Zinc in pigmented cells and structures, interactions and possible roles
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 . Feature trace element of melanoprotein pigments is zinc.
1. ZINC IN PIGMENT CELLS AND TISSUES
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  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 , B16 melanoma [10,75] and Harding-Passey melanoma . Newsome and Rothman  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 . Dencker and Tjälve  mentioned retention of 65Zn in hair of pigmented C57BL6 mice.
2. ZINC IN MELANOSOMES
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
Harding-Passey mouse 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 inµ 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  or mass spectrometry  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 , in melanosomes of inner ear and uveal tract , in retinal and choroidal pig melanosomes  and in melanosomes of human retinal pigment epithelium . Only Takaya  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 . 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.  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) . 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 .
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. showing that Zn level was higher in premelanosomes than in melanosomes of Green's hamster melanoma.
3. NATURALLY OCCURRING ZINC LIGANDS IN PIGMENT CELLS AND STRUCTURES
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.
Melanin behaves as a natural cation exchange material  and is therefore able to incorporate various ions both in vitro and in vivo . 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 . 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 .
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 . 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 ; 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 . The importance of protein in melanin-protein complexes for zinc binding was emphasized already by Bowness and Morton  but their results are difficult to interpret due to the usage of phosphate buffers in their experiments.
Metallothionein is an important intracellular ligand for zinc and copper as well as for some other transition metals .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 . Further zinc redistribution during tumour-related stress can be induced by a rise in the amount of hepatic metallothionein [70,93]. Some authors suppose  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 . 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  but Krauter et al.  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  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  studied B16 melanoma cells with low and high metallothionein constitutive expression and concluded that metallothionein was associated with cisplatin resistance. Oliver et al.  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) , hence lysosomes may be involved in the accumulation of zinc . 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.
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 . 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 . 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 . 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 . There are related proteins in melanomas of other species . It has become obvious that the B700 protein is part of the serum albumin family of proteins . 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 . 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.
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 .
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 . No data on the concentration of ferritin in pigment cells have been available, though.
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  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 higha-D-mannosidase expression in melanomas , (this enzyme has been suggested as a possible general indicator of Zn status ), 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 . 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 . The presence of Zn cysteinate was cytochemically confirmed in cat tapetum lucidum rod-shaped paraplasmic inclusions considered by some authors as melanosomes . 1H and 13C NMR studies revealed that Zn2+ binds also with oxidized glutathione in aqueous medium with 1:1 stoichiometry . Taking into account a significant role of glutathione for pigment cell metabolism , 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 .
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  (Fig.1).
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.
4. FUNCTIONS OF ZINC
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  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 . 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 .
4.2. Excretory function of melanosomes and pigment tissues
Melanin can participate in excretion of some substances under physiological conditions . As hair melanosomes represent rich tissue reservoirs of zinc lost during removal of keratin structures, we tried to quantitate the Zn excretion via hair . The daily Zn loss in man by this way varies around 20µg 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  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  and the presence of H2O2  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  and the fact that also zinc levels in substantia nigra are markedly increased under these circumstances may indicate a physiological response to oxidative stress .
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 . 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 . 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 .
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 . 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 .
According to Pffeifer and Mailloux  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 .
Scavenging role in the elimination of metals, when they reach too high levels in the cell, was ascribed to neuromelanin granules .
The complexity of zinc intercellular transport can be illustrated by earlier work of O'Rourke et al  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 .
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 . 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 . Altered organ distribution and survival of melanoma cells were observed in the Zn depleted dietary groups of P51 melanoma-bearing mice .
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  and anchorage-independent growth  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 . Preincubation in vitro of cell suspensions with 10-4 mol/l zinc acetate prior to injecting tumour cells inhibited melanoma development in mice . 10-4 mol/l zinc sulphate was shown to decrease the i.v. but not s.c. transplantability of B16 melanoma .
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  and for targetting tumour cells with chemotherapeutic agents since zinc may act as a carrier for pharmacologically active ligands .
1. Ackland ML, Mc Ardle HJ: The significance of
extracellular zinc-binding ligands in the uptake of zinc by human fibroblasts. J Cell.
Physiol. 145, 1990, 409-413.
2. Andrzejczyk J, Buszman E: Interaction of Fe3+, Cu2+ and Zn2+ with melanin and melanoproteins from bovine eyes. Acta Biochim Pol. 39, 1992, 85-88.
3. Andrzejczyk J, Buszman E, Wilczok T: Metal ion binding to DOPA-melanin-HSA complexes. Studia biophys. 136, 1990, 27-33.
4. Anke M, Groppel B: Toxic actions of essential trace elements (Mo,Cu,Zn,Fe,Mn). In: Trace Element-Analytical Chemistry in Medicine and Biology, vol.4; P.Bratter, P.Schramel eds, Walter de Gruyter, Berlin & New York 1987, pp. 201-236.
5. Baker EN, Baker HM, Smith CA, Stebbins MR, Kahn M, Hellström KE, Hellström I: Human melanotransferrin (p97) has only one functional iron-binding site. FEBS Lett. 298, 1992, 215-218.
6. Benedeto JP, Ortonne JP, Voulot C, Khatchadourian C, Prota G, Thivolet J: Role of thiol compounds in mammalian pigmentation. Part I: Reduced and oxidized glutathione. J Invest Dermatol. 77, 1981, 402-405.
7. Bertrand D: Oligo-élements et pigments. Ann Nutr Alimentation. 26, 1972, B477-B492.
8. Bobilya DJ, Briske-Anderson M, Reeves PG: Ligands influence Zn transport into cultured endothelial cells. Proc Soc Exp Biol Med. 202, 1993, 159-166.
9. Bogacz A, Buszman E, Wilczok T: Competition between metal ions for DOPA-melanin. Studia biophys. 132, 1989, 189-195.
10. Borovanský J, Hearn PR, Bleehen SS, Russell RGG: Distribution of 65Zn in mice with melanomas and in the subcellular fractions of melanomas. Neoplasma. 27, 1980, 247-252.
11. Borovanský J, Hearn PR, Bleehen SS, Russell RGG: unpublished results.
12. Borovanský J, Hor_i_ko J, Ducho_ J: The hair melanosome: another tissue reservoir of zinc. Physiol bohemoslov. 25, 1976, 87-91.
13. Borovanský J, Mi_ejovský P, Riley PA: Possible relationship between abnormal melanosome structure and cytotoxic phenomena in malignant melanoma. Neoplasma. 38, 1991, 393-400.
14. Borovanský J, Riley PA: The effect of divalent cations on Cloudman melanoma cells. Eur J Cancer Clin Oncol. 19, 1983, 91-99.
15. Borovanský J, Riley PA: Cytotoxicity of zinc in vitro. Chem-Biol Interactions. 69, 1989, 279-291.
16. Borovanský J, Riley PA, Vránková E, Ne_as E: The effect of zinc on mouse melanoma growth in vitro and in vivo. Neoplasma. 32, 1985, 401-406.
17. Bowness JM, Morton RA: Distribution of copper and zinc in the eyes of fresh-water fishes and frogs. Occurence of metals in melanin fractions from eye tissues. Biochem J. 51, 1952, 530-535.
18. Bowness JM, Morton RA: The association of zinc and other metals with melanin and a melanin-protein complex. Biochem J. 53, 1953, 620-626.
19. Bowness JM, Morton RA, Shakir MH, Stubs AL: Distribution of copper and zinc in mammalian eye. Occurence of metals in fractions from eye tissues. Biochem J. 51, 1952, 521-530.
20. Bray TM, Bettger WJ: The physiological role of zinc as an antioxidant. Free Radical Biol & Med. 8, 1990, 281-291.
21. Bremner I, May PM: Systemic interactions of zinc. In: Zinc in Human Biology, CF Mills ed, Springer Verlag London,Berlin & Heidelberg 1989, pp. 95-108.
22. Brown JP, Woodbury RG, Hart CE, Hellström I, Hellström KE: Quantitative analysis of melanoma-associated antigen p97 in normal and neoplastic tissues. Proc Natl Acad Sci. USA 78, 1981, 539-543.
23. Bruenger FW, Stover BJ, Atherton DR: The incorporation of various ions in in vivo- and in vitro-produced melanin. Rad Res. 32, 1967, 1-12.
24. Bustamante J, Guerra L, Bredeston L, Mordoh J, Boveris A: Melanin content and hydroperoxide metabolism in human melanoma cells. Exp Cell Res. 196, 1992, 172-176.
25. Buszman E, Kwasniak B, Bogacz A: Binding capacity of metal ions to synthetic DOPA melanin. Studia biophys. 125, 1988, 143-153.
26. Chakravarty PK, Ghosh A, Chowdhury JR: Zinc in human malignancies. Neoplasma. 33, 1986, 85-90.
27. Cousins RJ: Towards a molecular understanding of zinc metabolism. Clin Physiol Biochem. 4, 1986, 20-30.
28. Dencker L, Tjälve H: An autoradiographic study on the fate of 65Zn in zinc-rich tissues in some rodents. Medical Biology. 57, 1979, 391-397.
29. Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, Lees AJ, Jenner P, Marsden CD: Alterations in the level of iron, ferritin and other trace elements in Parkinson's disease and other neurodegenerative diseases affecting basal ganglia. Brain. 114, 1991, 1953-1975.
30. Dexter DT, Carter CJ, Wells FR, Javoy-Agid F, Agid Y, Lees A, Jenner P, Marsden CD: Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease. J Neurochem. 52, 1989, 381-389.
31. Ebadi M, Swanson S :The status of zinc, copper and metallothionein in cancer patients. In: Nutrition, Growth and Cancer, GP Tryfiates, KN Prasad eds, Alan R Liss Inc, New York 1988, pp. 161-175.
32. Elleder M, Borovanský J, Mazánek J, Vosmík F: Enzyme histochemistry of human melanomas and pigmented naevi with special reference to Ó-D-mannosidase activity. Histochem J. 18, 1986, 472-480.
33. Erkell LJ, Ryd W, Hagmar B: Effects of zinc on tumor transplantability. Inv Metastasis. 6, 1986, 112-122.
34. Everett G, Apgar J: Enzymes as indicators of zinc status. In: Trace Elements Analytical Chemistry in Medicine and Biology, P. Bratter, P. Schramel eds, W.de Gruyter, Berlin & New York 1987, pp.283-288.
35. Ezzahir A: The influence of melanins on the photoperoxidation of lipids. J Photochem Photobiol B: Biol. 3, 1989, 341-349.
36. Galin MA, Nano HD, Hall T: Ocular zinc concentrations. Invest Ophthalmol. 1, 1962, 142-148.
37. Garratt RC, Jhotí H: A molecular model for the tumour associated antigen, p97, suggests a Zn-binding function. FEBS Lett. 305, 1992, 55-61.
38. Gersten DM, Bijwaard KE, Walden Jr TL, Hearing VJ: Serological demonstration of the albuminoid nature of the B700 murine melanoma antigen. Proc Soc Exp Biol Med. 197, 1991, 310-316.
39. Gersten DM, Hearing VJ: Demonstration of B700 cross-reactive antigens on human and other animal melanomas. Pigment Cell Res. 1, 1988, 434-438.
40. Giroux EL, Henkin RI: Competition for zinc amongst serum albumin and amino acids. Biochim Biophys Acta. 273, 1972, 64-72.
41. Gjesdal F: Investigations on the melanin granules with special consideration of the hair pigment. Acta Pathol Microbiol Scand. 47, suppl.133, 1959, 1-112.
42. Har-El R, Chevion M: Zinc(II) protects against metal-mediated free radical induced damage: studies on single and double-strand DNA breakage. Free Rad Res Commun. 12, 1991, 509-515.
43. Harley-Mason J, Bu'Lock JD: Synthesis of 5,6-dihydroxy-indole derivatives: an oxido-reduction rearrangement catalyzed by zinc ions. Nature. 166, 1950, 1036-1037.
44. Hearing VJ, Nicolson JM: Abnormal protein synthesis in malignant melanoma cells. Cancer Biochem Biophys. 4, 1980, 59-63.
45. Horiko J, Borovanský J, Ducho_ J: Verteilung von Zink und Kupfer in menschlicher Kopfhaar verschiedener Farbtöne. Derm Monatschrift 159, 1973, 206-209.
46. Horiko J, Borovanský J, Ducho_ J, Procházková B: Distribution of zinc and copper in pigmented tissues. Hoppe-Seyler's Z Physiol Chem 354, 1973, 203-204.
47. Horiko J, Pantuek M: Hypozincemia in patients with malignant melanoma. Clin Chim Acta. 130, 1983, 279-282.
48. Hu KH, Friede RL: Topographic distribution of zinc in human brain by atomic absorption spectrophotometry. J Neurochem. 15, 1968, 677-685.
49. Issaq HJ: The role of metals in tumor development and inhibition. In: Metal Ions in Biological Systems, vol.10: Carcinogenicity and Metal Ions. H.Sigel ed, M.Dekker Inc, New York & Basel 1980, pp. 55-93.
50. Jara JR, Garcia-Borron JC, Aroca P, Lozano AJ: Regulation of melanogenesis. II. The role of metal cations. Biochim Biophys Acta. 1035, 1990, 276-285.
51. Kiss T, Gergely A: Complexes of 3,4-dihydroxyphenyl derivatives. VI. Microprocesses of formation of proton and metal complexes of L-dopa. Inorg Chim Acta. 78, 1983, 247-254.
52. Koch WH, Chedekel MR: Photochemistry and photobiology of melanin metabolites: Formation of free radicals. Photochem Photobiol. 46, 1987, 229-238.
53. Kohler T: Histochemical and cytochemical demonstration of zinc cysteinate in the Tapetum lucidum of the cat. Histochemistry. 70, 1981, 173-178.
54. Korohoda W, Michalik M, Pietrzkowski Z, Zaporowska-Siwiak E: Addition of iron and zinc complexes to Eagle's Minimal Essential Medium is sufficient to induce and support the proliferation of B16 melanoma cells. Folia Histochem Cytobiol. 31, 1993, 3-7.
55. Koropatnick J, Pearson J: Zinc treatment, metallothionein expression and resistance to cisplatin in mouse melanoma cells. Somatic Cell & Molec Genetics. 16, 1990, 529-537.
56. Krauter B, Nagel W, Hartmann HJ, Weser U: Copper-thionein in melanoma. Biochim Biophys Acta. 1013, 1989, 212-217.
57. Kreutzfeld KL, Lei KY, Bregman MD, Meyskens Jr FL: Dexamethazone and zinc in combination inhibit the anchorage-independent growth of S91 Cloudman murine melanoma. Life Sci. 36, 1985, 823-827.
58. Kurus E: Über den histochemischen Nachweis von Zink als Spurenelement im Auge des Menschen. Klin Mbl Augenheilk. 134, 1959, 338-350.
59. Leiner M, Leiner G: Der Zinkgehalt in den Augen von Knochenfischen II. Biol Zbl 64, 1944, 293-305
60. Meyer zum Gottesberge AM: Microanalytical investigations of the inner ear, uveal tract and retinal pigment epithelium melanin. Adv Biosci. 62, 1987, 435-443.
61. Murray MJ, Erickson KL, Fisher GL: Effects of dietary zinc on melanoma growth and experimental metastasis. Cancer Lett. 21, 1983, 183-194.
62. Newsome DA, Oliver PD, Deupree DM, Miceli MV, Diamond JG: Zinc uptake by primate retinal pigment epithelium and choroid. Curr Eye Res. 11, 1992, 213-217.
63. Newsome DA, Rothman RJ: Zinc uptake in vitro by human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 28, 1987, 1795-1799.
64. O'Rourke J, Durrani J, Benson C, Bronzino J, Miller C: Studies in uveal physiology: III.Anterior chamber clearance, uveoretinal distribution and respiratory response associated with zinc 69m. Arch Opthalmol. 88, 1972, 185-188.
65. O'Rourke JF, Patton H, Bradley R: A study of the uptake of P32, Zn65 and I131serum albumen by experimental malignant melanoma. Am J Ophthalmol. 44, 1957, 190-197.
66. Oliver PD, Tate Jr DJ, Newsome DA: Metallothionein in human retinal pigment epithelial cells: expression, induction and zinc uptake. Curr Eye Res. 11, 1992, 183-188.
67. Palumbo A, d'Ischia M, Misuraca G, Prota G: Effect of metal ions on the rearrangement of dopachrome. Biochim Biophys Acta. 925, 1987, 203-209.
68. Palumbo A, d'Ischia M, Misuraca G, Prota G, Schultz TM: Structural modifications in biosynthetic melanins induced by metal ions. Biochim Biophys Acta. 964, 1988, 193-199.
69. Pffeifer CC, Mailloux RJ: Hypertension: heavy metals, useful cations and melanin as a possible repository. Med Hypotheses. 26, 1988, 125-130.
70. Philcox JC, Tilley MH, Coyle P, Rofe AM: Metallothionein and zinc homeostasis during tumor progression. Biol Trace Element Res. 40, 1994, 295-308.
71. Pierson HF, Meadows GG: Modulation of peroxidation in murine melanoma by dietary tyrosine-phenylalanine restriction, levodopa methylester chemotherapy, and sodium ascorbate supplementation. J Nat Cancer Inst. 75, 1985, 507-516.
72. Pohla H, Simonsberger P, Adam H: X-ray microanalysis of rainbow trout (Salmo gairdneri Rich.) melanosomes with special reference to analytical methods. Mikroskopie. (Wien) 40, 1983, 273-284.
73. Postal WP, Vogel EJ, Young CM, Greenway FT: The binding of copper (II) and zinc (II) to oxidized
glutathione. J Inorg Biochem. 25, 1985, 25-33.
74. Potts AM, Au PC: The affinity of melanin for inorganic ions. Exp Eye Res. 22, 1976, 487-491.
75. Prasad KN, Ahrens CR, Barrett JM: Homeostasis of zinc and iron in mouse B16 melanoma. Cancer Res. 29, 1969, 1019-1023.
76. Price D, Joshi DG: Ferritin: A zinc detoxicant and zinc ion donor. Proc Natl Acad Sci. USA 79, 1982, 3116-3119.
77. Procházková B, Ducho_ J, Veverková V: Protein constituent of melanosomes of tumourous origin. (In Czech) Sborník lék. 79, 1977, 329-334.
78. Procházková B., Obrusník I, Ducho J: Influence of selenium on activity of glutathione peroxidase in experimetal melanoma. In: Pigment Cell 1985. Biological, Molecular and Clinical Aspects of Pigmentation. J Bagnara, SN Klaus, E Paul, M Schartl eds, University of Tokyo Press, Tokyo 1985, pp. 539-544.
79. Richards MP: Role of metallothionein in copper and zinc metabolism. J Nutr. 119, 1989, 1062-1070.
80. Riley PA: Radicals in melanin biochemistry. Ann NY Acad Sci. 551, 1988, 111-120.
81. Rorsman H: Binding of simple chemicals in melanin producing cells. Progress in Org Biol & Med Chem. 3, 1972, 655-670.
82. Samuelson DA, Smith P, Ulshafer RJ, Hendricks DG, Whitley RD, Hendricks H, Leone NC: X-Ray microanalysis of ocular melanin in pigs maintained on normal and low zinc diets. Exp Eye Res. 56, 1993, 63-70.
83. Sarna T, Swartz HM: Identification and characterization of melanin in tissues and body fluids. Folia Histochem Cytochem. 16, 1978, 275-286.
84. Sauer GR, Watanabe N: Ultrastructural and histochemical aspects of zinc accumulation by fish scales. Tissue Cell. 21, 1989, 935-943.
85. Schlopak TV: Microelements in Ophthalmology (in Russian), Medicina Publ, Moscow 1969, pp.47-82.
86. Seiji M, Fitzpatrick TB, Simpson RT, Birbeck MSC: Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature. 197, 1963, 1082-1084.
87. Shibata T, Prota G, Mishima Y: Regulatory factors of melanin monomer and polymer formation in melanogenic subcompartments of pigment cells. XIVth Int Pigment Cell Conference, Kobe 1990, p.91.
88. Smit NPM, van Roermund CWT, Aerts HMFG, Heikoop JC, Van der Berg M, Pavel S, Wanders RJA: Subcellular fractionation of cultured normal human melanocytes. New insights into the relationship of melanosomes with lysosomes and peroxisomes. Biochim Biophys Acta. 1181, 1986, 1-6.
89. Song MK, Shin WY, Adham NF, Costea NC: Zinc, calcium, and magnesium metabolism: effects on plasmacytomas in Balb/c mice. Am J Clin Nutr. 49, 1989, 701-707.
90. Stein WD: Chemical composition of the melanin granule and its relation to the mitochondrion. Nature. 175, 1955, 256-257.
91. Takaya K: Electron microscopy of human melanosomes in unstained, fresh air-dried hair bulbs and their examination by electron probe microanalysis. Cell Tissue Res. 178, 1977, 169-173.
92. Thathachari YT: Structure of melanins. Pigment Cell. 1, 1973, 158-174.
93. Ujjami B, Krakower G, Bachowski G, Krezoski S, Shaw III CF, Petering DH: Host zinc metabolism and the Ehrlich ascites tumour. Zinc redistribution during tumour-related stress. Biochem J. 233, 1986, 99-105.
94. Ulshafer RJ, Allen CB, Rubin ML: Distribution of elements in the human retinal pigment epithelium. Arch Opthalmol. 108, 1990, 113-117.
95. Vallee BL, Falchuk KH: The biochemical basis of zinc physiology. Physiol Rev. 73, 1993, 79-118.
96. Van Rij AM, Pories WJ: Zinc and tumor growth. In: Metal Ions in Biological Systems, vol.10-Carcinogenicity and Metal Ions. H Sigel ed, M Dekker Inc, New York & Basel 1980, pp.207-251.
97. White LP: Melanin: a naturally occurring cation exchange material. Nature. 182, 1958, 1427-1428.
98. Williams RJP: Zinc: what is its role in biology? Endeavour. 8, 1984, 65-70
99. Willson RL: Iron, zinc, free radicals and oxygen in tissue disorder and cancer control. In: Iron Metabolism. R Porter ed, Elsevier-Excerpta Medica 1977, pp.333-354.
100. Willson RL: Zinc and iron in free radical pathology and cellular control. In: Zinc in Human Biology, CF Mills ed, Springer Verlag Berlin & Heidelberg 1989, pp. 147-172.
101. Zecca L, Micacci C, Seraglia R, Parati E: The chemical characterization of melanin contained in substantia nigra of human brain. Biochim Biophys Acta. 1138, 1992, 6-10.
102. Zhou BK, Boissy RE, Pifko-Hirst S, Moran DJ, Orlow SJ: Lysosome-associated membrane protein 1 is the melanocyte vesicular membrane glycoprotein band II. J Invest Dermatol. 100, 1993, 110-114.
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.