The effect of oxidative degradation of Dopa-melanin on its basic physicochemical properties and photoreactivity.

Melanin, particularly eumelanin, is commonly viewed as an efficient antioxidant and photoprotective pigment. Nonetheless, the ability of melanin to photogenerate reactive oxygen species and sensitize the formation of cyclobutane pyrimidine dimers may contribute to melanin-dependent phototoxicity. The phototoxic potential of melanin depends on a variety of factors, including molecular composition, redox state, and degree of aggregation. Using complementary spectroscopic and analytical methods we analyzed the physicochemical properties of Dopa-melanin, a synthetic model of eumelanin, subjected to oxidative degradation induced by aerobic photolysis or exposure to 0.1 M hydrogen peroxide. Both modes of oxidative degradation were accompanied by dose-dependent bleaching of melanin and irreversible modifications of its paramagnetic, ion- and electron-exchange and antioxidant properties. Bleached melanin exhibited enhanced efficiency to photogenerate singlet oxygen in both UVA and short-wavelength visible light. Although chemical changes of melanin subunits, including a relative increase of DHICA content and disruption of melanin polymer induced by oxidative degradation were considered, these two mechanisms may not be sufficient for a satisfactory explanation of the elevated photosensitizing ability of the bleached eumelanin. This study points out possible adverse changes in the photoprotective and antioxidant properties of eumelanin that could occur in pigmented tissues after exposure to high doses of intense solar radiation.

Antioxidant and Neuroprotective Activity of Vitamin E Homologues: In Vitro Study.

Here we present comparative data on the inhibition of lipid peroxidation by a variety of tocochromanols in liposomes. We also show for the first time the potential neuroprotective role of all the vitamin E homologues investigated on the neuronally differentiated human neuroblastoma SH-SY5Y cell line. α-Tocopherol had nearly no effect in the inhibition of lipid peroxidation, while ß-, γ-, and δ-tocopherols inhibited the reaction completely when it was initiated in a lipid phase. Similar effects were observed for tocotrienol homologues. Moreover, in this respect plastochromanol-8 was as effective as ß-, γ-, and δ-tocochromanols. When the prenyllipids were investigated in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) test and incorporated into different lipid carriers, the radical oxidation was most pronounced in liposomes, followed by mixed micelles and the micellar system. When the reaction of tocochromanols was examined in niosomes, the oxidation was most pronounced for α-tocopherol and plastochromanol-8, followed by α-tocotrienol. Next, using retinoic acid-differentiated SH-SY5Y cells, we tested the protective effects of the compounds investigated on hydrogen peroxide (H2O2)-induced cell damage. We showed that tocotrienols were more active than tocopherols in the oxidative stress model. Plastochromanol-8 had a strong inhibitory effect on H2O2-induced lactate dehydrogenase (LDH) release and H2O2-induced decrease in cell viability. The water-soluble α-tocopherol phosphate had neuroprotective effects at all the concentrations analyzed. The results clearly indicate that structural differences between vitamin E homologues reflect their different biological activity and indicate their potential application in pharmacological treatments for neurodegenerative diseases. In this respect, the application of optimal tocochromanol-carrying structures might be critical.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

Comparison of ultrasmall IONPs and Fe salts biocompatibility and activity in multi-cellular in vitro models.

In the paper, the results of the first regular studies of ultra-small iron oxide nanoparticles (IONPs) toxicity in vitro were presented. The influence of PEG-coated NPs with 5 nm magnetite core on six different cell lines was examined. These were: human bronchial fibroblasts, human embryonic kidney cells (HEK293T), two glioblastoma multiforme (GBM) cell lines as well as GBM cells isolated from a brain tumor of patient. Additionally, mouse macrophages were included in the study. The influence of IONPs in three different doses (1, 5 and 25 µg Fe/ml) on the viability, proliferation and migration activity of cells was assessed. Moreover, quantifying the intracellular ROS production, we determined the level of oxidative stress in cells exposed to IONPs. In the paper, for the first time, the effect of Fe in the form of IONPs was compared with the analogical data obtained for iron salts solutions containing the same amount of Fe, on the similar oxidation state. Our results clearly showed that the influence of iron on the living cells strongly depends not only on the used cell line, dose and exposure time but also on the form in which this element was administered to the culture. Notably, nanoparticles can stimulate the proliferation of some cell lines, including glioblastoma multiforme. Compared to Fe salts, they have a stronger negative impact on the viability of the cells tested. Ultra-small NPs, also, more often positively affect cell motility which seem to differ them from the NPs with larger core diameters.

The influence of iron on selected properties of synthetic pheomelanin.

It is believed that while eumelanin plays photoprotective and antioxidant role in pigmented tissues, pheomelanin being more photoreactive could behave as a phototoxic agent. Although the metal ion-sequestering ability of melanin might be protective, transition metal ions present in natural melanins could affect their physicochemical properties. The aim of this research was to study iron binding by pheomelanin and analyze how such a binding affects selected properties of the melanin. Synthetic pheomelanin (CDM), prepared by enzymatic oxidation of DOPA in the presence of cysteine was analyzed by electron paramagnetic resonance (EPR) spectroscopy, spectrophotometry, chemical analysis, and time-resolved measurements of singlet oxygen phosphorescence. Iron broadened EPR signal of melanin and increased its optical absorption. Iron bound to melanin exhibited EPR signal at g = 4.3, typical for high-spin iron (III). Iron bound to melanin significantly altered the kinetics of melanin photodegradation, which in turn modified the accessibility and stability of the melanin-iron complexes as indicated by the release of iron from melanin induced by diethylenetriaminepentaacetic acid and KCN. Although bound to melanin iron little affects initial stages of photodegradation of CDM, the effect of iron becomes more pronounced at later stages of melanin photolysis.

Analysis of photoreactivity and phototoxicity of riboflavin's analogue 3MeTARF.

Recent studies focus on usage of blue light of λ = 450 nm in combination with photosensitizers to treat surface skin disorders, including cancers. In search of convenient therapeutic factor we studied riboflavin analogue 3-methyl-tetraacetylriboflavin (3MeTARF) as potential sensitizer. Riboflavin (Rfl) itself, non -toxic in the darkness, upon absorption of UVA and blue light, may act as photosensitizer. However, Rfl efficiency is limited due to its susceptibility to photodecomposition. Riboflavin's acetylated analogue, 3MeTARF, bears substituents in ribose chain, which inhibit intramolecular processes leading to degradation. Upon excitation, this compound, reveals higher photochemical resistance, remaining a good singlet oxygen generator. Thus, being more stable as the sensitizer, might be much more efficient in photodynamic processes. The objective of undertaken study was to elucidate mechanisms of 3MeTARF photoreactivity under the irradiation with blue light in comparison to its mater compound, riboflavin. We approached this goal by using spectroscopic methods, like direct singlet oxygen phosphorescence detection at 1270 nm, EPR spin trapping and oximetry. Additionally, we tested both riboflavin and 3MeTARF phototoxicity against melanoma cells (WM115) and we studied mechanism of photodynamic cell death, as well. Moreover, 3MeTARF induces apoptosis in melanoma cells at ten times lower concentration than riboflavin itself. Our studies confirmed that 3MeTARF remains stable upon blue light activation and is more efficient photosensitizer than Rfl.

Interaction of iron ions with melanin.

One of the antioxidant roles of melanin is binding redox-active transition metal ions. The aim of this study was to examine the redox reactions accompanying iron binding by melanin. Two kinds of synthetic eumelanin were mixed with iron (II) and iron (III) in the presence and absence of citrate and ADP in the aerobic and anaerobic system. The iron binding was examined by electron paramagnetic resonance (EPR) spectroscopy and thiocyanate assay. Obtained results indicate that although melanin reduces iron (III) that is unbound to this polymer, binding of iron (II) is accompanied by its oxidation by melanin.

Melanin presence inhibits melanoma cell spread in mice in a unique mechanical fashion.

Melanoma is a highly aggressive cancer that exhibits metastasis to various critical organs. Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts, becoming heavily pigmented. Until now the role of melanin in melanoma, particularly the effect of melanin presence on the abilities of melanoma cells to spread and metastasize remains unknown. Recently, we have shown that melanin dramatically modified elastic properties of melanoma cells and inhibited the cells invasive abilities in vitro. Here, we inoculated human melanoma cells with different melanin content into nude mice and tested the hypothesis that cell elasticity is an important property of cancer cells for their efficient spread in vivo. The obtained results clearly showed that cells containing melanin were less capable to spread in mice than cells without the pigment. Our findings indicate that the presence of melanin inhibits melanoma metastasis, emphasizing possible clinical implications of such an inhibitory effect.

The Ability of Functionalized Fullerenes and Surface-Modified TiO2 Nanoparticles to Photosensitize Peroxidation of Lipids in Selected Model Systems.

Photochemical properties of a new class of inorganic nanoparticles, namely a cationic C60 fullerene substituted with three quaternary pyrrolidinium groups (BB6) and a surface-modified nanocrystalline TiO2 with bromopyrogallol red (Brp@TiO2 ) were examined for their effectiveness in photogenerating singlet oxygen and free radicals. In particular, their ability to photosensitize peroxidation of unsaturated lipids was analyzed in POPC:cholesterol liposomes and B16 mouse melanoma cells employing a range of spectroscopic and analytical methods. Because melanoma cells typically are pigmented, we examined the effect of melanin on the photosensitized peroxidation of lipids in liposomes and B16 melanoma cells, mediated by BB6 and Brp@TiO2 nanoparticles. The obtained results suggest that peroxidation of unsaturated lipids, photosensitized by BB6 occurs mainly, although not exclusively, via Type II mechanism involving singlet oxygen. On the other hand, if surface-modified TiO2 is used as a photosensitizer, Type I mechanism of lipid peroxidation dominates, as indicated by the predominant formation of the free radical-dependent cholesterol oxidation products. The protective effect of melanin was particularly evident when BB6 was used as a photosensitizer, suggesting that melanin could efficiently interfere with Type II processes.

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis.

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5-S-cysteinyldopa (5SCD-M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV-visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD-M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD-M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin-eumelanin ratio, such undesirable changes could occur in individual of all skin types.

Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells.

Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells' metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells' metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of melanoma.

The nanomechanical role of melanin granules in the retinal pigment epithelium.

Nanomechanical properties of cells and tissues, in particular their elasticity, play an important role in different physiological and pathological processes. Recently, we have demonstrated that melanin granules dramatically modify nanomechanical properties of melanoma cells making them very stiff and, as a result, less aggressive. Although the mechanical effect of melanin granules was demonstrated in pathological cells, it was never studied in the case of normal cells. In this work, we analyzed the impact of melanin granules on nanomechanical properties of primary retinal pigment epithelium tissue fragments isolated from porcine eyes. The obtained results clearly show that melanin granules are responsible for the exceptional nanomechanical properties of the tissue. Our findings suggest that melanin granules in the retinal pigment epithelium may play an important role in sustaining the stiffness of this single cell layer, which functions as a natural mechanical barrier separating the retina from the choroid.

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion.

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6-dihydroxyindole-2-carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5-S-cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time-resolved near-infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450-nm, quantum yield of singlet oxygen was very low (~10-4 ) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.

Roles of reactive oxygen species in UVA-induced oxidation of 5,6-dihydroxyindole-2-carboxylic acid-melanin as studied by differential spectrophotometric method.

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA-induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA-induced degradation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA-induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole-2,3,5-tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA-melanin was confirmed by direct measurements of singlet oxygen phosphorescence.

Curcumin does not switch melanin synthesis towards pheomelanin in B16F10 cells.

Melanin, the basic skin pigment present also in the majority of melanomas, has a huge impact on the efficiency of photodynamic, radio- or chemotherapies of melanoma. Moreover, the melanoma cells produce more melanin than normal melanocytes in adjacent skin do. Thus, attention has been paid to natural agents that are safe and effective in suppression of melanogenesis. B16F10 cells were studied by electron paramagnetic resonance (EPR) spectroscopy. The cells were cultured for 24-72 h in RPMI or DMEM with or without curcumin. The results confirmed that curcumin has no significant effect on B16F10 cells viability at concentrations of 1-10 µM. Curcumin at concentration of 10 µM significantly inhibited their proliferation and stimulated differentiation. We have not stimulated melanogenesis hormonally but we found a strong increase in melanogenesis in DMEM, containing more L-Tyr, as compared to RPMI. The EPR studies revealed that the effect of curcumin on melanogenesis in RPMI-incubated cells was not significant, and only in DMEM was curcumin able to inhibit melanogenesis. The effect of curcumin was only quantitative, as it did not switch eumelanogenesis towards pheomelanogenesis under any conditions. Interestingly, we observed elevation of production of hydrogen peroxide in DMEM-incubated cells, in parallel to the facilitation of melanogenesis. Curcumin significantly but transiently intensified the already pronounced generation of H2O2 in DMEM. We conclude that the quantitative effect of curcumin on melanogenesis in melanoma is intricate. It depends on the basic melanogenetic efficiency of the cells, and can be observed only in strongly pigmented cells. Qualitatively, curcumin does not switch melanogenesis towards pheomelanogenesis, either in strongly, or in weakly melanized melanoma cells.

Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells.

The relationship between melanin pigmentation and metastatic phenotype of melanoma cells is an intricate issue, which needs to be unambiguously determined to fully understand the process of metastasis of malignant melanoma. Despite significant research efforts undertaken to solve this problem, the outcomes are far from being satisfying. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells. This prompted us to examine the mechanical effects of melanosomes on the transmigration abilities of melanoma cells. Here, we show for the first time that melanin granules inhibit transmigration abilities of melanoma cells in a number of granules dependent manner. Moreover, we demonstrate that the inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may play a key role in the efficiency of melanoma cells spread in vivo. Our findings may also contribute to better understanding of the process of metastasis of malignant melanoma.

Nanomechanical analysis of pigmented human melanoma cells.

Based on hitherto measurements of elasticity of various cells in vitro and ex vivo, cancer cells are generally believed to be much softer than their normal counterparts. In spite of significant research efforts on the elasticity of cancer cells, only few studies were undertaken with melanoma cells. However, there are no reports concerning pigmented melanoma cells. Here, we report for the first time on the elasticity of pigmented human melanoma cells. The obtained data show that melanin significantly increases the stiffness of pigmented melanoma cells and that the effect depends on the amount of melanin inside the cells. The dramatic impact of melanin on the nanomechanical properties of cells puts into question widely accepted paradigm about all cancer cells being softer than their normal counterparts. Our findings reveal significant limitations of the nanodiagnosis approach for melanoma and contribute to better understanding of cell elasticity.

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin.

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age-related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) to pyrrole-2,3,5-tricarboxylic acid (PTCA) is a marker for the heat-induced cross-linking of eumelanin. In this study, we examined UVA-induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo-oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life-long exposure to blue light.

Melanosome-iron interactions within retinal pigment epithelium-derived cells.

Melanosomes were recently shown to protect ARPE-19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound-to-melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H(2) O(2) , but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease.

In Parkinson's disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton's reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron-neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron-neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron-neuromelanin is phagocytosed by microglia and iron-neuromelanin degradation releases reactive/toxic iron.