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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.
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Continuous exposure of human skin to air pollution can result in a range of undesirable skin conditions. In our recent study, UV and visible light were found to increase cytotoxicity of fine particulate matter (PM2.5 ) against human keratinocytes. Since it is impossible to avoid exposure of human skin to PM2.5 , effective strategies are needed to reduce their damaging effects. l-ascorbic acid and resveratrol were tested as potential topical agents against pollution-related skin impairment. Although these agents were previously found to ameliorate PM-dependent damage, the effect of light and seasonal variation of particles were not previously studied. EPR spin-trapping, DPPH assay, and singlet oxygen phosphorescence were used to determine the scavenging activities of the antioxidants. MTT, JC-10 and iodometric assays were used to analyze the effect on PM2.5 -induced cytotoxicity, mitochondrial damage and oxidation of lipids. Live-cell imaging was employed to examine wound-healing properties of cells. Light-induced, PM2.5 -mediated oxidative damage was examined by immunofluorescent staining. Both antioxidants effectively scavenged free radicals and singlet oxygen produced by PM2.5 , reduced cell death and prevented oxidative damage to HaCaT cells. l-ascorbic acid and resveratrol, especially when applied in combination, can protect HaCaT cells against the dark and light induced toxicity of PM2.5 .
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Poluentes Atmosféricos , Material Particulado , Humanos , Material Particulado/toxicidade , Material Particulado/análise , Resveratrol/farmacologia , Células HaCaT , Oxigênio Singlete/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Estresse Oxidativo , Ácido Ascórbico/farmacologia , Poluentes Atmosféricos/farmacologia , Poluentes Atmosféricos/toxicidadeRESUMO
Staphylococcus aureus is a key pathogen in atopic dermatitis (AD) pathogenicity. Over half of AD patients are carriers of S. aureus. Clinical isolates derived from AD patients produce various staphylococcal enterotoxins, such as staphylococcal enterotoxin C or toxic shock syndrome toxin. The production of these virulence factors is correlated with more severe AD. In this study, we propose cationic heme-mimetic gallium porphyrin (Ga3+CHP), a novel gallium metalloporphyrin, as an anti-staphylococcal agent that functions through dual mechanisms: a light-dependent mechanism (antimicrobial photodynamic inactivation, aPDI) and a light-independent mechanism (suppressing iron metabolism). Ga3+CHP has two additive quaternary ammonium groups that increase its water solubility. Furthermore, Ga3+CHP is an efficient generator of singlet oxygen and can be recognized by heme-target systems such as Isd, which improves the intracellular accumulation of this compound. Ga3+CHP activated with green light effectively reduced the survival of clinical S. aureus isolates derived from AD patients (>5 log10 CFU/mL) and affected their enterotoxin gene expression. Additionally, there was a decrease in the biological functionality of studied toxins regarding their superantigenicity. In aPDI conditions, there was no pronounced toxicity in HaCaT keratinocytes with both normal and suppressed filaggrin gene expression, which occurs in â¼50% of AD patients. Additionally, no mutagenic activity was observed. Green light-activated gallium metalloporphyrins may be a promising chemotherapeutic to reduce S. aureus colonization on the skin of AD patients.
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BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disorder of unknown etiology characterized by organ fibrosis and microcirculation dysfunction. Emerging evidence suggests that SSc is related to increased oxidative stress, which contributes to further tissue and vascular damage. METHODS: Oxidative stress response in the peripheral blood was assessed in patients with SSc (nâ¯=â¯55) and well-matched controls (nâ¯=â¯44) using real-time monitoring of protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also analyzed the relationship between HP generation and SSc clinics, systemic inflammation, and cellular fibronectin, an emerging biomarker of endothelial damage. RESULTS: SSc was characterized by a significantly faster (2-fold) fluorescent product generation in the CBA assay and higher cumulative HP formation (3-fold) compared to controls (p<0.001, both). The dynamics of HP generation were not associated with the form of the disease (diffuse vs. limited SSc), current immunosuppressive therapy use, presence of abnormal nailfold capillaries, and autoantibody profile. Still, it was enhanced in patients with more severe illness and certain clinical manifestations (i.e., pulmonary hypertension, digital ulcers, and cyclophosphamide treatment) and in smokers (current or past). Higher serum CRP, blood eosinophil count, and cellular fibronectin with lower hemoglobin levels were independent determinants of increased HP formation. CONCLUSIONS: Our data indicate a pro-oxidant imbalance in SSc, likely related to systemic inflammation and endothelial injury. However, extensive prospective studies are needed to verify whether it is also associated with clinical disease progression.
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Endotélio , Inflamação , Escleroderma Sistêmico , Humanos , Estresse Oxidativo , Escleroderma Sistêmico/sangue , Microcirculação , Biomarcadores , Endotélio/lesões , Estudos de Casos e Controles , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , IdosoRESUMO
Photoreceptor membranes have a unique lipid composition. They contain a high level of polyunsaturated fatty acids including the most unsaturated fatty acid in nature, docosahexaenoic acid (22:6), and are enriched in phosphatidylethanolamines. The phospholipid composition and cholesterol content of the subcellular components of photoreceptor outer segments enables to divide photoreceptor membranes into three types: plasma membranes, young disc membranes, and old disc membranes. A high degree of lipid unsaturation, extended exposure to intensive irradiation, and high respiratory demands make these membranes sensitive to oxidative stress and lipid peroxidation. Moreover, all-trans retinal (AtRAL), which is a photoreactive product of visual pigment bleaching, accumulates transiently inside these membranes, where its concentration may reach a phototoxic level. An elevated concentration of AtRAL leads to accelerated formation and accumulation of bisretinoid condensation products such as A2E or AtRAL dimers. However, a possible structural impact of these retinoids on the photoreceptor-membrane properties has not yet been studied. In this work we focused just on this aspect. The changes induced by retinoids, although noticeable, seem not to be significant enough to be physiologically relevant. This is, however, an positive conclusion because it can be assumed that accumulation of AtRAL in photoreceptor membranes will not affect the transduction of visual signals and will not disturb the interaction of proteins engaged in this process.
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Even though melanin is commonly viewed as natural photoprotectant, the pigment demonstrates residual photoreactivity, which under certain conditions could contribute to UVA-dependent melanomagenesis. Skin melanin is constantly exposed to external stressors, including solar radiation, which could induce photodegradation of the pigment. Although photodegradation of melanin pigments was studied in synthetic models and RPE melanosomes, photochemical and photobiological effects of experimental photodegradation of human skin melanin of different chemical composition remain unknown. In this work, melanosomes isolated from hair of individuals of different skin phototypes (I-III, V) were exposed to high-intensity violet light and its impact on physical and chemical properties of the pigments were analyzed using electron paramagnetic resonance (EPR), spectrophotometry and dynamic light scattering (DLS). Photoreactivity of photodegraded melanins was examined by EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Antioxidant potential of the pigments was measured using the EPR DPPH assay. Cellular effect of the exposure of melanosome-loaded HaCaT cells to UV-Vis light was determined by MTT assay, JC-10 assay, and iodometric assay. The data revealed that experimental photodegradation increased photoreactivity of natural melanins, while decreasing their antioxidant capacity. Photodegraded melanin was responsible for higher cell death, a decrease in mitochondrial membrane potential and elevated levels of lipid hydroperoxides.
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Antioxidantes , Melaninas , Humanos , Antioxidantes/metabolismo , Melaninas/metabolismo , Luz , Melanossomas/metabolismo , Melanossomas/efeitos da radiação , CabeloRESUMO
The mechanism of very efficient relaxation of the melanin-photoexcited states, responsible for the photoprotective action of the pigment, remains a subject for intense investigation. The most recent study by C. Grieco, F. Kohl, and B. Kohler, entitled "Ultrafast radical photogeneration pathways in eumelanin," addresses key issues of melanin photophysics and photochemistry. By using femtosecond broad-band pump probe-transient absorption measurements, the researchers were able to identify the absorption spectrum of DOPA melanin radicals for the first time and proposed two distinct mechanisms of radical formation-photoionization and photoinduced charge separation. The observed photodynamic of melanin radicals suggests a new paradigm in which the ultrafast excited state deactivation is due to the efficient recombination of melanin radicals created promptly by photoexcitation.
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Melaninas , Melaninas/metabolismo , Radicais LivresRESUMO
Dopamine (DA) is a precursor of neuromelanin (NM) synthesized in the substantia nigra of the brain. NM is known to contain considerable levels of Fe and Cu. However, how Fe and Cu ions affect DA oxidation to DA-eumelanin (DA-EM) and modify its structure is poorly understood. EMs were prepared from 500 µM DA, dopaminechrome (DAC), or 5,6-dihydroxyindole (DHI). Autoxidation was carried out in the absence or presence of 50 µM Fe(II) or Cu(II) at pH 7.4 and 37 â. EMs were characterized by Soluene-350 solubilization analyzing absorbances at 500 nm (A500) and 650 nm (A650) and alkaline hydrogen peroxide oxidation (AHPO) yielding various pyrrole carboxylic acids. Pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) served as a molecular marker of cross-linked DHI units. Importantly, Fe and Cu accelerated DA oxidation to DA-EM and DHI oxidation to DHI-EM several-fold, whereas these metals only weakly affected the production of DAC-EM. The A500 values indicated that DA-EM contains considerable portions of uncyclized DA units. Analysis of the A650/A500 ratios suggests that Fe and Cu caused some degradation of DHI units of DA-EM during 72-h incubation. Results with AHPO were consistent with the A500 values and additionally revealed that (1) DA-EM is less cross-linked than DAC-EM and DHI-EM and (2) Fe and Cu promote cross-linking of DHI units. In conclusion, Fe and Cu not only accelerate the oxidation of DA to DA-EM but also promote cross-linking and degradation of DHI units. These results help to understand how Fe and Cu in the brain affect the production and properties of NM.
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Dopamina , Ferro , Dopamina/metabolismo , Ferro/metabolismo , Cobre , Melaninas/metabolismo , Oxirredução , Peróxido de Hidrogênio/químicaRESUMO
Neuromelanins are compounds accumulating in neurons of human and animal brain during aging, with neurons of substantia nigra and locus coeruleus having the highest levels of neuromelanins. These compounds have melanic, lipid, peptide, and inorganic components and are contained inside special autolysosomes. Neuromelanins can participate in neuroprotective or toxic processes occurring in Parkinson's disease according to cellular environment. Their synthesis depends on the concentration of cytosolic catechols and is a protective process since it prevents the toxic accumulation of catechols-derived reactive compounds. Neuromelanins can be neuroprotective also by binding reactive/toxic metals to produce stable and non-toxic complexes. Extraneuronal neuromelanin released by dying dopamine neurons in Parkinson's disease activates microglia which generate reactive oxygen species, reactive nitrogen species, and proinflammatory molecules, thus producing still neuroinflammation and neuronal death. Synthetic neuromelanins have been prepared with melanic, protein structure, and metal content closely mimicking the natural brain pigment, and these models are also able to activate microglia. Neuromelanins have different structure, synthesis, cellular/subcellular distribution, and role than melanins of hair, skin, and other tissues. The main common aspect between brain neuromelanin and peripheral melanin is the presence of eumelanin and/or pheomelanin moieties in their structure.
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Doença de Parkinson , Animais , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doenças Neuroinflamatórias , Encéfalo/metabolismo , Melaninas/química , Melaninas/metabolismo , Neurônios Dopaminérgicos/metabolismoRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds derived mostly from the incomplete combustion of fossil fuels and biomass. Human skin can absorb PAHs and the uptake increases with their molar mass and lipophilicity. Benzopyrene is high molecular weight PAH frequently appearing in ambient pollution. It exists in two isomeric forms: benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP), which exhibit different biological activity. Although certain properties of benzopyrenes suggested photoreactivity of the compounds, no direct measurements were previously conducted to characterize their photochemical activity. In this study, quantum yield and action spectra of singlet oxygen photogeneration by BaP and BeP were measured by time-resolved near-infrared phosphorescence, and the ability of both compounds to photogenerate superoxide anion was assessed by electron paramagnetic resonance (EPR) spin-trapping. The measurements revealed high efficiency of benzopyrenes to photogenerate singlet oxygen and their ability to photogenerate superoxide anion. Using HaCaT cells as single-layer skin model, we demonstrated concentration-dependent and light-dependent cytotoxicity of BaP and BeP. The compounds induced damage to the cell mitochondria and elevated the levels of intracellular reactive oxygen species.
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Benzo(a)pireno , Hidrocarbonetos Policíclicos Aromáticos , Humanos , Benzo(a)pireno/toxicidade , Benzo(a)pireno/química , Superóxidos , Oxigênio Singlete , Benzopirenos/farmacologia , QueratinócitosRESUMO
In the retina, retinoids involved in vision are under constant threat of oxidation, and their oxidation products exhibit deleterious properties. Using pulse radiolysis, this study determined that the bimolecular rate constants of scavenging cation radicals of retinoids by taurine are smaller than 2 × 107 M-1s-1 whereas lutein scavenges cation radicals of all three retinoids with the bimolecular rate constants approach the diffusion-controlled limits, while zeaxanthin is only 1.4-1.6-fold less effective. Despite that lutein exhibits greater scavenging rate constants of retinoid cation radicals than other antioxidants, the greater concentrations of ascorbate in the retina suggest that ascorbate may be the main protectant of all visual cycle retinoids from oxidative degradation, while α-tocopherol may play a substantial role in the protection of retinaldehyde but is relatively inefficient in the protection of retinol or retinyl palmitate. While the protection of retinoids by lutein and zeaxanthin appears inefficient in the retinal periphery, it can be quite substantial in the macula. Although the determined rate constants of scavenging the cation radicals of retinol and retinaldehyde by dopa-melanin are relatively small, the high concentration of melanin in the RPE melanosomes suggests they can be scavenged if they are in proximity to melanin-containing pigment granules.
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Retinoides , Vitamina A , Melaninas , Retinaldeído , Luteína , Zeaxantinas , Taurina , CátionsRESUMO
Airway inflammation in asthma is related to increased reactive oxygen species generation, potentially leading to tissue injury and subsequent airway remodeling. We evaluated oxidative stress in peripheral blood from asthmatic subjects (n = 74) and matched controls (n = 65), using recently developed real-time monitoring of the protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also investigated the relation of the systemic oxidative stress response in asthma to disease severity, lung function, airway remodeling indices (lung computed tomography and histology), and blood and bronchoalveolar lavage fluid (BAL) inflammatory biomarkers. We documented enhanced systemic oxidative stress in asthma, reflected by 35% faster and 58% higher cumulative fluorescent product generation in the CBA assay (p < 0.001 for both). The dynamics of HP generation correlated inversely with lung function but not with asthma severity or histological measures of airway remodeling. HP generation was associated positively with inflammatory indices in the blood (e.g., C-reactive protein) and BAL (e.g., interleukin [IL]-6, IL-12p70, and neutrophil count). Bronchial obstruction, thicker airway walls, increased BAL IL-6, and citrullinated histone 3 in systemic circulation independently determined increased HP formation. In conclusion, a real-time CBA assay showed increased systemic HP generation in asthma. In addition, it was associated with inflammatory biomarkers, suggesting that proper disease control can also lead to a decrease in oxidative stress.
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Melanin pigment plays a critical role in the protection against the harmful effects of ultraviolet radiation and other environmental stressors. It is produced by the enzymatic transformation of L-tyrosine to dopaquinone and subsequent chemical and biochemical reactions resulting in the formation of various 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI) oligomers-main constituents of eumelanin, and benzothiazine and benzothiazole units of pheomelanin. The biosynthesis of melanin is regulated by sun exposure and by many hormonal factors at the tissue, cellular, and subcellular levels. While the presence of melanin protects against the development of skin cancers including cutaneous melanoma, its presence may be necessary for the malignant transformation of melanocytes. This shows a complex role of melanogenesis in melanoma development defined by chemical properties of melanin and the nature of generating pathways such as eu- and pheomelanogenesis. While eumelanin is believed to provide radioprotection and photoprotection by acting as an efficient antioxidant and sunscreen, pheomelanin, being less photostable, can generate mutagenic environment after exposure to the short-wavelength UVR. Melanogenesis by itself and its highly reactive intermediates show cytotoxic, genotoxic, and mutagenic activities, and it can stimulate glycolysis and hypoxia-inducible factor 1-alpha (HIF-1α) activation, which, combined with their immunosuppressive effects, can lead to melanoma progression and resistance to immunotherapy. On the other hand, melanogenesis-related proteins can be a target for immunotherapy. Interestingly, clinicopathological analyses on advanced melanomas have shown a negative correlation between tumor pigmentation and diseases outcome as defined by overall survival and disease-free time. This indicates a "Yin and Yang" role for melanin and active melanogenesis in melanoma development, progression, and therapy. Furthermore, based on the clinical, experimental data and diverse effects of melanogenesis, we propose that inhibition of melanogenesis in advanced melanotic melanoma represents a realistic adjuvant strategy to enhance immuno-, radio-, and chemotherapy.
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One of the factors determining efficient antimicrobial photodynamic inactivation (aPDI) is the accumulation of a light-activated compound, namely, a photosensitizer (PS). Targeted PS recognition is the approach based on the interaction between the membrane receptor on the bacterial surface and the PS, whereas the compound is efficiently accumulated by the same mechanism as the natural ligand. In this study, we showed that gallium mesoporphyrin IX (Ga3+MPIX) provided dual functionalityâiron metabolism disruption and PS properties in aPDI. Ga3+MPIX induced efficient (>5log10 reduction in CFU/mL) bacterial photodestruction with excitation in the area of Q band absorption with relatively low eukaryotic cytotoxicity and phototoxicity. The Ga3+MPIX is recognized by the same systems as haem by the iron-regulated surface determinant (Isd). However, the impairment in the ATPase of the haem detoxification efflux pump was the most sensitive to the Ga3+MPIX-mediated aPDI phenotype. This indicates that changes within the metalloporphyrin structure (vinyl vs ethyl groups) did not significantly alter the properties of recognition of the compound but influenced its biophysical properties.
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Anti-Infecciosos , Gálio , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Gálio/farmacologia , Heme/metabolismo , Humanos , Mesoporfirinas , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus aureusRESUMO
Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging and is associated with retinal degeneration encountered in age-related macular degeneration (AMD) and Stargardt disease (SD). Currently, treatment for lipofuscin-induced retinal degeneration is unavailable. Here, we report that Remofuscin (INN: soraprazan, a tetrahydropyridoether small molecule) reverses lipofuscin accumulation in aged primary human RPE cells and is non-cytotoxic in aged SD mouse RPE cells in vitro. In addition, we show that the removal of lipofuscin after a single intravitreal injection of Remofuscin results in a rescue from retinal degeneration in a mouse model of advanced SD which is even accompanied by an amelioration of the retinal dysfunction. Finally, we demonstrate that the mechanism causing lipofuscinolysis may involve the reactive oxygen species generated via the presence of Remofuscin. These data suggest a possible therapeutic approach to untreatable lipofuscin-mediated diseases like AMD, SD and lipofuscinopathies in neurodegenerative diseases.
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Lipofuscina , Degeneração Retiniana , Animais , Camundongos , Espécies Reativas de Oxigênio , Epitélio Pigmentado da Retina , Doença de StargardtRESUMO
Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.
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Luz , Epitélio Pigmentado da Retina , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Cumarínicos , Lipídeos , Epitélio Pigmentado da Retina/metabolismo , c-Mer Tirosina Quinase/metabolismoRESUMO
Cholesterol (Ch) is one of the most important components of biological membranes, which has a significant impact on their biophysical properties. As a key component of lipid membranes, Ch along with other unsaturated lipids present in a biological membrane undergoes oxidation reaction during oxidative stress. Cholesterol oxidation products, cholesteryl esters and metabolites are also localise in lipid membranes, where they may modify membrane properties. In this work the impact of cholesterol, selected cholesteryl esters, cholesterol oxidation products and metabolites on lipid peroxidation induced by photodynamic action has been studied using EPR oximetry and direct detection of singlet oxygen phosphorescence at 1270 nm. The obtained rate constants values of interaction of selected lipids and sterols with singlet oxygen indicate that the tested compounds are not efficient singlet oxygen quenchers. Nevertheless, the presence of sterols modifies to different extend the oxygen photoconsumption rate in peroxidisable liposomes.
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Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Peroxidação de Lipídeos , Membrana Celular/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipossomos/metabolismo , Medições Luminescentes/métodos , Oxirredução , Estresse Oxidativo , Oximetria/métodos , Oxigênio/metabolismo , Oxigênio Singlete/metabolismoRESUMO
The human skin is exposed to various environmental factors including solar radiation and ambient air pollutants. Although, due to its physical and biological properties, the skin efficiently protects the body against the harm of environmental factors, their excessive levels and possible synergistic action may lead to harmful effects. Among particulate matter present in ambient air pollutants, PM2.5 is of particular importance for it can penetrate both disrupted and intact skin, causing adverse effects to skin tissue. Although certain components of PM2.5 can exhibit photochemical activity, only a limited amount of data regarding the interaction of PM2.5 with light and its effect on skin tissue are available. This study focused on light-induced toxicity in cultured human keratinocytes, which was mediated by PM2.5 obtained in different seasons. Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM) were employed to determine sizes of the particles. The ability of PM2.5 to photogenerate free radicals and singlet oxygen was studied using EPR spin-trapping and time-resolved singlet oxygen phosphorescence, respectively. Solar simulator with selected filters was used as light source for cell treatment to model environmental lightning conditions. Cytotoxicity of photoexcited PM2.5 was analyzed using MTT assay, PI staining and flow cytometry, and the apoptotic pathway was further examined using Caspase-3/7 assay and RT-PCR. Iodometric assay and JC-10 assay were used to investigate damage to cell lipids and mitochondria. Light-excited PM2.5 were found to generate free radicals and singlet oxygen in season-dependent manner. HaCaT cells containing PM2.5 and irradiated with UV-Vis exhibited oxidative stress features-increased peroxidation of intracellular lipids, decrease of mitochondrial membrane potential, enhanced expression of oxidative stress related genes and apoptotic cell death. The data indicate that sunlight can significantly increase PM2.5-mediated toxicity in skin cells.
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Poluentes Atmosféricos/efeitos da radiação , Poluentes Atmosféricos/toxicidade , Células HaCaT/efeitos dos fármacos , Luz/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/efeitos da radiação , Material Particulado/toxicidade , Poluentes Atmosféricos/química , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Radicais Livres/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , Tamanho da Partícula , Material Particulado/química , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Envelhecimento da Pele/efeitos dos fármacosRESUMO
Melanopsin, a member of the G protein-coupled receptors family, is involved in non-image-forming functions including circadian rhythm, sleep regulation and pupil response. In spite of significant research efforts, the signaling cascade involving melanopsin photoactivation remains poorly characterized. Here, we analyzed the effects of photoactivation of melanopsin on phospholipase C (PLC) and diacylglycerol. As an in vitro model, HEK293 cells with stable expression of human melanopsin were used. Although both the PLCß1 and PLCß4 subtypes were activated by the cell exposure to blue light, only PLCß4 appeared to play a significant role in the studied melanopsin signaling pathway. We have demonstrated, for the first time, that cells expressing human melanopsin and enriched with 11-cis-retinal exhibited significantly increased diacylglycerol level. To determine the role of phospholipase C and involvement of diacylglycerols, two approaches were employed: inhibition of the G protein and phospholipase C (using the BIM-46187 and U73122 inhibitors, respectively), and gene silencing using siRNA of PLCß1 and PLCß4 . While silencing the PLCß4 gene and using U73122 inhibited the diacylglycerol and calcium ion responses, the FOS gene expression level was only partially reduced. These results may facilitate a better understanding of the role of phospholipase C and diacylglycerols in the melanopsin signaling pathway.
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Diglicerídeos , Opsinas de Bastonetes , Células HEK293 , Humanos , Luz , Fosfolipase C beta/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Transdução de SinaisRESUMO
Photoreactivity of melanin has become a major focus of research due to the postulated involvement of the pigment in UVA-induced melanoma. However, most of the hitherto studies were carried out using synthetic melanin models. Thus, photoreactivity of natural melanins is yet to be systematically analyzed. Here, we examined the photoreactive properties of natural melanins isolated from hair samples obtained from donors of different skin phototypes (I, II, III, and V). X-band and W-band electron paramagnetic resonance (EPR) spectroscopy was used to examine the paramagnetic properties of the pigments. Alkaline hydrogen peroxide degradation and hydroiodic acid hydrolysis were used to determine the chemical composition of the melanins. EPR oximetry and spin trapping were used to examine the oxygen photoconsumption and photo-induced formation of superoxide anion, and time-resolved near infrared phosphorescence was employed to determine the singlet oxygen photogeneration by the melanins. The efficiency of superoxide and singlet oxygen photogeneration was related to the chemical composition of the studied melanins. Melanins from blond and chestnut hair (phototypes II and III) exhibited highest photoreactivity of all examined pigments. Moreover, melanins of these phototypes showed highest quantum efficiency of singlet oxygen photogeneration at 332 nm and 365 nm supporting the postulate of the pigment contribution in UVA-induced melanoma.