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In this review, we summarized the current literature on the impact of phenothiazine derivatives on autophagy in vitro. Phenothiazines are antipsychotic drugs used in the treatment of schizophrenia, which is related to altered neurotransmission and dysregulation of neuronal autophagy. Thus, phenothiazine derivatives can impact autophagy. We identified 35 papers, where the use of the phenothiazines in the in vitro autophagy assays on normal and cancer cell lines, Caenorhabditis elegans, and zebrafish were discussed. Chlorpromazine, fluphenazine, mepazine, methotrimeprazine, perphenazine, prochlorperazine, promethazine, thioridazine, trifluoperazine, and novel derivatives can modulate autophagy. Stimulation of autophagy by phenothiazines may be either mammalian target of rapamycin (mTOR)-dependent or mTOR-independent. The final effect depends on the used concentration as well as the cell line. A further investigation of the mechanisms of autophagy regulation by phenothiazine derivatives is required to understand the biological actions and to increase the therapeutic potential of this class of drugs.

Drug repositioning is an approach that could accelerate the clinical use of compounds in different diseases. The goal is to take advantage of the fact that approved drugs have been tested on humans and detailed information is available on their pharmacology, toxicity and formulation. It can significantly reduce the costs and time needed to implement necessary therapies on the market. In recent years, phenothiazines are being tested for cancer, viral, bacterial, fungal and other diseases. Most research focuses on chlorpromazine as a model drug in this class, but other drugs such as fluphenazine, perphenazine and prochlorperazine have been proven to inhibit the viability of different cancer cell lines. In this study, we performed an extensive literature search to find and summarize all papers on the chosen phenothiazines and their potential in treating different types of cancerin vitro for further animal/clinical trials. Fluphenazine, perphenazine and prochlorperazine possess anticancer activity towards different types of human cancer. The antitumor activity is mainly mediated by an effect of the drugs on the cell cycle, proliferation or apoptosis. Possible molecular targets of phenothiazine derivatives are the drug's efflux pumps (ABCB1 and P-glycoprotein) and two parallel pathways (AKT and Wnt) regulated by the D2 receptor antagonists. The drugs have the potential to reduce the viability of human cancer cell lines, fragment the DNA, stimulate apoptosis, inhibit cell migration and invasiveness as well as impair the production of reactive oxygen species. In addition, due to the sedative and antiemetic properties antipsychotics can be used as an adjuvant for the treatment of chemotherapy side effects.

Glioblastoma multiforme is the most frequent type of malignant brain tumor, and is one of the most lethal and untreatable human tumors with a very poor survival rate. Therefore, novel and effective strategies of treatment are required. Integrins play a crucial role in the regulation of cellular adhesion and invasion. Integrins and α-tubulin are very important in cell migration, whereas E-cadherin plays a main role in tumor metastasis. Notably, drugs serve a crucial role in glioblastoma treatment; however, they have to penetrate the blood-brain barrier (BBB) to be effective. ABC transporters, including ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily G member 2 (ABCG2), are localized in the brain endothelial capillaries of the BBB, have a crucial role in the development of multidrug resistance and are modulated by phenothiazine derivatives. The impact of perphenazine and prochlorperazine on the motility of human Uppsala 87 malignant glioma (U87-MG) cells was evaluated using a wound-healing assay, cellular migration and invasion were assessed by Transwell assay, and the protein expression levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins were determined by western blotting. The present study explored the effects of perphenazine and prochlorperazine on the levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins (α3, α5, and β1), as well as on the migratory and invasive ability of U87-MG cells. The results suggested that perphenazine and prochlorperazine may modulate the expression levels of multidrug resistance proteins (they decreased ABCB1 and increased ABCG2 expression), E-cadherin, α-tubulin and integrins, and could impair the migration and invasion of U-87 MG cells. In conclusion, the decrease in migratory and invasive ability following treatment with phenothiazine derivatives due to the increase in ABCG2 and E-cadherin expression, and decrease in α-tubulin and integrins expression, may suggest that research on perphenazine and prochlorperazine in the treatment of glioblastoma is worth continuing.

Purpose: Glioblastoma multiforme is the most frequent malignant brain tumor as well as one of the most lethal and untreatable human tumors with a very poor survival rate (up to 18 months). Thus, novel and effective strategies of treatment are still required since resistance and metastasis are major problems of anticancer chemotherapy. Interestingly, ABC transporters, which play a crucial role in the development of multidrug resistance, are modulated by phenothiazine derivatives, while cancer metastasis, migration, and invasion are regulated by cadherins, α-tubulin, and integrins. Methods: The impact on the motility of human glioblastoma U87-MG was performed by wound healing assay, cellular migration and invasion were performed by transwell assay, while ABCB1, ABCG2, E-cadherin, α-tubulin, and integrins (α3, α5, and β1) content were determined by Western blot.Results: The present study explores the effect of perphenazine and prochlorperazine on ABCB1, ABCG2, E-cadherin, α-tubulin, and integrins (α3, α5, and β1) amount as well as migration and invasion ability of human glioblastoma (U87-MG) cells suggesting that phenothiazine derivatives impair multidrug resistance proteins (ABCB1 and ABCG2), E-cadherin, α-tubulin, and integrins amount as well as impair migration and invasion of the U87-MG cell line. Conclusions: The study demonstrated that an increase of ABCG2 and E-cadherin as well as a decrease of α-tubulin, and integrins amount may explain the decrease of migration and invasion ability after phenothiazine derivatives treatment. Moreover, only prochlorperazine significantly reduces the rate of cell migration. Thus, the drug may be considered for the development of new and effective glioblastoma therapy.

Cutaneous melanoma is least common (only about 1% of skin cancers) but is the deadliest malignant tumor. Moreover, amelanotic types of melanoma are very difficult for clinical diagnosis. The standard therapy can cause a lot of side effects, e.g., nausea, vomiting, and headaches, which means that novel and effective strategies are required. Interestingly, phenothiazine derivatives possess sedative, antiemetic, and anticancer activity. Our goal was to determine the effect of perphenazine and prochlorperazine on cell viability, motility, microphthalmia-associated transcription factor (MITF) and tyrosinase content in melanotic and amelanotic melanoma cells. The viability of C32 and COLO829 melanoma cells was evaluated by the WST-1 colorimetric assay; impact on motility of human melanoma was performed by wound-healing assay, while tyrosinase and MITF content were determined by Western blot. In the present study, we explore the anticancer effect of perphenazine and prochlorperazine in human melanotic (COLO829) and amelanotic (C32) melanoma cells concluding that prochlorperazine inhibits cell viability in a concentration-dependent manner, impairs motility, and decreases tyrosinase and MITF amounts. Moreover, the analyzed drugs decrease/increase MITF amount depending on the type of melanoma. We demonstrated that the decrease of MITF and tyrosinase protein induces motility inhibition of C32 cells, which suggests the ability of those drugs to restore cancer cell sensitivity to treatment. The ability of prochlorperazine to contain the spread of the amelanotic melanoma in vivo may be helpful in the development of a new and effective antimelanoma therapies.

Small molecules are routinely used to inhibit protein kinases, but modulators capable of enhancing kinase activity are rare. We have previously shown that the small molecule INR119, designed as an inhibitor of MEK1/2, will enhance the activity of its fission yeast homologue, Wis1, under oxidative stress. To investigate the generality of these findings, we now study the effect of INR119 in human cells under similar conditions. Cells of the established breast cancer line MCF-7 were exposed to H2O2 or phenothiazines, alone or combined with INR119. In line with the previous results in fission yeast, the phosphorylation of the MAPKs ERK and p38 increased substantially more with the combination treatment than by H2O2 or phenothiazines, whereas INR119 alone did not affect phosphorylation. We also measured the mRNA levels of TP53 and BAX, known to be affected by ERK and p38 activity. Similarly, the combination of INR119 and phenothiazines increased both mRNAs to higher levels than for phenothiazines alone. In conclusion, the mechanism of action of INR119 on its target protein kinase may be conserved between yeast and humans.

Polyphenols have recently gained popularity among the general public as products and diets classified as healthy and containing naturally occurring phenols. Many polyphenolic extracts are available on the market as dietary supplements, functional foods, or cosmetics, taking advantage of clients’ desire to live a healthier and longer life. However, due to the difficulty of discovering the in vivo functions of polyphenols, most of the research focuses on in vitro studies. In this review, we focused on the cardioprotective activity of different polyphenols as possible candidates for use in cardiovascular disease therapy and for improving the quality of life of patients. Thus, the studies, which were mainly based on endothelial cells, aortic cells, and some in vivo studies, were analyzed. Based on the reviewed articles, polyphenols have a few points of action, including inhibition of acetylcholinesterase, decrease in reactive oxygen species production and endothelial tube formation, stimulation of acetylcholine-induced endothelium-derived mediator release, and others, which lead to their cardio- and/or vasoprotective effects on endothelial cells. The obtained results suggest positive effects of polyphenols, but more long-term in vivo studies demonstrating effects on mechanism of action, sensitivity, and specificity or efficacy are needed before legal health claims can be made.

In 2020 the whole world focused on antivirus drugs towards SARS-CoV-2. Most of the researchers focused on drugs used in other viral infections or malaria. We have not seen such mobilization towards one topic in this century. The whole situation makes clear that progress needs to be made in antiviral drug development. The first step to do it is to characterize the potential antiviral activity of new or already existed drugs on the market. Phenothiazines are antipsychotic agents used previously as antiseptics, anthelminthics, and antimalarials. Up to date, they are tested for a number of other disorders including the broad spectrum of viruses. The goal of this paper was to summarize the current literature on activity toward RNA-viruses of such drugs like chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine. We identified 49 papers, where the use of the phenothiazines for 23 viruses from different families were tested. Chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine possess anti-viral activity towards different types of viruses. These drugs inhibit clathrin-dependent endocytosis, cell-cell fusion, infection, replication of the virus, decrease viral invasion as well as suppress entry into the host cells. Additionally, since the drugs display activity at nontoxic concentrations they have therapeutic potential for some viruses, still, further research on animal and human subjects are needed in this field to verify cell base research., Graphical abstract Diagrammatic summary of phenothiazine derivatives antiviral activity. All of the phenothiazines (chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine) possess antiviral activity towards RNA viruses. These drugs inhibit clathrin-dependent endocytosis, cell-cell fusion, infection, replication of the virus, decrease viral invasion as well as suppress entry into the host cells.Image 1, Highlights • Phenothiazines possess antiviral activity towards RNA viruses. • An antiviral activity can be achieved below toxic serum concentration. • Phenothiazines are characterized by multidirectional points of action.

Purpose Fluphenazine and prochlorperazine as phenothiazine-class antipsychotic drugs are widely used to treat schizophrenia, however their use is associated with significant side effects such as extrapyramidal symptoms, as well as ocular and skin disorders. Our goal was to determine the effect of fluphenazine and prochlorperazine on cell viability and melanogenesis in lightly pigmented normal human melanocytes. Methods The viability of melanocytes was evaluated by the WST-1 colorimetric assay, while melanin content and tyrosinase activity were tested spectrophotometrically. Results It has been shown that both phenothiazines induce the concentration-dependent loss in cell viability. The EC50 values were calculated to be 6.13 and 0.63 μM for fluphenazine and prochlorperazine, respectively. Fluphenazine in the concentration of 5.0 μM and prochlorperazine in concentrations of 0.5 and 0.75 μM decreased melanin content and tyrosinase activity. The observed inhibition of melanogenesis may be explained by the decrease of enzyme activity. Conclusions The demonstrated changes in melanization process in lightly pigmented cells exposed to fluphenazine and prochlorperazine in vitro suggest a significant role of melanin and melanocytes in the mechanisms of undesirable side effects of these drugs in vivo. Graphical abstractFluphenazine and prochlorperazine significantly inhibits melanogenesis in lightly pigmented melanocytes HEMn-LP.

E-cigarettes are used by millions of people despite the fact that the harmful effect of aerosol emitted from these products to the human organism is still not clear. In this paper, toxicity of vapor generated using different solutions and battery output voltage on A549 cells viability is presented. The obtained EC50 values for commercially available propylene glycol/glycerol solution 1:1 e-liquids based on 3.2 V (0.127%), 4.0 V (0.112%) and 4.8 V (0.038%) were about 1.5-4.5 times higher than in tobacco smoke (0.0086%). Furthermore, it was shown that the increase of battery output voltage decreased A549 cell viability. In addition, commercially available extracts were more cytotoxic than laboratory made extracts. Owing to the expansiveness of e-cigarettes, it is very important to estimate their impact on public health. Our results not only confirm less cytotoxicity of e-liquid aerosol than cigarette smoke, but also demonstrate that solutions used in e-liquids and, for the first time, battery output voltage have a significant impact on cytotoxicity of e-cigarette vapor. Thus, the results of this study are very important for the current and future legal regulations on e-cigarettes.

Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells.

E-cigarettes are used by millions of people despite the fact that the harmful effect of aerosol emitted from these products to the human organism is still not clear. In this paper, toxicity of vapor generated using different solutions and battery output voltage on A549 cells viability is presented. The obtained EC

Chlorpromazine is used in the treatment of schizophrenia and psychotic disorders and belongs to phenothiazine class of neuroleptic drugs. It shows severe side effects such as extrapyramidal symptoms as well as ocular and skin disorders, but the mechanism is still not fully established. The aim of this study was to examine the effect of chlorpromazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. It has been demonstrated that chlorpromazine induces concentration dependent loss in cell viability. The value of EC(50) was calculated to be 2.53 μM. Chlorpromazine in lower concentrations (0.0001, 0.001 and 0.01 μM) increased the melanin and microphthalmia-associated transcription factor (MITF) content and tyrosinase activity, while changes of antioxidant enzymes activity were not observed. It suggests that long-term chlorpromazine therapy, even with low drug doses, may lead to hyperpigmentation disorders in skin and/or eye. The use of the analyzed drug in higher concentrations (0.1 and 1.0 μM) caused significant alterations of antioxidant enzymes activity in normal melanocytes, what may explain a potential role of chlorpromazine in the depletion of cellular antioxidant status leading to other adverse effects associated with the high-dose and/or long-term therapy.

Oxytetracycline is a broad-spectrum antibiotic, used in dermatology and veterinary medicine. Like other tetracyclines, it may evoke skin phototoxic reactions related to generation of reactive oxygen species (ROS). Melanins are biopolymers synthesised in melanocytes - highly specialised cells, localised in the basal layer of epidermis. Production of melanin is a defence mechanism against harmful effects of UV radiation, ROS and many chemical substances, including drugs. In the present study the influence of oxytetracycline and UVA radiation on darkly pigmented melanocytes viability, the melanogenesis process and the activity of antioxidant enzymes were analysed. The obtained results show that oxytetracycline decreases cell viability in a dose-dependent manner. It has also been stated that UVA radiation as well as simultaneous exposure to oxytetracycline and UVA radiation reduce melanocytes viability. The tested drug alone exhibits little effect on antioxidant enzymes activity and has no influence on the synthesis of melanin. However, simultaneous exposure of the cells to oxytetracycline and UVA radiation causes an increase of SOD and GPx activity, a decrease of CAT activity as well as stimulates melanogenesis. The obtained results suggest that phototoxicity of oxytetracycline towards normal human melanocytes depends on both time of UVA exposure and the drug concentration.

Fluoroquinolone antibiotics provide broad-spectrum coverage for a number of infectious diseases, including respiratory as well as urinary tract infections. One of the important adverse effects of these drugs is phototoxicity which introduces a serious limitation to their use. To gain insight the molecular mechanisms underlying the fluoroquinolones-induced phototoxic side effects, the impact of two fluoroquinolone derivatives with different phototoxic potential, norfloxacin and moxifloxacin, on melanogenesis and antioxidant enzymes activity in normal human melanocytes HEMa-LP was determined. Both drugs induced concentration-dependent loss in melanocytes viability. The value of EC50 for these drugs was found to be 0.5 mM. Norfloxacin and moxifloxacin suppressed melanin biosynthesis; antibiotics were shown to inhibit cellular tyrosinase activity and to reduce melanin content in melanocytes. When comparing the both analyzed fluoroquinolones, it was observed that norfloxacin possesses greater inhibitory effect on tyrosinase activity in melanocytes than moxifloxacin. The extent of oxidative stress in cells was assessed by measuring the activity of antioxidant enzymes: SOD, CAT, and GPx. It was observed that norfloxacin caused higher depletion of antioxidant status in melanocytes when compared with moxifloxacin. The obtained results give a new insight into the mechanisms of fluoroquinolones toxicity directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various phototoxic activities of the analyzed fluoroquinolone derivatives in vivo.

Nicotine is a natural ingredient of tobacco plants and is responsible for the addictive properties of tobacco. Nowadays nicotine is also commonly used as a form of smoking cessation therapy. It is suggested that nicotine may be accumulated in human tissues containing melanin. This may in turn affect biochemical processes in human cells producing melanin. The aim of this study was to examine the effect of nicotine on melanogenesis and antioxidant status in cultured normal human melanocytes HEMn-LP. Nicotine induced concentration-dependent loss in melanocytes viability. The value of EC50 was determined to be 7.43 mM. Nicotine inhibited a melanization process in human light pigmented melanocytes and caused alterations of antioxidant defense system. Significant changes in cellular antioxidant enzymes: superoxide dismutase and catalase activities and in hydrogen peroxide content were stated. The obtained results may explain a potential influence of nicotine on biochemical processes in melanocytes in vivo during long term exposition to nicotine.

Hypo- and hyperpigmentation disorders are the most severe dermatological diseases observed in patients from all over the world. These disorders can be divided into melanoses connected with disorders of melanocyte function and melanocytoses connected with melanocyte development. The article presents some hereditary hypomelanocytoses, which are caused by abnormal melanoblast development, migration and proliferation as well as by abnormal melanocyte viability and proliferation. These disorders are represented by Waardenburg syndrome, piebaldism and Tietz syndrome, and are caused by different mutations of various or the same genes. The types of mutations comprise missense and nonsense mutations, frameshifts (in-frame insertions or deletions), truncating variations, splice alterations and non-stop mutations. It has been demonstrated that mutations of the same gene may cause different hypopigmentation syndromes that may have similar phenotypes. For example, mutations of the MITF gene cause Waardenburg syndrome type 2A as well as Tietz syndrome. It has also been demonstrated that mutations of different genes may cause an identical syndrome. For example, mutations of MITF, SNAI2 and SOX10 genes are observed in Waardenburg syndrome type II and mutations of EDNRB, EDN3 and SOX10 genes are responsible for Waardenburg syndrome type IV. In turn, mutation of the KIT gene and/or heterozygous deletion of the SNAI2 gene result in piebaldism disease. The knowledge of the exact mechanisms of pigmentary disorders may be useful in the development of new therapeutic approaches to their treatment.

Aminoglycosides, broad spectrum aminoglycoside antibiotics, are used in various infections therapy due to their good antimicrobial characteristics. However, their adverse effects such as nephrotoxicity and auditory ototoxicity, as well as some toxic effects directed to pigmented tissues, complicate the use of these agents. This study was undertaken to investigate the effect of aminoglycoside antibiotic—kanamycin on viability, melanogenesis and antioxidant enzymes activity in cultured human normal melanocytes (HEMa-LP). It has been demonstrated that kanamycin induces concentration-dependent loss in melanocytes viability. The value of EC50 was found to be ∼6.0 mM. Kanamycin suppressed melanin biosynthesis: antibiotic was shown to inhibit cellular tyrosinase activity and to reduce melanin content in normal human melanocytes. Significant changes in the cellular antioxidant enzymes: SOD, CAT and GPx were stated in melanocytes exposed to kanamycin. Moreover, it was observed that kanamycin caused depletion of antioxidant defense sytem. It is concluded that the inhibitory effect of kanamycin on melanogenesis and not sufficient antioxidant defense mechanism in melanocytes in vitro may explain the potential mechanisms of undesirable side effects of this drug directed to pigmented tissues in vivo. J. Cell. Biochem. 114: 2746–2752, 2013. © 2013 Wiley Periodicals, Inc.

Streptomycin is an aminoglycoside antibiotic with an antituberculosis activity commonly used in clinical practice due to its good antimicrobial characteristics. A well-known undesirable side effect of this drug is ototoxicity, which may be caused by overproduction of reactive oxygen species and loss of melanocytes in the inner ear. The aim of this study was to examine the effect of streptomycin on melanogenesis and antioxidant defense system in cultured normal human melanocytes (HEMa-LP). Streptomycin induced concentration-dependent loss in melanocytes viability. The value of EC50 was determined to be ~5.0 mM. It has been shown that streptomycin causes inhibition of tyrosinase activity and reduces melanin content in human melanocytes in a concentration-dependent manner. Significant changes in the activity of cellular antioxidant enzymes: superoxide dismutase, catalase, and glutathione peroxidase were also stated. The results obtained in vitro may explain a potential role of melanocytes and melanin in the causative mechanisms of aminoglycosides ototoxic effects in vivo.

Fluoroquinolones are one of the most commonly prescribed classes of antibiotics. However, their use is often connected with high risk of phototoxic reactions that lead to various skin or eye disorders. The aim of this study was to examine the effect of ciprofloxacin, lomefloxacin, moxifloxacin and fluoroquinolone derivatives with different phototoxic potential, on the viability and melanogenesis in melanocytes.Normal human epidermal melanocytes, dark pigmented (HEMn-DP) were used as an in vitro model system. The effect of the tested antibiotics on cell viability and melanization in pigmented cells was investigated using a spectrophotometric method. The WST-1 assay was used to detect the cytotoxic effect of antibiotics.Ciprofloxacin, lomefloxacin and moxifloxacin induced the concentration-dependent loss in melanocytes viability. The values of ECThe results obtained in vitro suggest that the phototoxic potential of fluoroquinolones in vivo depends on specific drug-melanin interaction, the ability of drugs to affect melanogenesis as well as on the degree of melanocytes pigmentation.

Lomefloxacin is a synthetic, broad-spectrum antibacterial antibiotic. The use of this drug in the treatment of various infections may be limited by inherent serious adverse effects on pigmented tissues. The exact mechanisms of lomefloxacin phototoxic side effects have not been well established yet. The aim of this work was to examine the effect of lomefloxacin on antioxidant enzymes activity in cultured human normal melanocytes (HEMa-LP). The demonstrated changes in the activity of antioxidant enzymes (SOD, CAT and GPx) in melanocytes in the presence of lomefloxacin indicate that the analyzed drug is responsible for generation of reactive oxygen species, mainly superoxide anion radical and hydrogen peroxide, what may be the reason of the reduction of melanocytes antioxidant status. Modulation of antioxidant defense system in melanocytes by lomefloxacin in vitro may explain a potential role of melanocytes and ROS in the mechanisms of lomefloxacin phototoxic effects in vivo.

The aim of this work was to examine the impact of ketoprofen on melanogenesis in cultured human normal melanocytes (HEMn-DP). The WST-1 assay was used to detect ketoprofen cytotoxic effect. It has been demonstrated that ketoprofen induces the loss of melanocytes viability in a concentration–dependent manner. The value of EC50 was found to be ~ 1 mM. It has also been shown that ketoprofen causes inhibition of tyrosinase activity and reduces melanin content in human epidermal melanocytes. The demonstrated inhibitory effect of ketoprofen on melanization process in melanocytes in vitro may explain the potential role of melanin biopolymer in the mechanisms of undesirable phototoxic effects of this drug in vivo, as a result of its accumulation in pigmented tissues.

Aminoglycoside antibiotics, including gentamicin, are widely used clinically in treatment of bacterial infections. Unfortunately, their side effects, especially nephrotoxicity and ototoxicity remain a problem. It is known that aminoglycoside antibiotics bind well to melanin biopolymer, but the relation between their affinity to melanin and ototoxicity is not well documented. The aim of this work was to examine the impact of gentamicin on antioxidant enzymes activity in cultured dark pigmented normal human melanocytes (HEMn-DP). The WST-1 assay was used to detect gentamicin cytotoxic effect. The analyzed antibiotic induced concentration-dependent loss in melanocytes viability. The value of EC50 was found to be 7.5 mM. Significant changes in the cellular antioxidant enzymes: SOD, CAT and GPx were stated in melanocytes exposed to gentamicin, what may indicate the depletion of antioxidant defense system. It is concluded, that the results obtained in vitro may explain a potential role of melanocytes and melanin in the causative mechanisms of aminoglycosides ototoxic effects in vivo.

Doxycycline is a commonly used tetracycline antibiotic showing the broad spectrum of antibacterial action. However, the use of this antibiotic is often connected with the risk of phototoxic reactions that lead to various skin disorders. One of the factors influencing the photosensitivity reactions is the melanin content in melanocytes. In this study, the impact of doxycycline and UVA irradiation on cell viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn-DP) was examined. The exposure of cells to doxycycline and UVA radiation resulted in concentration-dependent loss in melanocytes viability and induced melanin biosynthesis. Significant changes were stated in cellular antioxidant enzymes activity: SOD, CAT and GPx, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain the mechanisms of phototoxic reactions that occur in normal human epidermal melanocytes in vivo after exposure of skin to doxycycline and UVA radiation.

Tetracycline is a semisynthetic antibiotic and is used in several types of infections against both gram-positive and gram-negative bacteria. This therapy is often associated with phototoxic reactions that occur after exposure to UV radiation and lead to photo-onycholysis, pseudoporphyria, solar urticaria and the fixed drug eruption in the skin. The phototoxic reactions may be related to the melanin content which, on one side may bind drugs - leading to their accumulation, and on the other side, they have photoprotective and antioxidant properties. In this study the effect of tetracycline and UVA irradiation on cell viability, biosynthesis of melanin and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn-DP) was analyzed. The viability of the cells treated with tetracycline and exposed to UVA radiation decreased in a drug concentration-dependent manner. At the same time, the induction of the melanization process was observed. The significant alterations in antioxidant defense system, on the basis of changes in SOD, CAT and GPx activities, were stated. The obtained results may give explanation for the phototoxic effects of tetracycline therapy observed in skin cells exposed to UVA radiation.

Aminoglycoside antibiotics, including gentamicin, despite their ability to induce adverse effects on pigmented tissues, remain valuable and sometimes indispensable for the treatment of various infections. It is known that gentamicin binds to melanin biopolymers, but the relation between this drug affinity to melanin and its toxicity is not well documented. The aim of this work was to examine the impact of gentamicin on viability and melanogenesis in HEMa-LP (light pigmented) and HEMn-DP (dark pigmented) normal human melanocytes.The effect of gentamicin on cell viability was determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay; melanin content and tyrosinase activity were measured spectrophotometrically. It has been demonstrated that gentamicin induces concentration-dependent loss in melanocytes viability. The application of antibiotic in concentration of 10 mM causes higher reduction in viability of the light pigmented melanocytes (by about 74%) when compared with the dark pigmented ones (by about 62%). The value of the concentration of a drug that produces loss in cell viability by 50% (EC50) for both cell lines was found to be ∼7.5 mM. It has been shown that gentamicin causes inhibition of tyrosinase activity and reduces melanin content in light pigmented melanocytes significantly more than in the dark pigmented cells.We have found that gentamicin modulates melanization process in melanocytes in vitro, what may explain the potential role of melanin biopolymer in the mechanisms of undesirable toxic effects of this drug in vivo, as a result of its accumulation in pigmented tissues. We have also stated that the melanogenesis process in light pigmented melanocytes is more sensitive to the inhibitory effect of gentamicin than in the dark pigmented cells.

Background Fluoroquinolones are a group of broad spectrum bactericidal antibiotics used to treat various infections of urinary and respiratory systems, as well as in ophthalmology and dermatology. This class of antibiotics causes toxic effects directed to pigmented tissues, what introduces a serious limitation to their use. The aim of this work was to examine the impact of sparfloxacin on melanogenesis and the antioxidant defense system in normal human epidermal melanocytes, adult, lightly pigmented (HEMa-LP). Methods The effect of sparfloxacin on cell viability was determined by WST-1 assay; melanin content, tyrosinase activity as well as antioxidant enzymes activity were measured spectrophotometrically. Results Sparfloxacin induced the concentration – dependent loss in melanocytes viability. The value of EC 50 was determined to be ∼0.25 mM. Sparfloxacin inhibited tyrosinase activity and reduced the melanin content in human melanocytes. To study the antioxidant defense system in melanocytes, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in cells exposed to sparfloxacin were determined. It was observed that sparfloxacin caused depletion of the antioxidant status of melanocytes. Conclusions The observed sparfloxacin-dependent inhibition of melanogenesis and changes of antioxidant enzymes activities in human melanocytes give a new insight into the mechanism of fluoroquinolones toxicity directed to pigmented tissues.

Aminoglycosides, broad spectrum aminoglycoside antibiotics, are used in various infections therapy due to their good antimicrobial characteristics. However, their adverse effects such as nephrotoxicity and auditory ototoxicity, as well as some toxic effects directed to pigmented tissues, complicate the use of these agents. This study was undertaken to investigate the effect of aminoglycoside antibiotic-kanamycin on viability, melanogenesis and antioxidant enzymes activity in cultured human normal melanocytes (HEMa-LP). It has been demonstrated that kanamycin induces concentration-dependent loss in melanocytes viability. The value of EC50 was found to be ~6.0 mM. Kanamycin suppressed melanin biosynthesis: antibiotic was shown to inhibit cellular tyrosinase activity and to reduce melanin content in normal human melanocytes. Significant changes in the cellular antioxidant enzymes: SOD, CAT and GPx were stated in melanocytes exposed to kanamycin. Moreover, it was observed that kanamycin caused depletion of antioxidant defense sytem. It is concluded that the inhibitory effect of kanamycin on melanogenesis and not sufficient antioxidant defense mechanism in melanocytes in vitro may explain the potential mechanisms of undesirable side effects of this drug directed to pigmented tissues in vivo.

Amikacin is principally used to treat infections caused by microorganisms resistant to other aminoglycosides. Ototoxicity is one of the side effects of amikacin, but the causative mechanism of damage to the ear has not been fully established. Thus, the aim of this work was to examine the impact of amikacin on the melanogenesis and antioxidant defense system in cultured human normal melanocytes (HEMa-LP). Amikacin induced the concentration - dependent loss in melanocytes viability. The value of EC50 was determined to be ~7.5 mM. The analyzed antibiotic inhibited melanin biosynthesis in concentration-dependent manner. Increasing the amikacin concentration also resulted in a decrease in cellular tyrosinase activity. To study the antioxidant defense system in melanocytes, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in cells exposed to amikacin were determined. Significant changes in cellular antioxidant enzymes activities were observed. Modulation of melanogenesis and the antioxidant status of melanocytes resulting from the use of amikacin in vitro may explain a potential role of melanin and melanocytes in the mechanisms of aminoglycosides ototoxic effects in vivo.

The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP) and microphthalmia transcription factor (MITF) in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme), tyrosine hydroxylase isoform I (THI) and tyrosinase related proteins (TRP1 and TRP2). Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP) by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI) transcription and phenylalanine hydroxylase (PAH) phosphorylation at Ser16 by protein kinase A (PKA). Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

Backround Lomefloxacin is a potent bactericidal antibiotic. The use of this drug in treatment of various infections is accompanied by serious adverse effects on pigmented tissues. The exact mechanisms of lomefloxacin side effects have not been well established yet. The aim of this study was to characterize the interaction between lomefloxacin and ś, and to examine how this interaction affects the cell viability and melanization in melanocytes. Methods Normal human epidermal melanocytes and the model DOPA-ś were used. The binding parameters of lomefloxacin-ś complexes as well as the antibiotic effect on cell viability and melanization in pigmented cells were investigated using a spectrophotometric method. Results Our results indicate that lomefloxacin forms stable complexes with ś. The analysis of drug binding to ś has shown that at least two classes of independent binding sites are involved in formation of these complexes. The WST-1 assay was used to detect the antibiotic cytotoxic effect. The value of ED 50 for lomefloxacin was about 0.75 mmol/l. It has been shown that lomefloxacin causes inhibition of tyrosinase activity, and reduces ś content in human skin melanocytes in a dose-dependent manner. Conclusion The ability of the analyzed fluoroquinolone to form complexes with ś, and the demonstrated inhibitory effect on a melanization process in melanocytes in vitro may explain a potential role of ś biopolymer in the mechanisms of undesirable side effects of lomefloxacin in vivo resulting from its accumulation in pigmented tissues.

There have been described serious adverse events caused by ciprofloxacin in pigmented tissues. It is known that some fluoroquinolones bind well to melanin rich tissues, but the relation between their affinity to melanin and the skin or eye toxicity is not well documented. The aim of this study was to examine whether ciprofloxacin binds to melanin, and how this interaction affects the proliferation and melanization in melanocytes. We have demonstrated that complexes which ciprofloxacin forms with melanin possess at least two classes of independent binding sites. Their association constants are K(1)~10(5) M(-1) and K(2)~10(2) M(-1), respectively. Ciprofloxacin has induced evident concentration-dependent loss in melanocytes viability. The value of ED(50) was found to be ~0.5 mM. It has also been shown that ciprofloxacin reduces melanin content, and decreases tyrosinase activity in human skin melanocytes. The ability of ciprofloxacin to interact with melanin and its inhibitory effect on melanization in melanocytes in vitro may explain a potential role of melanin in the mechanisms of ciprofloxacin toxic effects in vivo.

Aminoglycosides, such as amikacin and kanamycin, are powerful broad-spectrum antibiotics used for the treatment of many bacterial infections. The widely used aminoglycosides have the unfortunate side effects of targeting sensory hair cells of the inner ear, so that treatment often results in permanent hair cell loss. The aim of the study was to evaluate the influence of incubation time and drug concentration on viability of melanocytes cultured in the presence of amikacin or kanamycin. The normal human melanocytes HEMa-LP and the different concentrations of amikacin (0.075, 0.75 and 7.5 mmol/l) and kanamycin (0.06, 0.6 and 6.0 mmol/l), were used. The estimations were performed after 24, 48 and 72 h. The observed decrease in melanocytes viability may be an explanation for the mechanisms involved in aminoglycosides toxicity on pigmented tissues during high-dose and/or long-term therapy.

Thioridazine as an antipsychotic agent was extensively used to treat various psychotic disorders, e.g. schizophrenia. However, the therapy with this drug can induce serious side effects such as extrapyramidal symptoms or ocular and skin disorders, which mechanisms are still not fully established. To gain inside the molecular mechanisms underlying thioridazine toxicity, we examined the effect of this drug on cell viability, antioxidant defence system as well as melanogenesis in normal human melanocytes. It was demonstrated that thioridazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 2.24 μM. To study the effect of thioridazine on antioxidant defence system in melanocytes, the level of hydrogen peroxide and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were determined. The drug in concentrations of 0.1, 0.25, 1.0 and 2.5 μM caused changes in cellular antioxidant defence system indicating the induction of oxidative stress. It was also shown that the analysed neuroleptic in concentrations of 1.0 and 2.5 μM significantly inhibited melanogenesis. The observed changes in cell viability, antioxidant defence system and melanization in normal human melanocytes after thioridazine treatment may explain an important role of reactive oxygen species as well as melanin in mechanisms involved in this drug side effects directed on pigmented tissues.

Nicotine is a compound of tobacco plants and is responsible for addictive properties of tobacco which is used by about one billion of smokers all over the world. Recently, nicotine has drawn even more attention due to its presumed neuroprotective and antioxidant features as far as common use in various forms of smoking cessation therapies. It is suggested that nicotine may be accumulated in human tissues containing melanin. This may in turn influence biochemical processes in human cells producing melanin. The aim of this study was to examine the impact of nicotine on melanogenesis and antioxidant defense system in cultured normal human melanocytes (HEMn-DP). Nicotine induced concentration-dependent loss in melanocytes viability. The value of EC50 was determined to be 2.52 mM. Nicotine modulated melanin biosynthesis in normal human melanocytes. Significant changes in hydrogen peroxide content and cellular antioxidant enzymes: SOD, CAT, and GPx activities were stated in melanocytes exposed to nicotine, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain a potential influence of nicotine on biochemical processes in melanocytes in vivo during long-term exposition to nicotine.

The purpose of this study was to estimate the effect of chlorpromazine on free radical concentration in HEMn-DP melanocytes using electron paramagnetic resonance (EPR) spectroscopy. It was found that chlorpromazine at concentrations of 1 × 10(-7) and 1 × 10(-6) M contributed to the formation of free radicals (g values ~2) in a dose-dependent manner. The increase in free radical formation was accompanied by an increase in cytotoxicity, as shown by a tetrazolium assay. Homogeneous broadening of EPR lines, slow spin-lattice relaxation processes, and strong dipolar interactions characterized all the tested cellular samples. The performed examination of free radical formation in cells exposed to different chlorpromazine concentrations confirmed the usefulness of electron paramagnetic resonance spectroscopy to determine the effect of a drug on free radical production in a cellular model system in vitro.