January, 2026
International Journal of Molecular Sciences
Pmel17 Deficiency Affects Melanogenesis and Promotes Tumor Vascularization
Sopel J., Sarad K., Kozińska A., Mokrzyński K., Szczygieł D., Murzyn A., Drzał A., Słomiński A., Szczygieł M., Elas M.
Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.
August, 2025
Antimicrobial Agents and Chemotherapy
Allelopathic compound 2-methoxy-1,4-naphthoquinone is broadly effective against pathogenic Prototheca species in vitro and in vivo
Aliramezani A., Szewczyk G., Mokrzyński K., Ciastoń I., Nowicka B., Kwieciński JM.
Unpigmented, yeast-like Prototheca algae are emerging pathogens responsible for unusual infections in humans and animals. Their treatment is often unsuccessful, as traditional antifungals and antibacterials show unsatisfactory activity against Prototheca. Therefore, the development of improved drugs that consider the peculiarities of algal biology is needed. Here, we describe a novel anti-Prototheca compound, the 2-methoxy-1,4-naphthoquinone, identified among molecules known to mediate plant and algal allelopathy. The 2-methoxy-1,4-naphthoquinone was highly active in vitro against a range of Prototheca isolates from different species, both planktonically and in biofilms. Its mode of action included the induction of toxic reactive oxygen species, and it targeted energy generation in the pathogen’s mitochondria. It effectively treated Prototheca bovis skin infection in a mouse model, demonstrating translational potential. Altogether, this study not only identified a new effective anti-Prototheca compound, but also identified 1,4-naphthoquinone backbone as a lead structure for further therapeutic development and demonstrated the feasibility of exploring known anti-plant compounds as a source of anti-Prototheca treatments.
May, 2025
Photochemistry and Photobiology
The (un)known issue with using rose bengal as a standard of singlet oxygen photoproduction
Mokrzyński K., Szewczyk G.
Rose bengal (RB) is a widely used photosensitizer for determining quantum yields of singlet oxygen generation. While it is known to aggregate in polar environments at concentrations above 2 μM, the relationship between RB concentration and singlet oxygen photogeneration remains unclear. This study investigates the shift from monomeric to dimeric RB with increasing concentration and its impact on singlet oxygen generation in D2O-based solutions and DMPC liposomes. Absorbance maxima for RB were observed at 514 nm (dimer) and 549 nm (monomer), with ionic environments influencing aggregation rates. Singlet oxygen phosphorescence showed non-linear dependency above 2 μM, indicating the effects of aggregation. Results suggest that RB concentrations should be kept at 1 μM or lower in photochemical studies to avoid aggregation-related discrepancies in singlet oxygen yield determination. These findings highlight the importance of considering RB aggregation in photochemical research and medical applications.
April, 2025
International Journal of Molecular Sciences
Photoreactive Properties of Melanin Obtained from Human Induced Pluripotent Stem Cell-Derived Melanocytes
Mokrzyński K., Wojtała M., Sułkowski M., Ito S., Wakamatsu K., Żądło A., Majka M., Sarna T., Sarna M.
Although melanin is viewed as a natural sunscreen that protects pigmented cells against the adverse effects of solar radiation, recent studies have demonstrated that, under certain conditions, the pigment can actually contribute to light-induced oxidative damage of the cells. However, the main issue with such studies is finding natural pigments without photooxidative modifications. Recently, melanin obtained from melanocytes, generated from human induced pluripotent stem cells (hiPSC-Mel), was suggested as a promising source of the pigment without significant photooxidation. Although different studies have demonstrated the feasibility of the above-mentioned technique to obtain melanin-producing cells, no thorough analysis of the physicochemical properties of the pigment has been performed. To address this issue, we examined the key physicochemical parameters, including the aerobic photoreactivity of melanin isolated from hiPSC-Mel and compared them with those of melanin from other known sources of the pigment, such as bovine retinal pigment epithelium (bRPE) and phototype V (PT-V) hair. Electron paramagnetic resonance (EPR) spectroscopy, dynamic light scattering, UV–Vis absorption and HPLC analysis of melanin degradation products were used. The ability of the examined melanins to photogenerate reactive oxygen species was determined by employing EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Although the results of such measurements demonstrated that melanin obtained from hiPSC-Mel exhibited the physicochemical properties typical for eumelanin, a contribution from pheomelanin with a substantial presence of benzothiazine subunits, was also evident. Importantly, the hiPSC-Mel pigment had significantly lower photoreactivity compared to bRPE melanin and PT-V hair melanin. Our findings indicate that hiPSC-Mel could be an excellent source of high-quality pigment for photoprotection studies.
March, 2025
Molecules
Concentration-Dependent Photoproduction of Singlet Oxygen by Common Photosensitizers
Szewczyk G., Mokrzyński K.
Singlet oxygen quantum yield (ΦΔ) is a critical parameter in photodynamic studies, particularly for evaluating photosensitizers’ efficiency in diverse applications such as photodynamic therapy and environmental remediation. Standard photosensitizers, including Rose Bengal, Methylene Blue, and porphyrins, are widely employed as benchmarks for determining ΦΔ. However, accurate determination of ΦΔ relies not only on the intrinsic properties of these photosensitizers but also on their experimental conditions, such as concentration. This study investigated the influence of photosensitizer concentration on singlet oxygen quantum yield using several standard photosensitizers. Our findings revealed a significant decrease in ΦΔ with increasing photosensitizer concentrations across all tested compounds. This decline was attributed to self-quenching effects and molecular aggregation, which reduced the efficiency of energy transfer from the excited triplet state of the photosensitizer to molecular oxygen. The results emphasize the importance of optimizing photosensitizer concentration to ensure reliable ΦΔ measurements and avoid underestimations. This work underscores the need to consider concentration-dependent effects in future studies to ensure accurate and reproducible outcomes.
January, 2025
Scientific Reports
Synthesis and characterization of allomelanin model from 1,8-dihydroxynaphthalene autooxidation
Pukalski J.*, Mokrzyński K.*, Chyc M., Potrzebowski MJ. Makowski T., Dulski M., Latowski D.
In this work a novel method for synthesis of 1,8-dihydroxynaphthalene melanin was presented, as well as the physicochemical properties, molecular structure, and characteristics of the pigment. The proposed synthesis protocol is simple and cost-effective with no enzymes or catalysts needed. The final product is not adsorbed on any surface, since the pigment is the result of autooxidation of 1,8-dihydroxynaphthalene. Performed analyses revealed that the solubility, optical and paramagnetic properties are typical for melanins, and in the EPR spectra an unusual hyperfine structure was observed. The molecular structure of the pigment consists of three different layers forming polar and non-polar surfaces. Additionally, the presence of ether bonds presence was revealed. The developed method creates new opportunities for melanin research and eliminates the need to extract melanins from biological samples, which often lead to structural changes in isolated melanins, which undermines the reliability of analyses of the properties and structure of these polymers. On the other hand, the ubiquity of melanins in living organisms and the diversity of their biological functions have let to the growing interest of researchers in this group of pigments. The analyses carried out show that the obtained synthetic DHN polymer can be considered as a model DHN-melanin in mycological studies and material research.
September, 2024
Photochemical & Photobiological Sciences
Generation of singlet oxygen inside living cells: correlation between phosphorescence decay lifetime, localization and outcome of photodynamic action
Szewczyk G., Mokrzyński K., Sarna T.
Photodynamic therapy (PDT) is a promising alternative treatment for localized lesions and infections, utilizing reactive oxygen species (ROS) generated by photosensitizers (PS) upon light activation. Singlet oxygen (1O2) is a key ROS responsible for photodynamic damage. However, the effectiveness of PS in biological systems may not correlate with the efficiency of singlet oxygen generation in homogeneous solutions. This study investigated singlet oxygen generation and its decay in various cellular microenvironments using liposome and ARPE-19 cell models. Rose Bengal (RB), methylene blue (MB), and protoporphyrin IX (PpIX) were employed as selected PS. Lifetimes of singlet oxygen generated by the selected photosensitizers in different cellular compartments varied, indicating different quenching rates with singlet oxygen. RB, located near cell membranes, exhibited the highest phototoxicity and lipid/protein peroxidation, followed by PpIX, while MB showed minimal cytotoxicity in similar conditions. Singlet oxygen decay lifetimes provide insights into PS localization and potential phototoxicity, highlighting the importance of the lipid microenvironment in PDT efficacy, providing useful screening method prior to in vivo applications.
June, 2024
Chemistry of Materials
Water-Soluble Type I Radical Photoinitiators Dedicated to Obtaining Microfabricated Hydrogels
Tomak W., Petko F., Gałek M., Świeży A., Tyszka-Czochara M., Środa P., Mokrzyński K., Ortyl J.
Herein, five previously unknown benzoin ketal-based derivatives are presented as water-soluble, type I photoinitiators of free-radical photopolymerization processes for biomedical applications. Excellent spectroscopic properties of these compounds in the longwave ultraviolet and visible range up to 500 nm, enhanced solubility in water, and low cytotoxicity have been successfully proved, marking a significant advance over commonly used photoinitiators for biomedical applications. The developed new benzoin derivatives have been found to be effective in photoinitiating the free-radical polymerization of acrylate monomers, including the formation of hydrogel materials in aqueous media. In addition, these compounds can be used in advanced applications, including manufacturing of a hydrogel extracellular matrix with complex geometries using VAT printing. The described research represents a significant advance in the development of water-soluble photoinitiators for biomedical applications.
May, 2024
Featured
Pigment Cell & Melanoma Research
The effect of oxidative degradation of Dopa-melanin on its basic physicochemical properties and photoreactivity
Mokrzyński K., Żądło A., Szewczyk G., Sarna M., Sarna T., Ito S., Wakamatsu K., Sarna T.
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.
May, 2024
Science of The Total Environment
Photoreactivity of polycyclic aromatic hydrocarbons (PAHs) and their mechanisms of phototoxicity against human immortalized keratinocytes (HaCaT)
Mokrzyński K., Szewczyk G.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic compounds in the environment. They are produced by many anthropogenic sources of different origins and are known for their toxicity, carcinogenicity, and mutagenicity. Sixteen PAHs have been identified as Priority Pollutants by the US EPA, which are often associated with particulate matter, facilitating their dispersion through air and water. When human skin is exposed to PAHs, it might occur simultaneously with solar radiation, potentially leading to phototoxic effects. Phototoxic mechanisms involve the generation of singlet oxygen and reactive oxygen species, DNA damage under specific light wavelengths, and the formation of charge transfer complexes. Despite predictions of phototoxic properties for some PAHs, there remains a paucity of experimental data. This study examined the photoreactive and phototoxic properties of the 16 PAHs enlisted in the Priority Pollutants list. Examined PAHs efficiently photogenerated singlet oxygen and superoxide anion in simple solutions. Furthermore, singlet oxygen phosphorescence was detected in PAH-loaded HaCaT cells. Phototoxicity against human keratinocytes was evaluated using various assays. At 5 nM concentration, examined PAHs significantly reduced viability and mitochondrial membrane potential of HaCaT cells following the exposure to solar simulated light. Analyzed compounds induced a substantial peroxidation of cellular proteins after light treatment. The results revealed that a majority of the examined PAHs exhibited substantial reactive oxygen species photoproduction under UVA and violet-blue light, with their phototoxicity corresponding to their photoreactive properties. These findings improve our comprehension of the interactions between PAHs and human skin cells under environmental conditions, particularly when exposed to solar radiation.
January, 2024
Photochemistry and Photobiology
Can l-ascorbic acid and trans-resveratrol protect HaCaT cells from fine particulate matter toxicity?
Mokrzyński K., Krzysztyńska-Kuleta O., Wojtala M., Wnuk D., Sarna M., Sarna T.
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.
June, 2023
Featured
Journal of Photochemistry and Photobiology B: Biology
Photoreactivity and phototoxicity of experimentally photodegraded hair melanosomes from individuals of different skin phototypes
Mokrzyński K., Sarna M., Sarna T.
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.
May, 2023
Photochemistry and Photobiology
Benzo[A]Pyrene and Benzo[E]Pyrene: Photoreactivity and Phototoxicity toward Human Keratinocytes
Mokrzyński K., Szewczyk G., Sarna T.
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.
February, 2022
Molecules
Preliminary Studies of Antimicrobial Activity of New Synthesized Hybrids of 2-Thiohydantoin and 2-Quinolone Derivatives Activated with Blue Light
Kania A., Tejchman W., Pawlak AM., Mokrzyński K., Różanowski B., Musielak BM., Greczek-Stachura M.
Thiohydantoin and quinolone derivatives have attracted researchers’ attention because of a broad spectrum of their medical applications. The aim of our research was to synthesize and analyze the antimicrobial properties of novel 2-thiohydantoin and 2-quinolone derivatives. For this purpose, two series of hybrid compounds were synthesized. Both series consisted of 2-thiohydantoin core and 2-quinolone derivative ring, however one of them was enriched with an acetic acid group at N3 atom in 2-thiohydantoin core. Antibacterial properties of these compounds were examined against bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The antimicrobial assay was carried out using a serial dilution method to obtain the MIC. The influence of blue light irradiation on the tested compounds was investigated. The relative yield of singlet oxygen (1O2*, 1Δg) generation upon excitation with 420 nm was determined by a comparative method, employing perinaphthenone (PN) as a standard. Antimicrobial properties were also investigated after blue light irradiation of the suspensions of the hybrids and bacteria placed in microtitrate plates. Preliminary results confirmed that some of the hybrid compounds showed bacteriostatic activity to the reference Gram-positive bacterial strains and a few of them were bacteriostatic towards Gram-negative bacteria, as well. Blue light activation enhanced bacteriostatic effect of the tested compounds.
January, 2022
Photochemistry and photobiology
The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells
Olchawa MM., Herrnreiter AM., Skumatz CMB., Krzysztyńska-Kuleta OI., Mokrzyński KT., Burkę JM., Sarna TJ.
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 β5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvβ5 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.
December, 2021
European Polymer Journal
Visible light-induced photopolymerization of Deep Eutectic Monomers, based on methacrylic acid and tetrabutylammonium salts with different anion structures
Wierzbicki S., Mielczarek K., Topa-Skwarczyńska M., Mokrzyński K., Ortyl J., Bednarz S.
Deep Eutectic Monomers were prepared by dissolution of selected tetrabutylammonium salts serving as hydrogen bond acceptors (HBA) in methacrylic acid (MAA) acting both as hydrogen bond donor (HBD) and vinyl monomer. In this study, we systematically investigate the influence of chloride, nitrate, hydrogensulphate and fluoroborate anions on properties of DEMs and the course of radical photopolymerization. By Combining FTIR, 1H and 13C NMR spectroscopy we found the that degree of coordination of MAA in DEMs by anions follows the order: Cl− > NO3– > HSO4− ≫ BF4−. Moreover, the studies indicated that the strength of hydrogen bonds between HBD and HBA in DEMs is lower than in MAA dimers. Real-time FT-IR spectroscopy studies of photopolymerization of DEMs indicate that the mixtures formed from MAA and tetrabutylammonium fluoroborate show significant lower initial photopolymerization rate in comparison with other investigated DEMs, which polymerize with similar rates.
September, 2021
Featured
International Journal of Molecular Sciences
Fine Particulate Matter-Induced Oxidative Stress Mediated by UVA-Visible Light Leads to Keratinocyte Damage
Mokrzyński K., Krzysztyńska-Kuleta O., Zawrotniak M., Sarna M., Sarna T
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.
June, 2021
International Journal of Molecular Sciences
Photoreactivity of Hair Melanin from Different Skin Phototypes—Contribution of Melanin Subunits to the Pigments Photoreactive Properties
Mokrzyński K., Ito S., Wakamatsu K., Camenisch TG., Sarna T., Sarna M.
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.
June, 2020
Photochemical & Photobiological Sciences
Quercetin protects ARPE-19 cells against photic stress mediated by the products of rhodopsin photobleaching
Olchawa MM, Krzysztyńska-Kuleta OI, Mokrzyński KT, Sarna PM, Sarna TJ
Although the primary biological function of retina photoreceptors is to absorb light and provide visual information, exposure to intense light could increase the risk of phototoxic reactions mediated by rhodopsin photobleaching products (RPBP) that might accumulate in photoreceptor outer segments (POS). Here we investigated whether quercetin can modify the phototoxic potential of RPBP under in vitro photic stress conditions. ARPE-19 cells or quercetin enriched cultures pre-loaded with rhodopsin-rich POS isolated from bovine retinas were irradiated with green light to photobleach rhodopsin, and subsequently with blue light. Survival of cells was determined by MTT assay and propidium iodide staining. Changes in mitochondrial membrane potential (MMP) were assessed by JC-1 staining. Protein hydroperoxides, formed by photosensitized oxidation, mediated by RPBP, were analyzed in cells and in a model system with bovine serum albumin (BSA), using the coumarin boronic acid fluorogenic probe. The effect of photic stress on specific phagocytosis of RPE cells was determined by flow cytometry. Photoreactivity of POS with and without quercetin was analyzed by EPR oximetry and EPR spin trapping. Cytotoxicity measurements and MMP analyses confirmed that supplementation with quercetin protected ARPE-19 cells against photic stress mediated by rhodopsin-rich POS. Quercetin significantly reduced the inhibitory effect of RPBP-mediated stress on POS phagocytosis and the RPBP ability to photooxidize cellular proteins or BSA. The data support the hypothesis that quercetin may efficiently diminish the phototoxic action of retinoids, necessary for restoring the phagocytic function of ARPE-19 cells.
May, 2020
Cell Biochemistry and Biophysics
The influence of iron on selected properties of synthetic pheomelanin
Żądło A., Mokrzyński K., Ito S., Wakamatsu K., Sarna T
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.
April, 2020
Photochemical & Photobiological Sciences
The role of hydrogen peroxide and singlet oxygen in the photodegradation of melanin
Żądło A., Ito S., Sarna M., Wakamatsu K., Mokrzyński K., Sarna T.
When aging, melanin in human retinal pigment epithelium (RPE) undergoes oxidative modifications, which increase its photoreactivity and reduce its antioxidant capacity, elevating the risk of chronic phototoxicity to the retina. The aim of this research was to examine the effect of iron on the degradation of melanin induced by hydrogen peroxide and light, and to elucidate the role of hydrogen peroxide and singlet oxygen in the photodegradation of melanin. A water-soluble synthetic model of eumelanin with and without iron ions was treated either with exogenous hydrogen peroxide or with intense violet light. Oxidative modifications of melanin were analyzed by electron paramagnetic resonance (EPR) spectroscopy, absorption spectrophotometry, dynamic light scattering (DLS) and by chemical analysis of melanin subunits. The results showed that although iron strongly accelerated melanin degradation induced by hydrogen peroxide, it had very little influence on the rate of photodegradation of melanin. On the other hand, the photodegradation of melanin was partly inhibited by NaN3. The determination of hydrogen peroxide together with oxygen uptake indicates that irradiated melanin generates similar amounts of singlet oxygen and hydrogen peroxide. Analysis of melanin samples exhibiting comparable reduction of their EPR signal revealed that the loss of the representative melanin subunits was much higher in irradiated samples than in those treated with hydrogen peroxide in the dark. In conclusion, hydrogen peroxide, formed during the aerobic photolysis of melanin, is not responsible for the accompanying oxidative modifications of melanin. On the other hand, singlet oxygen can be considered as a key oxidizing agent involved in the photodegradation of melanin.
July, 2019
Journal of Pineal Research
Melatonin exerts oncostatic capacity and decreases melanogenesis in human MNT-1 melanoma cells
Kleszczyński K., Kim T-K., Bilska B., Sarna M., Mokrzyński K., Stagemann A., Pyza E., Reiter RJ, Steinbrink K., Böhm M., Słomiński AT
Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time-dependent manner (24, 48, 72 hours) in highly pigmented MNT-1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk-Mel-28 cells. Subsequently, MNT-1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N-phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10-3 mol/L, triggered the appearance of premelanosomes (stage I-II of melanosome) and MNT-1 cells synthesize de novo endogenous melatonin shown by LC-MS. In conclusion, these studies show a melanogenic-like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.