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Isolation of melanosomes from hair or feathers is difficult due to the need to break keratin structures using a strong chemical treatment. All recently published isolation approaches were examined in the isolation of melanosomes from human and dog hair. The best results were achieved from immediate separation of melanosomes by centrifugation after controlled NaOH hydrolysis of the hair. The crucial factor for successful separation is the choice of the optimal amount of time required for disintegration of the hair: if the time chosen is too long, melanosomes are damaged; if it is too short, contamination by residual keratin ensues. High temperatures must be avoided. Attempts to break hair structure by a number of milder reagents have failed. [ABSTRACT FROM AUTHOR]

Objectives of the study: To evaluate whether serum enzyme activities reflect both tumour progression and regression in a hereditary melanoma minipig MeLiM model /Cell mol Biol 45,1999,1119/ and to compare the enzyme profiles with those in murine melanomas and human disease. Material and methods: Tyrosinase activity was measured by a dopa oxidase method /Neoplasma 28,1981,59/, γ -glutamyltransferase (GGT) was monitored by means of the GGT100 set/Lachema, Czech Rep./ and neuron-specific enolase /NSE/ was analyzed with a radioimmunoassay kit /Cis-Bio Int., France/ in sera of minipigs with localized/n = 35/, metastatic /n = 22/ and cryptogenic /n = 14/ melanomas and also in animals with a spontaneous melanoma regression/n = 13/ or regression induced by devitalization therapy/n = 44/ and in control minipigs /n = 36/. Data were statistically analyzed by the Student's t -test. Results: A significant increase in tyrosinase activity (P < 0.05) was found not only in the group of animals with cryptogenic melanoma, but also in animals with spontaneous and experimentally induced melanoma regression, probably due to a better enzyme release from tumour cells. Similar results were obtained as for the GGT: There was a significant increase of activity in the groups of minipigs with regressing melanomas. NSE in experimental melanoma models has not yet been studied. The upper physiological NSE limit in minipigs (deduced as x + 2σ = 18 ng/ml) was overcome in 8 of 54 sera = in 18% of minipigs in advanced stage of melanoma progression. Conclusions: Our results document that enzyme profiles in melanoma-bearing minipigs faithfully resemble those in human counterpart (i.e. serum enzyme activities begin to be of marker value only in later stages) demonstrating that the MeLiM model is close to human melanoma unlike murine melanoma models, in which a steep increase of marker enzymes at an early stage of melanoma progression, is typical. Acknowledgements: Supported by GACR 524/01/0162 and CAV S5045113 grants. [ABSTRACT FROM AUTHOR]

Melanin production is the primary mechanism protecting human skin against the UV light-induced damage. The polymeric compound melanin is synthesized within melanocytes in the specialized subcellular organelles, termed melanosomes, which are then transferred to surrounding keratinocytes. The genes for melanin synthesis and deposition are coordinately expressed in melanocytes. The transcription factor MITF, which has been reported to activate more than 25 genes in pigment cells, has emerged as an essential regulator not only for melanocyte development, proliferation and survival, but also for the expression of enzymes and structural proteins ensuring the production of melanin. MITF is a transcriptional activator of several genes which encode melanosome-localized proteins involved both in melanin synthesis and in melanosome biogenesis and transport, including genes whose mutations are associated with human oculocutaneous and ocular forms of albinism. Here, we outline the mechanisms of transcriptional regulation of genes associated with the biosynthesis of melanin in melanocytes and melanoma cells. MITF is crucial in this process, while several other factors seem to have only an auxiliary role to play under specific circumstances.

The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant downregulation of MITF targets and decreased cell proliferation. Although Brm was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.

The transcription factor microphthalmia (MITF) is required for the formation of normal melanocytes during embryonic development and for the expression of pigment cell-specific markers, which are the downstream transcriptional targets of MITF. It also seems to be crucial for the survival of malignant melanocytes. The special interest of this review is the possible utility of MITF as a marker of malignant melanoma. Melanocyte-specific isoform of MITF appears to be a unique molecule in the differential diagnosis of melanocytic tumors.

Hereditary minipig melanomas, which have many histopathological and other features in common with their human counterparts, have recently become preferred melanoma models. The MeLiM (Melanoma-bearing Libĕchov Minipig) strain was selected by partial inbreeding. A high tumour incidence and malignant behaviour on the one hand together with the occurrence of spontaneous regression and high susceptibility to the devitalization technique as a new strategy in melanoma therapy, make the MeLiM strain a superior melanoma model to other hereditary swine melanomas. Biochemical analyses of the tumours revealed: (i) a high concentration of eumelanin and low concentration of phaeomelanin in melanoma cells, which makes the probability of photochemical regression of MeLiM melanomas negligible; (ii) an extremely high level of melanosomes (almost half of the melanoma dry weight), which suggests a high differentiation of the MeLiM melanoma and is consistent with its mechanical rigidity; (iii) the presence of typical melanoma enzymes--tyrosinase, alpha-mannosidase and gamma-glutamyltransferase; and (iv) in the tumours regressing as a consequence of devitalization treatment, alpha-mannosidase activity was reduced and tyrosinase activity approached the detection threshold, which is in accordance with the substitution of melanoma tissue for connective tissue in devitalized tumours observed histologically. (Immuno)histochemical comparisons (based on dopaoxidase reaction, S100 and HMB-45 reactivities) of the skin from white and pigmented minipigs revealed the absence of melanocytes in white skin. This is the first direct evidence supporting a possible explanation for the absence of melanoma in white minipigs. The similarities and dissimilarities of the MeLiM model compared with human melanoma are highlighted.

Our mini review summarizes what is known about the (bio)degradation of melanosomes. Unlike melanosome biogenesis where our knowledge enables us to explain it in molecular terms posing many interesting questions on the relation between lysosomes and melanosomes, melanosome degradation has remained 'terra incognita'. Observations at optical and ultrastructural levels describe the disintegration of melanosomes in the lysosomal compartment (in auto- and heterophagosomes). Histochemical studies suggest the participation of acid hydrolases in the process of melanosome degradation. Biochemical data confirm the ability of lysosomal hydrolases to degrade melanosome constituents except the melanin moiety. The similarity of melanin structure to that of polycyclic aromatic hydrocarbons suggests that melanin should be sensitive mainly, if not exclusively, to oxidative breakdown. In vitro melanin can indeed be decomposed by an oxidative attack and the degradation is accompanied by fluorescence and decreasing absorbance. From enzymes engaged in the biotransformation of polycyclic hydrocarbons only phagosomal NADPH oxidase meets the criteria (particularly as for compartmental and catalytic properties) to be involved in melanin biodegradation. The in vivo biodegradation of melanin has so far been clearly demonstrated in Aspergillus and fungi melanins.

The induction of autofluorescence of melanins by UV radiation (330-380 nm) and near UV (400-440 nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow autofluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of autofluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H2O2, Na2O2 or HIO4. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary autofluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the autofluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in autofluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.

"Leucine aminopeptidase" (LAP, aminopolypeptidase, EC 3.4.11) activity has been recommended and widely used as a histochemical marker for the identification of contaminating LAP-positive fibroblasts in pigment cell cultures. Using a sensitive biochemical assay with L-leucyl-p-nitroanilide as a substrate we demonstrated that in vitro melanoma cells also exhibit LAP activity. Our comparison of four melanoma cell lines with four fibroblast lines showed that the differences in the enzyme activity were not qualitative but only quantitative. For this reason the specific antibodies, karyological analysis and electron microscopy are recommended as more reliable means in distinguishing fibroblasts from poorly differentiated pigment cells than the LAP-cytochemistry.

In addition to tyrosinase and its related proteins melanosomes contain a variety of further enzyme activities. Using spectrophotometric methods alpha-mannosidase and gamma-glutamyl-transferase (GGT) were studied in B 16 melanoma, in isolated melanosomes and in tumour host (mice C57BL6J) sera. When compared to the original melanoma tissue (12.8-26.7 nkat/g TP) isolated melanosomes exhibited much higher alpha-mannosidase activity [227-420 nkat/g of total proteins (TP)]. Strong activation by Zn2+ and no influence of Co2+ ions suggested that the dominant form of alpha-mannosidase of the enzyme present in melanosomes was of the acid (lysosomal) type. The GGT activity of isolated melanosomes (168-244 nkat/g TP) was comparable with that of the whole melanoma tissue (203-375 nkat/g TP) . Treatment of melanosomes with detergents (0.1% Triton X-100, 0.5% deoxycholate) revealed striking extractibility differences between the two enzymes investigated in relation to their localization: alpha-mannosidase remained immobilized in the melanized matrix of melanosomes whereas the membrane bound GGT was easily released. Unlike the alpha-mannosidase the GGT serum levels were increasing in relation to the melanoma growth. The demonstration of acid form of alpha-mannosidase in melanosomes is consistent with their lysosomal ranking; the presence of GGT is in keeping with its expected roles both in protection against oxidative stress and in melanogenesis.

Contradiction between repeatedly reported electron microscopic and histochemical observation of melanosome disintegration in the presence of lysosomal enzymes in vivo and failing attempts to induce such degradation by biochemical means in vitro with the aim to explain chemical mechanism(s) of this process belongs to chronically challenging problems in biochemistry of melanin structures. Attempts have been made to bring about melanosome disintegration in vivo by inoculating melanosomes isolated from dog hair into the tissue of amelanotic Bomirski melanoma and into peritoneal cavities of DBA/2 and C57BL/6J mice, and by repeated injection of melanosomes isolated from Bomirski pigmented melanoma into Syrian hamster foot pads. Both histological and electron microscopic observations demonstrated that melanosomes were phagocytized by macrophages and sporadically by fibroblasts. In peritoneal cavities the injected foreign melanosomes remained mostly extracellularly, were surrounded by foreign body multinuclear cells and formed granulomas. There were no convincing signs of degradation of the hair melanosomes, which behaved like inert foreign bodies. Only some phagocytized Bomirski hamster melanoma melanosomes tended to lose their integrity. Our data suggest that melanosomes devoid of their limiting membranes are not necessarily prone to extensive disintegration in vivo. The earlier reported association of lysosomal enzymes with disintegrating melanosomes does not constitute an evidence for their participation in melanosome degradation. Considering the structure of melanins, redox mechanisms (analogous with the metabolism of polycyclic hydrocarbons (DePierre and Ernster, 1978)) seem to be more probably involved in pigment degradation than hydrolytic reactions.

The total serum activity of gamma-glutamyl transpeptidase (GGT) was shown to increase with the growth of transplantable B16 and S91 melanomas in inbred mice. In an effort to define the source of the GGT shed into the bloodstream the physicochemical characteristics of the partially purified GGT isoforms from liver, serum and B16 melanoma were compared. The molecular weights of the serum and melanoma isoforms were identical (86 kDa) and differed from that of the liver isoform (69 kDa). In polyacrylamide gel electrophoresis the serum and melanoma isoforms had a similar mobility which exceeded that of the liver enzyme. Treatment of the enzyme preparations with neuraminidase removed the differences in the electrophoretic mobility of the three GGT isoforms studied. On ion exchange chromatography on a DEAE-Spheron 300 LC column the melanoma and serum isoforms had an affinity to the sorbent unlike the liver isoform. Our observations suggest that melanoma cells express a sialoform of GGT which is responsible for an increase in the total GGT serum activity. Biochemical and histochemical analyses did not reveal any increase in liver GGT production associated with melanoma development. Detection of the GGT isoform of tumour origin in sera ranks GGT among the specific melanoma markers.

Previous studies have shown that sensitivity to high extracellular levels of Zn2+ is a general feature of cells in vitro and that a prerequisite of the toxic action of zinc is entry into cells via channels that are shared with iron or calcium. As the biochemical and toxicological behaviour of zinc chelate complexes could be different from that of free Zn2+, the effect of chelating agents on zinc transport into human melanoma cell lines was tested. EDTAcal and tetracycline reduced the toxic action of zinc ions in vitro, whereas phenytoin and diethyldithiocarbamate potentiated its effects. D-penicillamine, an effective chelator of zinc in vivo, also exerted a protective action in vitro. Comparison of sensitivity to Zn2+ in vitro between human melanoma lines and several lines of pigment cells from skin of various origins demonstrated that melanoma cells are killed by zinc ions at concentrations which are only partially toxic for normal pigment cells. This is consistent with the repeatedly observed high uptake of 65Zn by melanoma cells.

The current state of art in the detection of metals in tissues, cells and their subcellular fractions has been reviewed. Both classic chemical techniques (including titration, spectrophotometric, electrochemical and isotope methods, neutron activation analysis, mass spectrometry, inductively coupled plasma) and cell biology techniques (i.e. conventional histochemical methods, autometallography, autoradiography and microanalytical techniques using beam of electrons or protons, laser, X-ray or ion beam) were discussed. This survey of analytical techniques is, wherever possible, illustrated by examples documenting how our knowledge on metals in pigment cells and their subcellular fractions has been growing.

There has been a discrepancy between promising results of experimental chemotherapy in animal melanoma models and clinical response rates. This inconsistency seems to reflect weak points of the assays used so far to monitor the response of melanoma cells to chemotherapeutic agents. Therefore a less usual approach was chosen in the present study: Tumor cells were cultured in peritoneal cavity (B16 melanoma in inbred C57BL/6J mice and Cloudman S91 melanoma in inbred DBA2 mice) to maintain normal in vivo conditions; the animals were receiving the tested agents in i.p. injections and the prolongation of their life span was considered as the principle parameter of therapeutic efficiency of the compounds tested. Previously described therapeutic potency both of vitamins (C, alpha-tocopherol acid succinate) and some phenols (hydroquinone, 4-hydroxyanisole) was confirmed. Benzoate, spin trap N-butyl-alpha-phenyl-nitrone and ammonium chloride as a lysosomotropic agent failed to increase the survival of melanoma-bearing mice. Free radical scavenger methimazole exerted a therapeutic effect in mice with pigmented B16 melanoma. Only classic cytostatic agents--cisplatin and cyclophosphamide--proved its therapeutic effect in both melanoma models studied. These results are in accord with the known resistance of human melanoma to conventional chemotherapy. Measurement of serum activity of gamma-glutamyltransferase was shown to be useful for monitoring therapeutic effect.

Human body contains a number of natural pigments-authentic melanins, soluble melanins, neuromelanins, lipofuscins-having a similar origin-oxidation and polymerization of precursor phenols, and sharing many common characteristics among which dominates their free radical activity. Related substances can be formed from exogenous compounds introduced at some occasions to the organism. The functional properties of the pigments seem to be determined by their localization, i.e. cellular or extracellular, and as regards those intracellular ones by their subcellular compartmentalization. All the intracellular pigments mentioned in this study are associated with lysosomal enzymes at some stage of their development. A special attention was paid to a neglected group of soluble melanins. It was proposed that methylation may decide about the fate of precursor molecules whether they will join the soluble melanin pool or will be excreted as melanogens. Participation of the pigments in various biochemical and pathobiochemical processes has been reviewed.

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

Increased total serum sialic acid levels have proved to be a useful non-specific marker of various neoplastic diseases including melanoma. In some malignancies, specific fractions of sialic acid were reported to be more reliable for monitoring disease progression. In this study various fractions of sialic acid were measured in the sera of Bomirski melanoma-bearing Syrian hamsters using the modified thiobarbituric acid method of Skoza and Mohos. Significantly higher serum levels of total sialic acid, 601.0 +/- 49.1 micrograms/ml, were demonstrated in animals with melanoma in comparison with control hamsters, 463.1 +/- 33.0 micrograms/ml (P < 0.01). Free sialic acid concentration was found to be negligible compared with that found in the sera of melanoma patients. Lipid bound sialic acid measurements carried out according to Bhatavdekar et al. were shown to be influenced by interfering substances. Hence, only the estimation of total sialic acid in sera can be recommended for monitoring melanoma progression. Bomirski melanoma proved to be a good model of human melanoma.

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.

Basic morphometric parameters of melanosomes isolated from mouse hair, hamster eye and hamster hair are given and tabelized with those published formerly to enlarge the widest published collection of melanosomes isolated from different types of pigmented tissues up to 17 types. The splitting of melanosomes isolated from mixed tissue of hamster eyes into two subpopulations confirmed the presence of double origin of melanosomes in the eye. The close correspondence of subpopulations of hamster eye melanosomes with hamster hair and hamster melanoma (type Bomirski) respectively supports the hypothesis of high structural stability of melanosomes even under pathological conditions.

Melanosome is a clear out organelle of its own. The experience of the authors in the melanosome investigation has been reviewed and put to context with the literature data. Melanin moiety, a prominent feature of melanosome, was found to vary between 20-60 weight %. Melanosome concentration in pigment tissues was demonstrated to be remarkably high which explains their capacity to perform various functions both cytoprotective and cytotoxic-ascribed to the presence of melanin. Putative relation of melanosomes to other cell organelles was critically analysed. Systems of melanosome maturation were explained. The ultrastructure of melanosomes determined by the quality of their protein matrix was described and basic morphometric data summarized. The importance of the compartmentalization of melanogenesis was underlined and the fragility of melanosomal limiting membrane shown. The melanosome degradation repeatedly observed by electron microscopy has not been confirmed by biochemical means.

Concentration of melanosomes in various tissues has been unknown because of the impracticability of their direct quantification. Using an indirect approach comprising the estimation of melanin both in freeze-dried tissue samples and in isolated melanosomes, we obtained data on the amount of melanosomes in various pigment tissues. The concentrations of melanosomes found in the tissues were relatively high, not only reflecting the dark color of pigment tissues but also explaining their capacity to perform various functions ascribed to the presence of melanin.

Melanogenesis has been regarded as a hazard for pigment cells which are endangered by reactive quinones and semiquinones generated by this process. Normally the potentially cytotoxic species are confined to melanosomes by a limiting membrane and thus separated from the rest of the cell. Our electron microscopic investigation has demonstrated the presence of abnormal and incomplete melanosomes in human melanomas from epidermal and mucosal sites, in melanoma metastases, and in B16 mouse melanoma. We conclude that significant leakage of reactive melanin precursors including free radical species may occur from aberrant melanosomes in pigmented tumors. This would be expected to be reflected by fully extended physiological scavenging mechanisms, and by local and distant manifestations of cytotoxicity: Among these manifestations is free radical damage to the liver, detected by a thiobarbituric acid-reactive substances assay, in B16 melanoma-bearing mice. The efflux of toxic species from abnormal melanosomes may explain both the observed frequent occurrence of necrosis in melanomas and the therapeutic efficacy of tyrosinase substrates and may also be one of the factors influencing the extent of melanogenuria.

A histochemical study of alpha-D-mannosidase revealed that normal human melanocytes (resting state, activated, lentigo simplex) exhibit either no or just detectable activity, as do melanocytes in the initial phase of lentigo maligna. Junctional, or occasionally zone A naevocytes displayed a very low enzyme activity. On the other hand, melanocytes in the initial stage of neoplastic transformation (dysplastic naevi, advanced stage of lentigo maligna) and also melanoma cells in disorders of low malignant potential (initial naevogenic melanoma, superficial spreading melanoma) displayed a high activity uniformly throughout the cell population. In the malignant forms (nodular melanoma, recurrences, metastases), the enzyme activity was remarkably heterogeneous, suggesting a breakdown of uniformity during malignant transformation. The significance of alpha-mannosidase activity induction in the course of melanocyte neoplastic transformation is not clear at present. The results of biochemical assays suggest that the lysosomal isoenzyme is mainly responsible. Other lysosomal enzymes, and dehydrogenases studied concomitantly, did not display any comparable phenomena of induction or similar behaviour. However, the results of a comparison of alpha-mannosidase with the melanocyte reference enzyme tyrosinase suggested activity patterns in the enzyme pair which may provide a better insight into the biochemical differentiation of human melanocytes in neoplastic disorders. The possible relationship of alpha-mannosidase to melanogenesis is also discussed.

The procedure for isolating soluble melanoprotein from human malignant melanoma was described. Electron microscopy confirmed the absence of any contaminating organized particles. The melanoprotein was brown hygroscopic substance with 8.8% of melanin, easily soluble in water. The solution revealed general absorption with a shoulder at 280 nm. Chemical composition was typical for a melanoprotein complex with characteristic high sulphur content (2.1%) and abundance in zinc (210 mug Zn/g dry sample). Sedimentation and electrophoretic properties indicated relative homogeneity. Similar substance could also be isolated from Harding-Passey mouse melanoma. Such soluble melanoproteins have been needed for their broad exploitation in tumor immunology.

Four human melanoma cell lines were characterized by evaluation of the morphology, ultrastructure, cytogenetics, plating efficiency, agar colony formation and alpha-mannosidase values. Biochemical studies demonstrate that all studied lines produced alpha-mannosidase even under condition of long-term in vitro growth. All respective lines were characterized by a great variations in biological markers studied and very few reliable comparisons could be obtained as regards the proliferation activity and capacity, cellular composition and clonogenic potential as well. Due to cellular selections during long-term in vitro cultivation, the range for using these experimental systems as models for the study of biology of human malignant melanoma seems to be rather limited.

Melanosomes were isolated from human and dog hairs and their Zn content was determined. The mean Zn concentrations, in mug Zn/g dry weight, were 687 (men's melanosomes), 641 (women's melanosomes) and 691 (dog melanosomes). These values rank melanosomes from pigmented keratinous structures among the structural elements with the highest Zn content. An interpretation of the possible causes of this accumulation was submitted. By means of the conversion factor between hair length and weight (determined at 0.004), it was estimated that daily Zn lossess via hair vary in the region of 19 mug. In detailed studies of Zn metabolism, excretion via pigmented keratinous structures ought not to be neglected.

Melanin concentration which represents one of biochemical parameters expressing the degree of pigment cell differentiation was studied by new gravimetric procedure in human, Bomirski hamster, Harding-Passey mouse and horse melanomas and also in several non-tumor pigment tissues. In amelanotic varieties of human and hamster melanomas relatively high melanin level was discovered. This finding (together with known electron microscopic evidence of pre-melanosomes presence in some amelanotic melanomas) suggests that the term "amelanotic" is not correct. Comparison of melanin content between melanomas and normal pigment tissues clearly demonstrates in quantitative way that as for melanomas their proliferation does not decrease the degree of their biochemical differentiation.

The effect of Zn2+ on mouse melanoma growth in vitro and in vivo was studied. Under in vitro conditions the proliferation of a Cloudman mouse melanoma cell line was inhibited by zinc ions at 10(-4) M, as measured by 3H-thymidine incorporation and optical density of NaOH cell digests. However, in vivo it was not possible to suppress both B16 and Cloudman S91 melanoma growth in mice by the administration of zinc ions. There were no significant differences in tumor growth after subcutaneous inoculation between mice constantly receiving 0.1% zinc acetate or 0.05% zinc sulphate in their drinking water and control groups, nor was it possible to decrease the number of lung metastases by zinc treatment after intravenous inoculation of tumor cells. The increased dietary supply of Zn failed to influence the survival time of mice in both melanoma types studied. Preincubation in vitro of cell suspensions in 10(-3) M zinc acetate prior to injection inhibited melanoma development in vivo. This implies that the in vivo zinc levels did not reach the necessary cytotoxic concentration.

The effect of zinc ions on B16 mouse melanoma lines, HeLa cells and I-221 epithelial cells was investigated in vitro in order to ascertain whether sensitivity to Zn2+ is a general feature of cells in vitro and in an attempt to elucidate the mechanism(s) of zinc cytotoxicity. The proliferation of B16, HeLa and I-221 cell lines was inhibited by 1.25 x 10(-4), 1.50 x 10(-4) and 1.50 x 10(-4) mol/l Zn2+, respectively. The free radical scavengers, methimazole and ethanol, did not suppress the toxicity of Zn2+, neither did superoxide dismutase or catalase. The addition of the chelating agent EDTA reduced the zinc cytotoxicity. It was possible to suppress the cytotoxicity of zinc by increasing the concentration of either Fe2+ or Ca2+ but not Mg2+, which suggests that a prerequisite for the toxic action of zinc is entry into cells using channels that are shared with iron or calcium. This view was supported by experiments in which transferrin intensified the cytotoxic action of zinc in serum-free medium. Another agent facilitating zinc transport, prostaglandin E2, inhibited the proliferation of the B16 melanoma cell line. There were no conspicuous differences in zinc toxicity to pigmented and unpigmented cells. The toxic effect of zinc in the cell systems studied exceeded that of iron, copper, manganese and cobalt in the same concentration range. In vitro, Zn2+ should be regarded as a dangerous cation.

Melanogens which are considered as probable precursors or their metabolites in the metabolic pathway toward melanin are excreted in an increasing amount during the melanotic melanoma growth in hamsters. Moreover, an increased excretion of zinc was observed. The evaluation of dependence between zinc and Thormählen positive melanogens excretions during the melanoma growth suggests an exponential type of dependence. Likewise as in the case of human melanoma the hyperzincuria proved to be a laboratory sign of tumor disease progression in Syrian hamsters as well. The cause of an increased zinc excretion during the melanoma growth seems to consist in an increased lysis of pigment cells. The presumed destruction of tumor cells was not accompanied by a higher excretion of magnesium, though. Concentration of creatinine proved to be a useful mean for expressing of standard excretion of various substances also in hamsters.