NO-induced apoptosis in human melanocytes from lightly and darkly pigmented skin.

Introduction: Nitric oxide (NO) is a potent modulator of programmed cell death, with the ability to both induce and prevent apoptosis. Some of the factors that are capable of triggering apoptosis of skin cells also cause NO overproduction in the epidermis. Unlike keratinocytes, melanin-producing melanocytes are highly resistant to apoptotic death. Aim: To investigate whether NO can induce apoptosis in normal human epidermal melanocytes and whether the pigmentation phenotype of the cells can affect their response to NO. Material and methods: Human epidermal melanocytes, derived from lightly and darkly pigmented neonatal foreskins, were cultured in the presence of various concentrations of SPER/NO. The effect of NO released from its donor on the cell morphology, viability, and proliferation was assessed. The ability of NO to induce cell apoptosis was evaluated by Hoechst 33342 staining, DNA fragmentation assay, flow cytometry with annexin V and propidium iodide staining, determination of caspase 3/7, 8, and 9 activities, and assessment of changes in the cell expression levels of BAX and BCL-2. Results: We have shown that NO is capable of inducing apoptosis in normal human epidermal melanocytes in vitro, with preferential activation of the intrinsic (mitochondrial) pathway. Melanocytes from darkly pigmented skin showed a strong increase in BCL-2 expression in response to NO and were significantly more resistant to apoptosis than those from lightly pigmented skin. Conclusions: The pigmentation phenotype may be an important factor modulating the response of human epidermal melanocytes to proapoptotic activity of extracellular NO.

The pigmentation phenotype of melanocytes affects their response to nitric oxide in vitro.

Introduction: It has been shown that nitric oxide (NO) can modulate the immune properties of epidermal melanocytes, and that overexpression of NO in the skin may contribute to inflammation-related pigmentary disorders. Little is known about whether constitutive cell pigmentation affects the sensitivity of melanocytes to NO. Aim: To compare the effect of NO on melanin synthesis and the expression of key melanogenesis-related genes in normal human melanocytes of various degrees of constitutive pigmentation. Material and methods: Human epidermal melanocytes derived from lightly and darkly pigmented skin (HEMn-LP and HEMn-DP, respectively) were cultured with or without a NO donor (SPER/NO). Then the total melanin content, the pheomelanin content, the activity and concentration of tyrosinase, and the expressions of TYR and DCT were assessed. Results: NO released from SPER/NO did not alter the total amount of melanin produced by cultured cells but increased the proportion of pheomelanin, especially in HEMn-DP. Transcriptional activity of the melanogenesis-related genes, in particular DCT, was downregulated in HEMn-DP and upregulated in HEMn-LP cultured with SPER/NO. Conclusions: NO can promote pheomelanogenesis in human epidermal melanocytes, and the cell response in this respect is associated with the pigmentation phenotype. During exposure to NO, melanocytes from dark skin produce much more pheomelanin than lightly pigmented cells. NO-induced overproduction of pheomelanin in the skin could be one of the factors responsible for the greater propensity to develop severe inflammatory dermatoses in dark-skinned individuals, or even melanoma de novo formation based on local chronic inflammation.

Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide-stimulated melanocytes from lightly and darkly pigmented skin.

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA-binding activity of NF-κB p50 and p65. We found substantial differences in the LPS-stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL-16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL-6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.

Detection and quantitation of a pheomelanin component in melanin pigments using pyrolysis-gas chromatography/tandem mass spectrometry system with multiple reaction monitoring mode.

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin-type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin-type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin.

Melanin from epidermal human melanocytes: study by pyrolytic GC/MS.

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.

Thermochemolysis as the useful method to assess the purity of melanin isolated from the human melanoma malignum.

Melanin formation in pigmented melanoma cells is considered as a target for the tumor therapy. The evaluation of potential correlation between melanin structure and the tumor type could be also of diagnostic and prognostic importance. One of the major problems in structural investigations of natural melanins is the lack of appropriate methods, which allow isolation of pure intact pigment. In this study the thermochemolysis technique was used to assess the purification grade of melanin isolated from the human melanoma malignum cells by two different enzymatic methods. Melanin samples were thermally degraded in the presence of tetramethylammonium hydroxide and the thermochemolysis products were analyzed by gas chromatography/mass spectrometry (GC/MS). Compounds of lipid origin, especially fatty acid methyl esters and aliphatic and cyclic hydrocarbons, were predominant among pyrolysis products of melanin isolated from the tumor cells by method I. In contrast, during thermochemolysis of the pigment sample isolated by the method II, mainly eumelanin markers (pyrrole and its methyl derivatives, toluene, styrene, phenol, benzyl nitrile and indole) were formed. The comparison of pyrolysis profiles of the analyzed samples indicate that method II is more efficient for melanoma pigment purification.

[The role of neuromelanin in Parkinson's disease--new concepts]. / Rola neuromelaniny w chorobie Parkinsona--nowe koncepcje.

Progressive degeneration of dopaminergic neurons of the substantia nigra and the resulting dopamine deficiency in the striatum are neuropathological basis of the movement disorders in Parkinson's disease (PD). Neuromelanin-containing neurons are particularly susceptible to degeneration and their depigmentation is the hallmark of the advanced disease. The proposed mechanisms underlaying the pathogenesis and progression of neurodegeneration in the substantia nigra include iron-catalyzed oxidative stress, mitochondrial dysfunctions, inflammation and disturbances of protein metabolism. This review presents some new concepts concerning important but ambiguous role of neuromelanin in the above mentioned processes. It seems that the imbalance between cytoprotective and cytotoxic action of the pigment may cause neuronal death via mitochondrial oxidative stress, inhibition of ubiquitine-proteasome system and alpha-synuclein accumulation. Extraneuronal melanin may contribute to chronic inflammation by excessive secretion of cytokines and nitric oxide due to prolonged microglia activation. Recent reports suggest a possible role of the lipid component of neuromelanin in pigment-dependent cytotoxicity.

GC/MS analysis of thermally degraded neuromelanin from the human substantia nigra.

Neuromelanin (NM) is a complex polymer pigment found in catecholaminergic neurons of the human brain. The structure, formation pathway, and physiological function of NM have not yet been clarified, but interest in this polymer has been sparked by the suggestion that NM is involved in cell death in Parkinson's disease. In the current study, pyrolysis-gas chromatography/mass spectrometry analysis was applied for structural investigation of NM isolated from the human substantia nigra, using synthetic eumelanin and pheomelanin-type pigments as reference materials. None of the heterocyclic, sulfur-containing compounds being characteristic thermal degradation products of cysteinyldopamine-derived units of synthetic pheomelanin standard was detected in the pyrolysates of natural NM. The results suggest that nigral pigment isolated from normal brain tissue does not contain benzothiazine-type monomer units. Pyrolytic experiments in the presence of a derivatizing agent allowed identification of high levels of saturated and monounsaturated straight-chain C14-C18 fatty acids and led to the conclusion that a part of a lipid component is chemically bound to the NM macromolecule. The nigral pigment was also shown to be tightly associated with an isoprenoid-type compound.