Histochemical differentiation of peroxidase-mediated from tyrosinase-mediated melanin formation in mammalian tissues

Summary Preincubation with the copper-chelator, sodium diethyldithiocarbamate (DDC) and the presence of catalase in the incubation media allowed an accurate and reproducible differentiation of the role of tyrosinase from that of peroxidase in the oxidation of tyrosine and dopa in melanocytes, mast cells and eosinophils. These studies indicated that mammalian peroxidase in melanocytes, mast cells and eosinophils can mediate the conversion of tyrosine to melanin in the presence of dopa co-factor, as well as the conversion of dopa to melanin. With the methods employed, there was no evidence that tyrosinase in the preparations studied had significant ability to mediate the oxidation of tyrosine to melanin (even in the presence of dopa co-factor), although there was abundant evidence that it can mediate the conversion of dopa to melanin. Mammalian peroxidase may have roles in initiating melanin synthesis and catechol amine synthesis in vivo.Supported by USPHS Grant T1 AM 5,220, The General Research Support Fund, Boston City Hospital, and The Pathology Research Fund, St. Vincent Hospital.     

Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation.

1. Melanosomal tyrosinase was isolated from normal C57B1 mice, and a comparison of the tyrosine-hydroxylation and dopa (3,4-dihydroxyphenylalanine)-oxidation activities of this enzyme was made. 2. The results indicate that in the absence of dopa cofactor, this enzyme is capable of tyrosine hydroxylation, but with very little subsequent dopa oxidation and melanin formation. 3. This mechanism of enzyme action may play an important role in the intracellular regulation of melanin formation. 4. Further, dopa appears to act as a positive allosteric effector for tyrosine hydroxylation by tyrosinase, in addition to its known activity as a hydrogen donor for the reaction.     

The Effect of Oxygen on Melanin Precursors Released from Retinal Pigment Epithelial Cells In Vitro

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240–275 nm and a maximum around 300 nm wth a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

The effect of oxygen on melanin precursors released from retinal pigment epithelial cells in vitro.

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240-275 nm and a maximum around 300 nm with a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins

Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.     

Limitations of the quantitative cytochemical assay of catechol oxidase in melanoma cells

Summary The cytochemical quantification of catechol oxidase activity in fixed B16 melanoma cells was investigated using dopa as the substrate. Inhibitors showed that peroxidases do not significantly interfere. The kinetics of melanin formation were studied initially in solution with purified catechol oxidase. Two key parameters were identified: lag-time and the rate of melanin formation. The lag-time was taken as the time required by intermediates to reach a critical concentration at which the polymerization process starts and melanin production becomes measurable (at 640 nm). In solution, the lag-time decreases as the enzyme activity increases, particularly when the activity is very low. The rate at which melanin is formed by pure enzyme in solution is independent of dopa concentration when its activity is low but increases linearly with dopa concentration when the activity is comparatively high.In fixed melanoma cells, the lag-time decreases linearly with increases of dopa concentrations up to 20mm; at concentrations higher than this, the lag decreases more slowly. In contrast, the rate of melanin production is unaffected by changes in dopa concentration. The lag-times of different cells lines incubated at the same substrate concentration decrease as the enzyme activity of the cells increases. The rate of melanin production seems to be affected by factors other than catechol oxidase activity, such as the intracellular organization and distribution of the enzyme.     

Involvement of reactive oxygen species in the oxidation of tyrosine and dopa to melanin and in skin tanning

The role of reactive oxygen (1O2 and O2-.) in skin photosensitization and tanning reaction has been examined. Riboflavin (RF), hematoporphyrin (HP), 3-carbethoxypsoralen (3-CP), and 8-methoxypsoralen (8-MOP), upon photoexcitation under aerobic conditions, produced singlet O2 (1O2). RF, 3-CP, and 8-MOP also produced superoxide anion (O2-.). Reactive O2 produced by photosensitized RF, 3-CP, and 8-MOP was found to oxidize tyrosine and dopa to dopachrome and subsequently their conversion to melanin. HP did not oxidize tyrosine to dopachrome, and 3-CP and RF revealed substantial oxidation of tyrosine. Dopa was oxidized to dopachrome and subsequently to melanin by all photosensitizers tested at a variable rate as follows: RF greater than 3-CP greater than HPD greater than 8-MOP. UVA alone and to a lesser extent UVB also produced 1O2 which induced the oxidation of tyrosine and dopa to dopachrome and subsequently to melanin. The production of dopachrome was higher with dopa compared to tyrosine under all irradiation conditions. These observations appear to have relevance to the O2-requiring immediate tanning reaction of the skin stimulated by solar radiation and in the induction of skin photosensitization.     

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.     

Analysis of the structure of synthetic and natural melanins by solid-phase NMR

The structures of one synthetic and two natural melanins are examined by solid-state NMR using cross polarization, magic angle sample spinning, and high-power proton decoupling. The structural features of synthetic dopa melanin are compared to those of melanin from malignant melanoma cells grown in culture and sepia melanin from squid ink. Natural abundance 13C and 15N spectra show resonances consistent with known pyrrolic and indolic structures within the heterogeneous biopolymer; 13C spectra indicate the presence of aliphatic residues in all three materials. These solid-phase experiments illustrate the promise of solid-phase NMR for elucidating structural information from insoluble biomaterials.     

Hydroxylation of tyrosine by plant peroxidase and mushroom tyrosinase, with and without hydrazine, to retard the oxidation of dopa.

Tyrosine was oxidized to dopa by horseradish peroxidase and mushroom tyrosinase. The first step of hydroxylation of tyrosine in the synthesis of melanin was demonstrated by isolation of dopa from the reaction mixture using hydrazine as a selective retardant.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.     

Role of estradiol and 2-hydroxyestradiol in melanin formation in vitro

Melanin formation from 3,4-dihydroxyphenylalanine (dopa) was studied in the presence of estradiol and 2-hydroxyestradiol by use of a tyrosinase isolated from B16-F10 melanoma cells grown in C57 black female mice. Both steroids were found incorporated into melanin, but the 2-hydroxy compound was incorporated to a higher extent. The melanin was also able to bind substantial amounts of the two steroids, and the more highly oxidized compound showed higher binding. Melanin isolated from incubates of dopa with mushroom tyrosinase has the ability to bind the steroids and to incorporate small amounts into its structure. It is suggested that melanin in mammalian tissues may function as a depository for estrogens, particularly for those which are more highly oxidized.     

Pigment Deposition in Viscera Associated with Prolonged Chlorpromazine Therapy

Twelve physically healthy young adult mental hospital patients died unexpectedly while on prolonged chlorpromazine therapy. Five of them had clinically obvious pigmentation of the exposed skin. Two of these had impairment of vision as well. Autopsies were performed on all 12 patients. Extensive deposits of pigment (exhibiting the physical and histochemical properties of melanin) were present in macrophages in the dermis and throughout the reticuloendothelial system, and in the parenchymal cells of internal organs. The dopa tyrosinase reaction indicated increased melanocyte activity in the epidermis.The possible mechanism of production of this pigment is discussed, and the belief is expressed that the increased melanin production is due, at least partly, to the effect of chlorpromazine on the autonomic nervous system, blocking the production of pigment-lightening factors, of which melatonin is the most important. A short outline of contemplated further investigation is given.     

Phosphorylated mixed isomers of L-dopa increase melanin content in skins of Skh-2 pigmented hairless mice

Dopa phosphates, a new class of compounds, contain phosphate-ester linkages at the 3- and/or 4- positions of the phenylalanine ring of L-dopa. Dopa phosphates have been shown to increase pigment production in the epidermis of hairless mice. Groups of Skh-2 pigmented hairless mice were treated topically with various concentrations of dopa phosphates daily for five weeks. Half of each group received suberythemal UVB radiation three times weekly for four weeks from a bank of filtered FS20 lamps. UVB and dopa phosphates alone each caused a modest increase in epidermal pigmentation. However, treatment of mice with dopa phosphates plus UVB radiation resulted in a marked increase in pigmentation, greater than with either treatment alone. The optimal concentration of dopa phosphates was 0.01% (100 micrograms/ml Tris-glycerol buffer) whether or not they were applied in conjunction with UVB radiation. Histological analyses revealed that dopa phosphates and UVB radiation each caused an increase in the number of pigmented melanocytes in the epidermis. Control groups treated with Tris-glycerol buffer alone, or buffer containing L-phenylalanine or L-dopa showed no significant changes in pigmentation. Our results indicate that dopa phosphates stimulate the production of melanin and affect the development and distribution of melanocytes in the skin of Skh-2 mice. By these criteria, dopa phosphates and UVB act in a similar manner to increase melanin content in the skin. The processes may be related to those recently observed in cultured mouse melanoma cells where dopa phosphates are incorporated into melanin, presumably following enzymatic hydrolysis by cellular phosphatases with the resultant production of L-dopa and inorganic phosphate.     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

Spleen and Liver Pigmented Macrophages of Rana Esculenta L. A New Melanogenic System?

The present study reports the results of a morpho‐functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin‐synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosine‐hydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p‐phenylenediamine‐pyrocatechol (PPD‐PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenol‐oxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue‐specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Mutational mapping of the catalytic activities of human tyrosinase.

Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.