced by the zinc release from eye melanosomes in the face of reduced amounts of bioavailable zinc, for example with a deficient diet [82].

4.5. Zinc and melanomas

Inhibition of tumour growth by dietary zinc deficiency appears to be a general effect irrespective of cell type, species or site of growth [49,89,96]. This may be mediated by the direct requirements for zinc for cellular proliferation as well as by indirect effects on immune function and the interaction with other trace elements.

As for melanoma, P51 mouse melanoma cells (derived from B16 melanoma) when grown in zinc-depleted media had longer doubling time and a decreased thymidine uptake [61]. On the contrary it was reported that the addition of zinc and iron tartrate complexes to Eagle's minimal essential medium was sufficient to support the proliferation of B16 melanoma cells in the absence of serum [54]. Altered organ distribution and survival of melanoma cells were observed in the Zn depleted dietary groups of P51 melanoma-bearing mice [61].

Zn2+ concentrations exceeding 10-4 mol/l are generally cytotoxic in vitro [14,15]. It is therefore not surprising that in vitro Zn2+ was shown to inhibit both the anchorage-dependent [14] and anchorage-independent growth [57] of Cloudman S91 melanoma . Attempts to suppress B16 and Cloudman S91 growth by zinc acetate administration in mice were unsuccessful because the necessary Zn2+ levels in vivo were difficult to reach [16]. Preincubation in vitro of cell suspensions with 10-4 mol/l zinc acetate prior to injecting tumour cells inhibited melanoma development in mice [16]. 10-4 mol/l zinc sulphate was shown to decrease the i.v. but not s.c. transplantability of B16 melanoma [33].

Strong homeostatic control of zinc levels [4,27,95] prevents direct therapeutic use of zinc. The increased zinc uptake by melanomas might be rendered suitable for tumour localization with 69mZn [10] and for targetting tumour cells with chemotherapeutic agents since zinc may act as a carrier for pharmacologically active ligands [96].


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This work was supported by Charles University grant No.240. The author is grateful to prof. J. Ducho (Charles University, Prague) and to prof. P.A. Riley (University College, London) for stimulating discussions.