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Impact of sparfloxacin on melanogenesis and antioxidant defense system in normal human melanocytes HEMa-LP – An in vitro study
Pharmacological Reports 67 (2015) 38–43
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Impact of sparfloxacin on melanogenesis and antioxidant defense system in normal human melanocytes HEMa-LP – An in vitro study Artur Beberok *, Dorota Wrzes´niok, Michał Otre˛ba, Ewa Buszman Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Sosnowiec, Poland
A R T I C L E I N F O
Article history: Received 25 March 2014 Received in revised form 25 July 2014 Accepted 28 July 2014 Available online 10 August 2014 Keywords: Sparfloxacin Melanocytes Melanization Tyrosinase Antioxidant enzymes
A B S T R A C T
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 EC50 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. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Fluoroquinolones are one of the most commonly prescribed classes of antibiotics. Their broad spectrum of antibacterial coverage and high tissue distribution facilitate the use against a wide variety of community acquired infections. Sparfloxacin, a third-generation fluoroquinolone derivative, is a potent antibacterial agent active against a wide range of Gram-positive and Gram-negative organisms [1,2]. Sparfloxacin acts by inhibiting two types of topoisomerase enzymes, DNA gyrase (topoisomerase II) and topoisomerase IV which are responsible for supercoilling, transcription, replication and chromosomal separation of prokaryotic DNA [3–5]. Although fluoroquinolones are generally well tolerated, they have been associated with a wide array of adverse events such as phototoxicity and photogenotoxicity, which have
* Corresponding author. E-mail address: [email protected] (A. Beberok).
been demonstrated in different in vitro [6–8], in vivo [9,10] and clinical studies [10–12]. Serious incidence of cutaneous toxicity (including erythema, pruritus, urticaria, rash or bullous eruptions) or eye toxicity (retinal degeneration including macular degeneration) has been reported for the use of sparfloxacin [7,13–15]. Moreover, Struwe et al. [16] demonstrated that sparfloxacin caused photogenotoxic effects in the skin and eye (retina and cornea) of rats. This fluoroquinolone belongs to the class of antibiotics known to exhibit severe phototoxicity [17] which may be associated with the production of reactive molecules i.e., reactive oxygen species (singlet oxygen, superoxide anion radical or hydroxyl radical) or highly reactive photodegradation products, able to modify the cell components, including lipids, proteins and nucleic acids [12,18,19]. Melanin, the end product of melanogenesis, determines the color of human skin, hair and eyes. This biopolymer is synthesized and deposited in the form of pigment granules in specialized organelles called melanosomes in the cytoplasm of the melanocytes. Human melanocytes synthesize two distinct types of
http://dx.doi.org/10.1016/j.pharep.2014.07.015 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
A. Beberok et al. / Pharmacological Reports 67 (2015) 38–43
melanin, the black/dark brown eumelanins and the lighter, yellowish/brown, sulphur-containing pheomelanins, which arise from the common biosynthetic pathway involving the tyrosinasecatalyzed oxidation of tyrosine [20–22]. Previously, we documented that ciprofloxacin [23], lomefloxacin [24], norfloxacin and sparfloxacin [25] formed stable complexes with model synthetic melanin in vitro. Moreover, we demonstrated that ciprofloxacin [23] and lomefloxacin [24] modulated biochemical processes in normal human melanocytes, suggesting a mechanism for the drug-induced toxicity on pigmented tissues. Whether the same mechanism can be attributed to another fluoroquinolone derivative, i.e., sparfloxacin, with relatively high toxic potential, remains to be determined. The results concerning the ability of drug-melanin complexes formation as well as the impact of drug on melanogenesis and antioxidant status in human melanocytes could serve as a screening pre-clinical tool to avoid the drug induced toxic effects on pigmented tissues. The objective of the present work was to examine the effect of sparfloxacin on melanogenesis and the antioxidant defense system in cultured normal human melanocytes (HEMa-LP).
Materials and methods Materials Sparfloxacin, L-3,4-dihydroxyphenylalanine (L-DOPA), Triton X-100 and mushroom tyrosinase were purchased from Sigma– Aldrich Inc. (USA). Penicillin was acquired from Polfa Tarchomin (Poland). A growth medium M-254, gentamicin, amphotericin B, and a human melanocyte growth supplement-2 (HMGS-2) were obtained from Cascade Biologics (UK). Trypsin/EDTA was obtained from Cytogen (Poland). Cell proliferation reagent WST-1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH S.A. (Poland). Cell culture Normal human epidermal melanocytes, adult, lightly pigmented (HEMa-LP, Cascade Biologics, UK) were grown according to the manufacturer’s instruction. The cells were cultured in M-254 medium supplemented with HMGS-2, penicillin (100 U/ml), gentamicin (10 mg/ml) and amphotericin B (0.25 mg/ml) at 37 8C in 5% CO2. All experiments were performed using cells from the passages 5–7. Cell viability assay The viability of melanocytes was evaluated by the WST-1 (4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) colorimetric assay. WST-1 is a water-soluble tetrazolium salt, the rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of viable cells. In brief, 5000 cells per well were placed in a 96-well microplate in a supplemented M-254 growth medium and incubated at 37 8C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with sparfloxacin solutions in a concentration range from 0.001 to 1.0 mM. After 21-h incubation, 10 ml of WST-1 were added to 100 ml of culture medium in each well, and the incubation was continued for 3 h. The absorbance of the samples was measured at 440 nm with a reference wavelength of 650 nm, against the controls (the same cells but not treated with sparfloxacin) using a microplate reader UVM 340 (Biogenet, Poland). The controls were normalized to 100% for each assay and treatments were expressed as the percentage of the controls.
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Measurement of melanin content The melanocytes were seeded in a 35 mm dish at a density of 1 105 cells per dish. Sparfloxacin treatment at concentrations of 0.0025 mM, 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM, respectively, began 48 h after seeding. After 24 h of incubation, the cells were washed three times and detached with trypsin/ EDTA. Cell pellets were placed into Eppendorf tubes, dissolved in 100 ml of 1 M NaOH at 80 8C for 1 h, and then centrifuged for 20 min at 16000 g. The supernatants were placed into a 96-well microplate, and absorbance was measured using a microplate reader at 405 nm, i.e. a wavelength at which melanin absorbs light [26]. A standard synthetic melanin curve (0–400 mg/ml) was performed in triplicate for each experiment. Melanin content in sparfloxacin treated cells was expressed as the percentage of the controls (untreated melanocytes). Tyrosinase activity assay Tyrosinase activity in HEMa-LP cells was determined by measuring the rate of oxidation of L-DOPA to dopachrome according to the method described by Kim et al. [27] and Busca et al. [28], with a slight modification. The cells were cultured at a density of 1 105 cells in a 35 mm dish for 48 h. After 24-h incubation with sparfloxacin (concentrations of 0.0025 mM, 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM) the cells were washed three times with PBS, lysed with phosphate buffer (pH 6.8) containing 0.1% Triton X-100, and lysates were clarified by centrifugation at 10000 g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the same lysis phosphate buffer (without Triton). 100 ml of each lysate were placed in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 ml of L-DOPA solution at 37 8C. Absorbance of dopachrome was measured every 10 min for at least 1 h at 475 nm using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls (untreated melanocytes). A cell-free assay system was used to test for direct effects on tyrosinase activity. 130 ml of phosphate buffer containing sparfloxacin at concentrations from 0.0025 to 1.0 mM were mixed with 20 ml of mushroom tyrosinase (1000 units), and 100 ml of L-DOPA solution (2 mg/ml) were added to each well. The assay mixtures were incubated at 37 8C for 20 min, and absorbance of dopachrome was measured at 475 nm in a microplate reader. The mushroom tyrosinase activities were calculated in the relation to the controls (samples without sparfloxacin). The value IC50 (the concentration of a drug that inhibits a standard response by 50%) was calculated on the basis of a dose-dependent inhibition curve, as described by Chung et al. [29]. Superoxide dismutase (SOD) assay Superoxide dismutase (SOD) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. This kit utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radical. SOD activity was expressed in U/mg protein. Catalase (CAT) assay Catalase (CAT) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. This kit utilizes the peroxidatic function of CAT for determination of enzyme activity. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration
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of H2O2. The produced formaldehyde is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as a chromogen. One unit of CAT was defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde per minute at 25 8C. CAT activity was expressed in nmol/min/mg protein. Glutathione peroxidase (GPx) assay Glutathione peroxidase (GPx) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. The measurement of GPx activity is based on the principle of a coupled reaction with glutathione reductase (GR). The oxidized glutathione (GSSG) formed after reduction of hydroperoxide by GPx is recycled to its reduced state by GR in the presence of NADPH. The oxidation of NADPH is accompanied by a decrease in absorbance at 340 nm. One unit of GPx was defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute at 25 8C. GPx activity was expressed in nmol/min/mg protein. Statistical analysis In all experiments, mean values of at least three separate experiments (n = 3) performed in triplicate standard error of the mean (SEM) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software. A value of p < 0.05 (*) or p < 0.005 (**), obtained with a Student’s t-test by comparing the data with those for the controls (cells without sparfloxacin), was considered statistically significant.
(i.e. the amount of a drug that produces loss in cell viability by 50%) was 0.25 mM. The effect of sparfloxacin on melanization process The effectiveness of a melanization process was estimated by measuring the melanin content and cellular tyrosinase activity in human melanocytes treated with sparfloxacin at concentrations from 0.0025 mM to 0.25 mM, for 24 h. After determining a calibration curve, the melanin content per cell was determined as 36.5 to 26.0 pg/cell for melanocytes treated with antibiotic and 37.1 3.4 pg/cell for a control sample. The obtained results, recalculated for culture (1 105 cells), were finally expressed as a percentage of the controls (Fig. 2). Sparfloxacin in concentration of 0.0025 mM had no effect on melanin content in melanocytes. In cells treated with sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM for 24 h, melanin production decreased in a concentration-dependent manner by about 5%, 17%, 22% and 29%, respectively. Tyrosinase activity in HEMa-LP cells treated with sparfloxacin also decreased in a manner correlating well with the inhibitory effect on melanin production (Fig. 3). After 24-h incubation with sparfloxacin, tyrosinase activity was suppressed to 87% at 0.0125 mM, 66% at 0.025 mM, 56% at 0.125 mM and to 41% at 0.25 mM when compared with the controls. The use of the analyzed antibiotic at the lowest concentration (0.0025 mM) had no effect on tyrosinase activity. The analyzed drug significantly decreased mushroom tyrosinase activity (Table 1) in a concentration-dependent manner. The concentration of sparfloxacin required for 50% inhibition of mushroom tyrosinase activity (IC50) was 0.8 mM.
Results
The effect of sparfloxacin on antioxidant enzymes activity
The effect of sparfloxacin on cell viability
To explain the effect of the tested antibiotic on reactive oxygen species metabolism, the activities of the antioxidative enzymes were characterized. Human melanocytes HEMa-LP were exposed to sparfloxacin at concentrations of 0.025 mM or 0.25 mM (EC50) for 24 h. The first enzyme measured was SOD, i.e. the enzyme which catalyzes the formation of hydrogen peroxide from a superoxide anion. Sparfloxacin enhanced the SOD activity in a
Melanocytes were treated with sparfloxacin in a range of concentrations from 0.001 mM to 1.0 mM for 24 h (Fig. 1). The cell viability was determined by the WST-1 test assay. At a relative low antibiotic concentration (0.001 mM) the loss in cell viability was not statistically significant. Cells treated with 0.01, 0.1, 0.25, 0.5, 0.75 and 1.0 mM of sparfloxacin for 24 h lost about 23%, 41%, 50%, 74%, 88% and 91% in viability, respectively. The value of EC50
Fig. 1. The effect of sparfloxacin on viability of melanocytes. Cells were treated with various sparfloxacin concentrations (0.001–1.0 mM) and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values SEM from three independent experiments (n = 3) performed in triplicate are presented. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
Fig. 2. The effect of sparfloxacin on melanin content in melanocytes. Cells were cultured with sparfloxacin in concentrations from 0.0025 mM to 0.25 mM for 24 h, and the melanin content was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples.
A. Beberok et al. / Pharmacological Reports 67 (2015) 38–43
Fig. 3. The effect of sparfloxacin on the tyrosinase activity in melanocytes. Cells were cultured with sparfloxacin in concentrations from 0.0025 mM to 0.25 mM for 24 h, and the tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
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Fig. 4. The superoxide dismutase (SOD) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples.
Table 1 The inhibitory effect of sparfloxacin on mushroom tyrosinase activity. Sparfloxacin concentration (mM)
Inhibition SEMa (%)
IC50 b (mM)
0.0025 0.025 0.25 1.0
76.9 4.4 68.7 4.0 56.8 2.1 47.2 2.4
0.80
a Samples contained phosphate buffer with different sparfloxacin concentrations, mushroom tyrosinase (1000 units) and L-DOPA solution (2 mg/ml). Tyrosinase activity was measured as described in ‘‘Materials and methods’’ section. b 50% inhibitory concentration
concentration-dependent manner (Fig. 4). The treatment of cells with 0.025 mM and 0.25 mM of sparfloxacin, increased the SOD activity by 12% and 26%, respectively, as compared with the controls. CAT and GPx work together to catalyze the breakdown of hydrogen peroxide, produced by SOD, to water. The intracellular CAT activity was significantly increased by 69% for cells treated with sparfloxacin at 0.025 mM concentration. Sparfloxacin at concentration of 0.25 mM (EC50) had no effect on CAT activity (Fig. 5). The activity of GPx increased by 13% for sparfloxacin concentration 0.025 mM and decreased by 27% for cells treated with 0.25 mM (EC50), in comparison to control cells (Fig. 6).
Fig. 5. The catalase (CAT) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. **p < 0.005 vs. the control samples.
Discussion Sparfloxacin is an important agent of a fluoroquinolone group. It is used clinically to treat urinary, skin, gastrointestinal and respiratory infections. Severe phototoxic reactions have been observed with sparfloxacin treatment in patients exposed to direct or indirect sunlight or UV light from sunlamps [2,30,31]. Thus, the use of sparfloxacin is markedly limited. Previously, we demonstrated that sparfloxacin interacted specifically with melanin, which might cause the drug accumulation in melanin-rich tissues. Slow release of sparfloxacin from binding sites may result in its high level and a long term build-up of this drug stored on melanin, and may lead to prolonged exposure of melanin containing cells and surrounding tissues to the toxicity of the tested fluoroquinolone antibiotic [25,32]. The aim of the present work was to investigate the effect of sparfloxacin on melanin formation (closely related to
Fig. 6. The glutathione peroxidase (GPx) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
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pigmentation) and on the antioxidant defense system in HEMa-LP melanocytes. It was observed that sparfloxacin at concentration of 0.001 mM did not have any significant effect on melanocytes viability. Higher drug concentrations (from 0.01 mM to 1.0 mM) resulted in the loss of cell viability in a concentration-dependent manner (Fig. 1). The value of EC50 was determined to be 0.25 mM. For the same cell line the value of EC50 established for ciprofloxacin was 0.5 mM [23] and 0.75 mM for lomefloxacin [24], which indicates that sparfloxacin is significantly more toxic. Melanin is synthesized in the presence of tyrosinase, a coppercontaining metalloglycoprotein which is though to possess multiple catalytic activities utilizing L-tyrosine, dihydroxyphenylalanine (L-DOPA) and 5,6-dihydroxyindole as substrates [33]. Since tyrosinase is a major regulator of melanin synthesis, we have examined a direct effect of sparfloxacin on the activity of this enzyme. Sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM decreased the tyrosinase activity in melanocytes by 13%, 34%, 44% and 59%, respectively (Fig. 3). We repeated the experiment with commonly used mushroom tyrosinase and observed that sparfloxacin at concentrations from 0.0025 to 1.0 mM decreased its activity by about 23–53%, respectively (Table 1). Our results indicate that an inhibitory effect of sparfloxacin on melanogenesis is probably due to its direct inhibition of tyrosinase activity. The results of our prior studies [23,24] and those obtained in this study reveal that sparfloxacin demonstrates a greater inhibitory effect on cellular tyrosinase activity than ciprofloxacin or lomefloxacin. The analysis of melanin formation in cells cultured in the presence or absence of a drug showed that sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM suppressed the melanin content to 95%, 83%, 78% and 71%, respectively (Fig. 2). In comparison to ciprofloxacin [23] and lomefloxacin [24], sparfloxacin causes higher reduction in melanin content. It has been suggested that free radicals generated after fluoroquinolones treatment play an important role in these antibiotics toxicity [12,18,34]. Cells have their own set of antioxidant defense mechanisms to reduce free radicals formation and to overcome the limit of damaging effects. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are the enzymatic defense system of cells against oxygen radicals [35,36]. Melanin is known to be a scavenger of free radicals and it has been suggested that it possesses the superoxide dismutase activity [37]. Moreover, this biopolymer acts as a biochemical dustbin, mopping up potentially toxic agents. Such properties may be important for protecting the pigment cells as well as surrounding tissues from the natural toxins, xenobiotics, oxygen and free radicals, including ROS [37,38]. In the present study, it has been observed for the first time that sparfloxacin causes significant changes in the activities of the antioxidant enzymes: SOD, CAT and GPx in melanocytes. SOD protects cells by dismutating superoxide into proradical hydrogen peroxide, which in turn is inactivated to oxygen and water by catalase or other H2O2-removing enzymes such as glutathione peroxidase [39]. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide [40]. If H2O2 is not effectively cleared, the level of reactive hydroxyl radicals may increase due to iron-catalyzed Fenton-type reactions [35]. It was observed that the SOD activity increased in a concentrationdependent manner after exposure of melanocytes to sparfloxacin (Fig. 4). This phenomenon might be the main reason of superoxide anion overproduction. Treatment of cells with sparfloxacin at concentration of 0.025 mM increased the CAT and GPx activity (Figs. 5 and 6), what may result from the increasing H2O2 level. At this concentration the cell viability was high enough to assure an appropriate functionality of melanocytes. The still high melanin
content (over 80%) and tyrosinase activity (about 70%) contribute to effective cell defense system. The use of sparfloxacin at concentration of 0.25 mM had no effect on CAT activity, as compared to the control, and decreased GPx activity. Thus, it may be assumed that alterations in the CAT and GPx activity at this concentration play a critical role in sparfloxacin toxicity as a result of redundant H2O2 level that cannot be eliminated. Li et al. [41] have demonstrated that another fluoroquinolone antibiotic, ofloxacin, induces oxidative stress by increasing ROS production and decreasing antioxidant enzymes activity in chondrocytes. The use of ofloxacin in concentration of 0.22 mM decreased CAT and GPx activity, what was connected with the H2O2 overproduction leading to the depletion of antioxidant status of chondrocytes. One has to take into consideration that sparfloxacin concentrations found to have an inhibitory effect on melanogenesis and antioxidant defense system in normal human melanocytes are about 10-fold and 100-fold higher than the concentrations normally observed in clinical trials [42]. However, we have previously demonstrated that sparfloxacin forms complexes with melanin, what may lead to the accumulation of this drug in melanin reach tissues. Such accumulation increases the drug concentration and may induce some toxic effects on melanin containing cells and surrounding tissues [27]. Thus, it is possible that sparfloxacin concentration in melanocytes may be significantly higher than that in serum and therefore the reduction of melanin content, the inhibition of tyrosinase activity as well as the depletion of antioxidant status in the presence of this drug could be observed. In conclusion, 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, especially during a high-dose and/or long-term therapy. Moreover, it may be concluded, that the demonstrated greater inhibitory effect of sprafloxacin on melanogenesis in normal human melanocytes in comparison to ciprofloxacin [23] and lomefloxacin [24] may be an explanation for various toxic activities of these fluoroquinolone derivatives.
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[26] Ozeki H, Ito S, Wakamatsu K, Thody AJ. Spectrophotometric characterization of eumelanin and pheomelanin in hair. Pigment Cell Res 1996;9:265–70. [27] Kim DS, Kim SY, Park SH, Choi YG, Kwon SB, Kim MK, et al. Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis. Biol Pharm Bull 2005;12:2216–9. [28] Busca R, Berlotto C, Ortonne JP, Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70(S6)-kinase pathway induces B16 melanoma cell differentiation. J Biol Chem 1996;271:31824–30. [29] Chung SW, Ha YM, Kim YJ, Song S, Lee H, Suh H, et al. Inhibitory effects of 6-(3hydroxyphenyl)-2-naphtol on tyrosinase activity and melanin synthesis. Arch Pharm Res 2009;32:289–94. [30] Leone R, Venegoni M, Motola D, Moretti U, Piazzetta V, Cocci A, et al. Adverse drug reactions related to the use of fluoroquinolone antimicrobials. Drug Saf 2003;26:109–20. [31] Schentag JJ. Sparfloxacin: a review. Clin Ther 2000;22:372–87. [32] Beberok A, Buszman E, Zdybel M, Pilawa B, Wrzes´niok D. EPR examination of free radical properties of DOPA–melanin complexes with ciprofloxacin, lomefloxacin, norfloxacin and sparfloxacin. Chem Phys Lett 2010;497:115–22. [33] Otre˛ba M, Rok J, Buszman E, Wrzes´niok D. Regulation of melanogenesis: the role of cAMP and MITF. Adv Clin Exp Med 2012;66:33–40. [34] Marrot L, Belaidi JP, Jones C, Perez P, Riou L, Sarasin A, et al. Molecular responses to photogenotoxic stress induced by the antibiotic lomefloxacin in human skin cells: from DNA damage to apoptosis. J Invest Dermatol 2003;121:596–606. [35] Finaud J, Lac G, Filaire E. Oxidative stress: relationship with exercise and training. Sports Med 2006;36:327–58. [36] Mari M, Colell A, Morales A, von Montfort C, Garcia-Ruiz C, Ferna´ndez-Checa JC. Redox control of liver function in health and disease. Antioxid Redox Signal 2010;12:1295–331. [37] Hoogduijn MJ, Cemeli E, Ross K, Anderson D, Thody AJ, Wood JM. Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+. Exp Cell Res 2004;294:60–7. [38] Rozanowska M, Sarna T, Land EJ, Truscott G. Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing oxidising radicals. Free Radic Biol Med 1999;26:518–25. [39] Kawamoto K, Sha SH, Minoda R, Izumikawa M, Kuriyama H, Schacht J, et al. Antioxidant gene therapy can protect hearing and hair cells from ototoxicity. Mol Ther 2004;9:173–81. [40] Meresca V, Flori E, Briganti S, Mastrofrancesco A, Fabbri C, Mileo AM, et al. Correlation between melanogenic and catalase activity in in vitro human melanocytes: a synergic strategy against oxidative stress. Pigment Cell Melanoma Res 2008;21:200–5. [41] Li Q, Penq S, Sheng Z, Wang Y. Ofloxacin induces oxidative damage to joint chondrocytes of juvenile rabbits: excessive production of reactive oxygen species, lipid peroxidation and DNA damage. Eur J Pharmacol 2010;626:146–53. [42] Trautmann M, Ruhnke M, Borner K, Wagner J, Koeppe P. Pharmacokinetics of sparfloxacin and serum bactericidal activity against pneumococci. Antimicrob Agents Chemother 1996;40:776–9.
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Short communication
Impact of sparfloxacin on melanogenesis and antioxidant defense system in normal human melanocytes HEMa-LP – An in vitro study Artur Beberok *, Dorota Wrzes´niok, Michał Otre˛ba, Ewa Buszman Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Sosnowiec, Poland
A R T I C L E I N F O
Article history: Received 25 March 2014 Received in revised form 25 July 2014 Accepted 28 July 2014 Available online 10 August 2014 Keywords: Sparfloxacin Melanocytes Melanization Tyrosinase Antioxidant enzymes
A B S T R A C T
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 EC50 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. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Fluoroquinolones are one of the most commonly prescribed classes of antibiotics. Their broad spectrum of antibacterial coverage and high tissue distribution facilitate the use against a wide variety of community acquired infections. Sparfloxacin, a third-generation fluoroquinolone derivative, is a potent antibacterial agent active against a wide range of Gram-positive and Gram-negative organisms [1,2]. Sparfloxacin acts by inhibiting two types of topoisomerase enzymes, DNA gyrase (topoisomerase II) and topoisomerase IV which are responsible for supercoilling, transcription, replication and chromosomal separation of prokaryotic DNA [3–5]. Although fluoroquinolones are generally well tolerated, they have been associated with a wide array of adverse events such as phototoxicity and photogenotoxicity, which have
* Corresponding author. E-mail address: [email protected] (A. Beberok).
been demonstrated in different in vitro [6–8], in vivo [9,10] and clinical studies [10–12]. Serious incidence of cutaneous toxicity (including erythema, pruritus, urticaria, rash or bullous eruptions) or eye toxicity (retinal degeneration including macular degeneration) has been reported for the use of sparfloxacin [7,13–15]. Moreover, Struwe et al. [16] demonstrated that sparfloxacin caused photogenotoxic effects in the skin and eye (retina and cornea) of rats. This fluoroquinolone belongs to the class of antibiotics known to exhibit severe phototoxicity [17] which may be associated with the production of reactive molecules i.e., reactive oxygen species (singlet oxygen, superoxide anion radical or hydroxyl radical) or highly reactive photodegradation products, able to modify the cell components, including lipids, proteins and nucleic acids [12,18,19]. Melanin, the end product of melanogenesis, determines the color of human skin, hair and eyes. This biopolymer is synthesized and deposited in the form of pigment granules in specialized organelles called melanosomes in the cytoplasm of the melanocytes. Human melanocytes synthesize two distinct types of
http://dx.doi.org/10.1016/j.pharep.2014.07.015 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
A. Beberok et al. / Pharmacological Reports 67 (2015) 38–43
melanin, the black/dark brown eumelanins and the lighter, yellowish/brown, sulphur-containing pheomelanins, which arise from the common biosynthetic pathway involving the tyrosinasecatalyzed oxidation of tyrosine [20–22]. Previously, we documented that ciprofloxacin [23], lomefloxacin [24], norfloxacin and sparfloxacin [25] formed stable complexes with model synthetic melanin in vitro. Moreover, we demonstrated that ciprofloxacin [23] and lomefloxacin [24] modulated biochemical processes in normal human melanocytes, suggesting a mechanism for the drug-induced toxicity on pigmented tissues. Whether the same mechanism can be attributed to another fluoroquinolone derivative, i.e., sparfloxacin, with relatively high toxic potential, remains to be determined. The results concerning the ability of drug-melanin complexes formation as well as the impact of drug on melanogenesis and antioxidant status in human melanocytes could serve as a screening pre-clinical tool to avoid the drug induced toxic effects on pigmented tissues. The objective of the present work was to examine the effect of sparfloxacin on melanogenesis and the antioxidant defense system in cultured normal human melanocytes (HEMa-LP).
Materials and methods Materials Sparfloxacin, L-3,4-dihydroxyphenylalanine (L-DOPA), Triton X-100 and mushroom tyrosinase were purchased from Sigma– Aldrich Inc. (USA). Penicillin was acquired from Polfa Tarchomin (Poland). A growth medium M-254, gentamicin, amphotericin B, and a human melanocyte growth supplement-2 (HMGS-2) were obtained from Cascade Biologics (UK). Trypsin/EDTA was obtained from Cytogen (Poland). Cell proliferation reagent WST-1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH S.A. (Poland). Cell culture Normal human epidermal melanocytes, adult, lightly pigmented (HEMa-LP, Cascade Biologics, UK) were grown according to the manufacturer’s instruction. The cells were cultured in M-254 medium supplemented with HMGS-2, penicillin (100 U/ml), gentamicin (10 mg/ml) and amphotericin B (0.25 mg/ml) at 37 8C in 5% CO2. All experiments were performed using cells from the passages 5–7. Cell viability assay The viability of melanocytes was evaluated by the WST-1 (4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) colorimetric assay. WST-1 is a water-soluble tetrazolium salt, the rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of viable cells. In brief, 5000 cells per well were placed in a 96-well microplate in a supplemented M-254 growth medium and incubated at 37 8C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with sparfloxacin solutions in a concentration range from 0.001 to 1.0 mM. After 21-h incubation, 10 ml of WST-1 were added to 100 ml of culture medium in each well, and the incubation was continued for 3 h. The absorbance of the samples was measured at 440 nm with a reference wavelength of 650 nm, against the controls (the same cells but not treated with sparfloxacin) using a microplate reader UVM 340 (Biogenet, Poland). The controls were normalized to 100% for each assay and treatments were expressed as the percentage of the controls.
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Measurement of melanin content The melanocytes were seeded in a 35 mm dish at a density of 1 105 cells per dish. Sparfloxacin treatment at concentrations of 0.0025 mM, 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM, respectively, began 48 h after seeding. After 24 h of incubation, the cells were washed three times and detached with trypsin/ EDTA. Cell pellets were placed into Eppendorf tubes, dissolved in 100 ml of 1 M NaOH at 80 8C for 1 h, and then centrifuged for 20 min at 16000 g. The supernatants were placed into a 96-well microplate, and absorbance was measured using a microplate reader at 405 nm, i.e. a wavelength at which melanin absorbs light [26]. A standard synthetic melanin curve (0–400 mg/ml) was performed in triplicate for each experiment. Melanin content in sparfloxacin treated cells was expressed as the percentage of the controls (untreated melanocytes). Tyrosinase activity assay Tyrosinase activity in HEMa-LP cells was determined by measuring the rate of oxidation of L-DOPA to dopachrome according to the method described by Kim et al. [27] and Busca et al. [28], with a slight modification. The cells were cultured at a density of 1 105 cells in a 35 mm dish for 48 h. After 24-h incubation with sparfloxacin (concentrations of 0.0025 mM, 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM) the cells were washed three times with PBS, lysed with phosphate buffer (pH 6.8) containing 0.1% Triton X-100, and lysates were clarified by centrifugation at 10000 g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the same lysis phosphate buffer (without Triton). 100 ml of each lysate were placed in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 ml of L-DOPA solution at 37 8C. Absorbance of dopachrome was measured every 10 min for at least 1 h at 475 nm using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls (untreated melanocytes). A cell-free assay system was used to test for direct effects on tyrosinase activity. 130 ml of phosphate buffer containing sparfloxacin at concentrations from 0.0025 to 1.0 mM were mixed with 20 ml of mushroom tyrosinase (1000 units), and 100 ml of L-DOPA solution (2 mg/ml) were added to each well. The assay mixtures were incubated at 37 8C for 20 min, and absorbance of dopachrome was measured at 475 nm in a microplate reader. The mushroom tyrosinase activities were calculated in the relation to the controls (samples without sparfloxacin). The value IC50 (the concentration of a drug that inhibits a standard response by 50%) was calculated on the basis of a dose-dependent inhibition curve, as described by Chung et al. [29]. Superoxide dismutase (SOD) assay Superoxide dismutase (SOD) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. This kit utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radical. SOD activity was expressed in U/mg protein. Catalase (CAT) assay Catalase (CAT) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. This kit utilizes the peroxidatic function of CAT for determination of enzyme activity. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration
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of H2O2. The produced formaldehyde is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as a chromogen. One unit of CAT was defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde per minute at 25 8C. CAT activity was expressed in nmol/min/mg protein. Glutathione peroxidase (GPx) assay Glutathione peroxidase (GPx) activity was measured using an assay kit (Cayman, MI, USA) according to the manufacturer’s instruction. The measurement of GPx activity is based on the principle of a coupled reaction with glutathione reductase (GR). The oxidized glutathione (GSSG) formed after reduction of hydroperoxide by GPx is recycled to its reduced state by GR in the presence of NADPH. The oxidation of NADPH is accompanied by a decrease in absorbance at 340 nm. One unit of GPx was defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute at 25 8C. GPx activity was expressed in nmol/min/mg protein. Statistical analysis In all experiments, mean values of at least three separate experiments (n = 3) performed in triplicate standard error of the mean (SEM) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software. A value of p < 0.05 (*) or p < 0.005 (**), obtained with a Student’s t-test by comparing the data with those for the controls (cells without sparfloxacin), was considered statistically significant.
(i.e. the amount of a drug that produces loss in cell viability by 50%) was 0.25 mM. The effect of sparfloxacin on melanization process The effectiveness of a melanization process was estimated by measuring the melanin content and cellular tyrosinase activity in human melanocytes treated with sparfloxacin at concentrations from 0.0025 mM to 0.25 mM, for 24 h. After determining a calibration curve, the melanin content per cell was determined as 36.5 to 26.0 pg/cell for melanocytes treated with antibiotic and 37.1 3.4 pg/cell for a control sample. The obtained results, recalculated for culture (1 105 cells), were finally expressed as a percentage of the controls (Fig. 2). Sparfloxacin in concentration of 0.0025 mM had no effect on melanin content in melanocytes. In cells treated with sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM for 24 h, melanin production decreased in a concentration-dependent manner by about 5%, 17%, 22% and 29%, respectively. Tyrosinase activity in HEMa-LP cells treated with sparfloxacin also decreased in a manner correlating well with the inhibitory effect on melanin production (Fig. 3). After 24-h incubation with sparfloxacin, tyrosinase activity was suppressed to 87% at 0.0125 mM, 66% at 0.025 mM, 56% at 0.125 mM and to 41% at 0.25 mM when compared with the controls. The use of the analyzed antibiotic at the lowest concentration (0.0025 mM) had no effect on tyrosinase activity. The analyzed drug significantly decreased mushroom tyrosinase activity (Table 1) in a concentration-dependent manner. The concentration of sparfloxacin required for 50% inhibition of mushroom tyrosinase activity (IC50) was 0.8 mM.
Results
The effect of sparfloxacin on antioxidant enzymes activity
The effect of sparfloxacin on cell viability
To explain the effect of the tested antibiotic on reactive oxygen species metabolism, the activities of the antioxidative enzymes were characterized. Human melanocytes HEMa-LP were exposed to sparfloxacin at concentrations of 0.025 mM or 0.25 mM (EC50) for 24 h. The first enzyme measured was SOD, i.e. the enzyme which catalyzes the formation of hydrogen peroxide from a superoxide anion. Sparfloxacin enhanced the SOD activity in a
Melanocytes were treated with sparfloxacin in a range of concentrations from 0.001 mM to 1.0 mM for 24 h (Fig. 1). The cell viability was determined by the WST-1 test assay. At a relative low antibiotic concentration (0.001 mM) the loss in cell viability was not statistically significant. Cells treated with 0.01, 0.1, 0.25, 0.5, 0.75 and 1.0 mM of sparfloxacin for 24 h lost about 23%, 41%, 50%, 74%, 88% and 91% in viability, respectively. The value of EC50
Fig. 1. The effect of sparfloxacin on viability of melanocytes. Cells were treated with various sparfloxacin concentrations (0.001–1.0 mM) and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values SEM from three independent experiments (n = 3) performed in triplicate are presented. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
Fig. 2. The effect of sparfloxacin on melanin content in melanocytes. Cells were cultured with sparfloxacin in concentrations from 0.0025 mM to 0.25 mM for 24 h, and the melanin content was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples.
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Fig. 3. The effect of sparfloxacin on the tyrosinase activity in melanocytes. Cells were cultured with sparfloxacin in concentrations from 0.0025 mM to 0.25 mM for 24 h, and the tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
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Fig. 4. The superoxide dismutase (SOD) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples.
Table 1 The inhibitory effect of sparfloxacin on mushroom tyrosinase activity. Sparfloxacin concentration (mM)
Inhibition SEMa (%)
IC50 b (mM)
0.0025 0.025 0.25 1.0
76.9 4.4 68.7 4.0 56.8 2.1 47.2 2.4
0.80
a Samples contained phosphate buffer with different sparfloxacin concentrations, mushroom tyrosinase (1000 units) and L-DOPA solution (2 mg/ml). Tyrosinase activity was measured as described in ‘‘Materials and methods’’ section. b 50% inhibitory concentration
concentration-dependent manner (Fig. 4). The treatment of cells with 0.025 mM and 0.25 mM of sparfloxacin, increased the SOD activity by 12% and 26%, respectively, as compared with the controls. CAT and GPx work together to catalyze the breakdown of hydrogen peroxide, produced by SOD, to water. The intracellular CAT activity was significantly increased by 69% for cells treated with sparfloxacin at 0.025 mM concentration. Sparfloxacin at concentration of 0.25 mM (EC50) had no effect on CAT activity (Fig. 5). The activity of GPx increased by 13% for sparfloxacin concentration 0.025 mM and decreased by 27% for cells treated with 0.25 mM (EC50), in comparison to control cells (Fig. 6).
Fig. 5. The catalase (CAT) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. **p < 0.005 vs. the control samples.
Discussion Sparfloxacin is an important agent of a fluoroquinolone group. It is used clinically to treat urinary, skin, gastrointestinal and respiratory infections. Severe phototoxic reactions have been observed with sparfloxacin treatment in patients exposed to direct or indirect sunlight or UV light from sunlamps [2,30,31]. Thus, the use of sparfloxacin is markedly limited. Previously, we demonstrated that sparfloxacin interacted specifically with melanin, which might cause the drug accumulation in melanin-rich tissues. Slow release of sparfloxacin from binding sites may result in its high level and a long term build-up of this drug stored on melanin, and may lead to prolonged exposure of melanin containing cells and surrounding tissues to the toxicity of the tested fluoroquinolone antibiotic [25,32]. The aim of the present work was to investigate the effect of sparfloxacin on melanin formation (closely related to
Fig. 6. The glutathione peroxidase (GPx) activity in HEMa-LP cells after 24-h incubation with 0.025 or 0.25 mM of sparfloxacin. Data are mean SEM of at least three independent experiments (n = 3) performed in triplicate. *p < 0.05 vs. the control samples; **p < 0.005 vs. the control samples.
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pigmentation) and on the antioxidant defense system in HEMa-LP melanocytes. It was observed that sparfloxacin at concentration of 0.001 mM did not have any significant effect on melanocytes viability. Higher drug concentrations (from 0.01 mM to 1.0 mM) resulted in the loss of cell viability in a concentration-dependent manner (Fig. 1). The value of EC50 was determined to be 0.25 mM. For the same cell line the value of EC50 established for ciprofloxacin was 0.5 mM [23] and 0.75 mM for lomefloxacin [24], which indicates that sparfloxacin is significantly more toxic. Melanin is synthesized in the presence of tyrosinase, a coppercontaining metalloglycoprotein which is though to possess multiple catalytic activities utilizing L-tyrosine, dihydroxyphenylalanine (L-DOPA) and 5,6-dihydroxyindole as substrates [33]. Since tyrosinase is a major regulator of melanin synthesis, we have examined a direct effect of sparfloxacin on the activity of this enzyme. Sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM decreased the tyrosinase activity in melanocytes by 13%, 34%, 44% and 59%, respectively (Fig. 3). We repeated the experiment with commonly used mushroom tyrosinase and observed that sparfloxacin at concentrations from 0.0025 to 1.0 mM decreased its activity by about 23–53%, respectively (Table 1). Our results indicate that an inhibitory effect of sparfloxacin on melanogenesis is probably due to its direct inhibition of tyrosinase activity. The results of our prior studies [23,24] and those obtained in this study reveal that sparfloxacin demonstrates a greater inhibitory effect on cellular tyrosinase activity than ciprofloxacin or lomefloxacin. The analysis of melanin formation in cells cultured in the presence or absence of a drug showed that sparfloxacin at concentrations of 0.0125 mM, 0.025 mM, 0.125 mM and 0.25 mM suppressed the melanin content to 95%, 83%, 78% and 71%, respectively (Fig. 2). In comparison to ciprofloxacin [23] and lomefloxacin [24], sparfloxacin causes higher reduction in melanin content. It has been suggested that free radicals generated after fluoroquinolones treatment play an important role in these antibiotics toxicity [12,18,34]. Cells have their own set of antioxidant defense mechanisms to reduce free radicals formation and to overcome the limit of damaging effects. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are the enzymatic defense system of cells against oxygen radicals [35,36]. Melanin is known to be a scavenger of free radicals and it has been suggested that it possesses the superoxide dismutase activity [37]. Moreover, this biopolymer acts as a biochemical dustbin, mopping up potentially toxic agents. Such properties may be important for protecting the pigment cells as well as surrounding tissues from the natural toxins, xenobiotics, oxygen and free radicals, including ROS [37,38]. In the present study, it has been observed for the first time that sparfloxacin causes significant changes in the activities of the antioxidant enzymes: SOD, CAT and GPx in melanocytes. SOD protects cells by dismutating superoxide into proradical hydrogen peroxide, which in turn is inactivated to oxygen and water by catalase or other H2O2-removing enzymes such as glutathione peroxidase [39]. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide [40]. If H2O2 is not effectively cleared, the level of reactive hydroxyl radicals may increase due to iron-catalyzed Fenton-type reactions [35]. It was observed that the SOD activity increased in a concentrationdependent manner after exposure of melanocytes to sparfloxacin (Fig. 4). This phenomenon might be the main reason of superoxide anion overproduction. Treatment of cells with sparfloxacin at concentration of 0.025 mM increased the CAT and GPx activity (Figs. 5 and 6), what may result from the increasing H2O2 level. At this concentration the cell viability was high enough to assure an appropriate functionality of melanocytes. The still high melanin
content (over 80%) and tyrosinase activity (about 70%) contribute to effective cell defense system. The use of sparfloxacin at concentration of 0.25 mM had no effect on CAT activity, as compared to the control, and decreased GPx activity. Thus, it may be assumed that alterations in the CAT and GPx activity at this concentration play a critical role in sparfloxacin toxicity as a result of redundant H2O2 level that cannot be eliminated. Li et al. [41] have demonstrated that another fluoroquinolone antibiotic, ofloxacin, induces oxidative stress by increasing ROS production and decreasing antioxidant enzymes activity in chondrocytes. The use of ofloxacin in concentration of 0.22 mM decreased CAT and GPx activity, what was connected with the H2O2 overproduction leading to the depletion of antioxidant status of chondrocytes. One has to take into consideration that sparfloxacin concentrations found to have an inhibitory effect on melanogenesis and antioxidant defense system in normal human melanocytes are about 10-fold and 100-fold higher than the concentrations normally observed in clinical trials [42]. However, we have previously demonstrated that sparfloxacin forms complexes with melanin, what may lead to the accumulation of this drug in melanin reach tissues. Such accumulation increases the drug concentration and may induce some toxic effects on melanin containing cells and surrounding tissues [27]. Thus, it is possible that sparfloxacin concentration in melanocytes may be significantly higher than that in serum and therefore the reduction of melanin content, the inhibition of tyrosinase activity as well as the depletion of antioxidant status in the presence of this drug could be observed. In conclusion, 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, especially during a high-dose and/or long-term therapy. Moreover, it may be concluded, that the demonstrated greater inhibitory effect of sprafloxacin on melanogenesis in normal human melanocytes in comparison to ciprofloxacin [23] and lomefloxacin [24] may be an explanation for various toxic activities of these fluoroquinolone derivatives.
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