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Effect of tetracycline and UV radiation on melanization and antioxidant status of melanocytes
Journal of Photochemistry and Photobiology B: Biology 148 (2015) 168–173
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Effect of tetracycline and UV radiation on melanization and antioxidant status of melanocytes Jakub Rok, Ewa Buszman ⇑, Marcin Delijewski, Michał Otre˛ba, Artur Beberok, Dorota Wrzes´niok ´ ska 4, PL 41-200 Sosnowiec, Poland Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellon
a r t i c l e
i n f o
Article history: Received 18 January 2015 Received in revised form 13 April 2015 Accepted 20 April 2015 Available online 27 April 2015
a b s t r a c t Tetracycline is a semisynthetic antibiotic and is used in several types of infections against both grampositive 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. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Tetracycline is one of the first generation tetracycline antibiotics, still in use today. The drug was synthesized by catalytic hydrogenation of chlortetracycline. The obtained C7-dechloro derivative had a better solubility profile and favorable pharmacological activity. This compound was approved by the FDA for clinical use in 1954, as the first novel tetracycline by modification of a natural product, and it was one of the first commercially successful semisynthetic antibiotics used in medicine [1]. Tetracyclines, despite the development of resistance by some bacterial species, are still effectively used against both gram-positive and gram-negative bacteria. These compounds are particularly useful in several types of infections, such as atypical pneumonias, community-acquired pneumonia, rickettsial and chlamydial infections, Lyme disease, cholera, syphilis and periodontal infections [2]. Tetracyclines bind primarily to the 30S subunit of bacterial ribosome where they inhibit protein synthesis by blocking the binding of aminoacylated tRNA (aa-tRNA) to the A site of the ribosome [3]. In addition to their antimicrobial activity, tetracyclines have a number of non-antibacterial effects, including the inhibition
⇑ Corresponding author. Tel.: +48 32 364 16 11. E-mail address: [email protected] (E. Buszman). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.009 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
of inflammation, proteolysis and angiogenesis, as well as anticancer and antimetastatic activity [2,4]. It has been reported that tetracycline causes phototoxic reactions. Although tetracycline is less photoactive than chlortetracycline and doxycycline, it may cause among others photoonycholysis, pseudoporphyria, solar urticaria and the fixed drug eruption [5–8]. In correlation with the clinical observations, it was found that tetracyclines cause singlet oxygen mediated oxidation, which leads to the damage of living cells [9]. Photosensitivity is an adverse cutaneous reaction that results when a certain chemical or drug is applied topically or taken systemically at the same time when a person is exposed to UV radiation or visible light [10]. This phenomenon has been recognized for hundreds of years. In the 13th century, Ibn El-Bitar – the Arab scholar noted that certain plant extracts could be applied in combination in exposure to sunlight to treat vitiligo. These herbal medicines were rediscovered in the 1940s and identified as furocoumarins (psoralens) – phototoxic substances, which have been used in modern photochemotherapy to treat chronic dermatoses, such as vitiligo, psoriasis and fungoides [5,11–13]. It was suggested, that phototoxic reactions may correspond to the binding of drug to melanin biopolymers and drug accumulation in pigmented tissues [14]. Melanins are produced by melanocytes – highly specialized cells residing primarily in the hair follicle, epidermis and eye.
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The melanogenesis process takes place in membrane-bound organelles called melanosomes. The active export of melanosomes from melanocytes to surrounding keratinocytes in the skin or to newly synthesized hair is the basis of skin and hair pigmentation [15]. The most well-known agent that enhances melanogenesis is UV radiation. UV-induced skin pigmentation plays a photoprotective role by preventing from DNA damages and mutations. The shielding effect of melanin, is achieved by its ability to serve as a physical barrier that scatters UV radiation, and as an absorbing filter that reduces the penetration of UV through the epidermis. Melanin, besides functioning as a broadband UV absorbent, has also antioxidant properties, acting as a free radical scavenger and having a superoxide dismutase properties [16]. Previously, we documented that aminoglycoside antibiotics: amikacin [17], kanamycin [18], netilmicin [19] and streptomycin [20] as well as fluoroquinolones: ciprofloxacin [21] and lomefloxacin [22], suppressed melanin biosynthesis and affected antioxidant enzymes activities in human light pigmented melanocytes. We also documented that nicotine modulated melanin biosynthesis and antioxidant enzymes activities in human dark pigmented melanocytes [23]. The purpose of this work was to estimate the effect of tetracycline and UV radiation on viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes, dark pigmented (HEMn-DP).
2. Materials and methods
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2.4. 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 °C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with tetracycline and exposed to UVA irradiation. After 21-h incubation since irradiation, 10 ll of WST-1 were added to 100 ll of culture medium in each well, and the incubation was continued for another 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 a drug) using a microplate reader UVM 340 (Biogenet). The controls were normalized to 100% for each assay and treatments were expressed as the percentage of the controls. 2.5. Melanin content Cell pellets were placed into Eppendorf tubes, dissolved in 100 ll of 1 M NaOH at 80 °C for 1 h, and then centrifuged for 20 min at 16,000g. The supernatants were placed into a 96-well microplate, and absorbance was measured using microplate reader at 405 nm – a wavelength at which melanin absorbs light [24]. Melanin content in tetracycline treated cells was expressed as the percentage of the controls (untreated melanocytes).
2.1. Materials 2.6. Tyrosinase activity Tetracycline hydrochloride, amphotericin B solution (250 lg/ ml), L-3,4-dihydroxyphenylalanine (L-DOPA) and phosphate buffered saline (PBS) were purchased from Sigma–Aldrich Inc. (USA). A growth medium M-254 and a human melanocyte growth supplement-2 (HMGS-2) were acquired from Cascade Biologics (UK). Neomycin sulfate was obtained from Amara (Poland). Penicillin was acquired from Polfa Tarchomin (Poland). Trypsin/EDTA was obtained from Cytogen (Poland). Cell Proliferation Reagent WST1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH SA (Poland).
2.2. Cell culture Human epidermal melanocytes, neonatal, dark pigmented (HEMn-DP, Cascade Biologics) were grown according to the manufacturer’s instruction. The cells were cultured in a M-254 medium supplemented with HMGS-2, penicillin (100 U/ml), neomycin (10 lg/ml) and amphotericin B (0.25 lg/ml) at 37 °C in 5% CO2. All experiments were performed using cells from the passages 5 to 10.
Tyrosinase activity in HEMn-DP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome according to the method described previously [25,26]. Cell lysates were clarified by centrifugation at 10,000g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the phosphate buffer. 100 ll of each lysate were put in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 ll of L-DOPA solution at 37 °C. Control wells contained 100 ll of lysis buffer and 40 ll of L-DOPA solution. Absorbance of dopachrome was measured every 10 min for at least 1.5 h at 475 nm using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls. 2.7. 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.
2.3. UVA irradiation procedure 2.8. Catalase (CAT) assay The ultraviolet light source used in this study was a filtered lamp BVL-8.LM (Vilber Lourmat, France). The intensity of UVA (kmax = 365 nm) radiation was 720 mW/cm2 at 15 cm. The cells, after 24-h incubation with a drug, were irradiated uncovered in petri dishes. Before irradiation the medium had been replaced by PBS. Time of UV exposure was 15 or 30 min. Simultaneously, the nonirradiated cell cultures (control samples) were kept in the dark at 37 °C and 5% CO2. After irradiation PBS was removed from the cells and melanocytes were incubated in the growth medium for 24 h. Then the cells were lysed.
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 of H2O2. The formaldehyde produced is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. One unit of CAT was defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde
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per minute at 25 °C. CAT activity was expressed in nmol/min/mg protein. 2.9. 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 (GSH) 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 °C. GPx activity was expressed in nmol/min/mg protein. 2.10. Statistical analysis In all experiments, mean values of at least three separate experiments (n = 3) performed in triplicate ± standard error of the mean (S.E.M.) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software by means of one-way ANOVA (the influence of UVA radiation or tetracycline) and twoway ANOVA (the influence of UVA radiation and tetracycline), as well as Dunnett’s multiple comparison test in both cases. In all cases the statistical significance was found at least at p < 0.05. 3. Results 3.1. The effect of tetracycline and UVA radiation on cell viability The impact of tetracycline on the viability of melanocytes, was evaluated using antibiotic in concentrations 0.5 lM, 5.0 lM and 50 lM for 24 h (Fig. 1). The concentration of tetracycline 0.5 lM did not cause statistically significant changes in the viability of cells. The exposure of melanocytes to tetracycline in concentrations 5.0 and 50 lM was associated with loss in cell viability by 13.6% and 49.2%, respectively. The effect of simultaneous exposure of melanocytes to tetracycline in concentrations 0.5 lM, 5.0 lM and 50 lM and UVA radiation for 15 min, resulted in cell loss by 6.4%, 14.2% and 47.8%,
Fig. 1. The effect of tetracycline and UVA radiation (15 or 30 min) on viability of melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM, 5.0 lM or 50.0 lM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values ± S.E.M. from three independent experiments (n = 3) performed in triplicate are presented. ⁄⁄ p < 0.005 vs. the control samples.
respectively. After prolongation of the UVA exposure time to 30 min, the changes in cell viability observed for these concentrations of tetracycline were expressed by the cell loss by 20.1%, 36.6% and 70.9%, respectively. The changes of the amount of tested melanocytes and their morphology caused by treatment with 50.0 lM of tetracycline and exposure to UVA radiation for 30 min are presented in Fig. 2. Observed changes show a similar pattern as the results of the WST-1 cell proliferation test. These changes involve mainly cell loss and a decrease of number and the length of dendrites. Although alterations caused by UVA radiation are minor (Fig. 2B) in comparison to the control, there was observed a significant melanocytes loss and changes in their morphology after exposure of cells to tetracycline (Fig. 2C) as well as simultaneous tetracycline and UVA radiation (Fig. 2D). 3.2. The effect of tetracycline and UVA radiation on melanization process The melanization process in cells incubated for 24 h with tetracycline in concentrations 0.5 lM and 5.0 lM was described by melanin content and activity of the main melanotic enzyme, tyrosinase. Tetracycline in these concentrations had no effect on both melanin content (Fig. 3) and the activity of tyrosinase (Fig. 4). In case of cells treated with the same tetracycline concentrations and exposed to UVA radiation for 15 min, melanin production was increased to 108.7% and 115.7%, respectively, when compared with the controls. Prolongation of the UVA exposure time to 30 min resulted in elevation of melanin content to 110.6% and 123.2%, respectively, when compared with the controls. The tyrosinase activity after 24-h incubation of melanocytes with tetracycline in concentrations 0.5 lM and 5.0 lM with simultaneous exposition to UVA radiation for 15 min resulted in the increase of tyrosinase activity to 103.9% (the value not statistically significant) and 114.6%, respectively, when compared with the controls. Prolongation of the UVA exposure time to 30 min resulted in rise of tyrosinase activity to 112.5% and 122.3%, respectively, when compared with the controls. 3.3. The effect of tetracycline and UVA radiation on antioxidant enzymes activities In order to estimate the impact of tetracycline and UVA radiation on antioxidant defense system in normal human melanocytes HEMn-DP, evaluation of the activities of superoxide dismutase, catalase and glutathione peroxidase was made. Melanocytes were exposed to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h. Only in the highest tested concentration, tetracycline alone increased the SOD activity by 20.2% (Fig. 5), when compared with the controls. The simultaneous exposure of cells to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 15 min resulted in increase of the activity of SOD by 13.5% and 32.1%, respectively, while prolongation of the irradiation time to 30 min produced increase in SOD activity by 22.8% and 35.2%, respectively, when compared with the controls. Tetracycline alone only in the highest tested concentration reduced the CAT activity by 8.1% (Fig. 6), when compared with the controls. The simultaneous exposure of melanocytes to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 15 min resulted in the decrease of the activity of CAT by 21.7% and 35.1%, respectively, while prolongation of the irradiation time to 30 min led to decrease in CAT activity by 31.7% and 38.2%, respectively, when compared with the controls. Tetracycline alone in the highest tested concentration (5.0 lM) increased the activity of GPx by 25.6% (Fig. 7), when compared
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Fig. 2. Morphological changes of human epidermal melanocytes HEMn-DP: untreated (A), exposed to UVA radiation for 30 min (B), treated with 50.0 lM of tetracycline (C), treated with 50.0 lM of tetracycline and exposed to UVA radiation for 30 min (D).
5.0 lM and 50 lM decreased the cell viability in a dose-dependent manner (Fig. 1). The value of EC50 was 50 lM. For all tested concentrations, the effect of simultaneous exposure of melanocytes to tetracycline and UVA radiation for 30 min, was associated with heightened cell loss in the examined concentrations, indicating the phototoxic effect of tetracycline. Melanins are the end-products of multistep L-tyrosine oxidation. The key enzyme involved in the synthesis of all types of melanins is tyrosinase (EC 1.14.18.1), which catalyses the rate-limiting initial step in melanogenesis – the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine
(DOPA) and its immediate subsequent oxidation to DOPAquinone [28]. Tetracycline in tested concentrations had no influence on the melanization process in cultured melanocytes. After exposure of melanocytes to UVA radiation, the induction of melanogenesis expressed by consistent increase in melanin content and Fig. 3. The effect of tetracycline and UVA radiation (15 or 30 min) on melanin content in melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM or 5.0 lM for 24 h, and melanin content was measured as described in Section 2. Results are expressed as percentages of the controls. Data are mean ± S.E.M. 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.
with the controls. In case of cells exposed to UVA radiation for 15 min, only tetracycline in concentration 0.5 lM caused significant increase of GPx activity by 15.6%. The exposure of cells to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 30 min increased the activity of GPx by 41.3% and 22.9%, respectively, when compared with the controls. 4. Discussion More than 300 types of topical and systemic medications are responsible for photosensitivity. Among these agents, one of the most commonly used is group of tetracycline antibiotics [27]. In the present study the simultaneous effect of tetracycline and UVA irradiation on cell viability, melanin content and tyrosinase activity, as well as antioxidant defense system in dark pigmented melanocytes was examined. We used the culture of normal human epidermal melanocytes HEMn-DP. Tetracycline in concentrations
Fig. 4. The effect of tetracycline and UVA radiation (15 or 30 min) on tyrosinase activity in melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM or 5.0 lM for 24 h, and tyrosinase activity was measured as described in Section 2. Results are expressed as percentages of the controls. Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
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Fig. 5. The superoxide dismutase (SOD) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
tyrosinase activity (Figs. 3 and 4) was observed. The increase was especially seen for the concentration of tetracycline 5.0 lM and UVA exposure for 30 min, reaching a rise by 27.4% in melanin content, corresponding well with increase by 31.1% in tyrosinase activity. Skin pigmentation is determined by over 100 genes including those that encode transcription factors, enzymes, hormones, autocrine and paracrine factors and their receptors [29]. Besides these biochemical factors, the induction of melanogenesis might be a result of the external and internal stress [30], also in form of phototoxicity. Clinically, a phototoxic reaction resembles a sunburn and is characterized by erythema and edema. The erythema usually starts a few hours after exposure, reaches a peak in several hours to a few days, and is followed by desquamation and hyperpigmentation [31]. Hyperpigmentation results from the induction of melanocyte proliferation and migration, enhanced melanin production, deposition of the drug or its photoproducts in the skin or from post-inflammatory changes secondary to subliminal phototoxicity. Other less common manifestations of phototoxicity that
Fig. 6. The catalase (CAT) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. 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.
have been described include slate-gray pigmentation, lichenoid eruptions, photo-onycholysis, and pseudoporphyria [6,32]. The observed specific increase in melanin content may occur in response to the phototoxic effect of the antibiotic which leads to induction of defense mechanisms in the cell, including production of melanin. This is consistent with the increasing activity of tyrosinase with the rising concentration of tetracycline. As melanin may interact with various chemical substances leading to the formation of drug-melanin complexes, thus protecting cells from exposure to harmful compounds, the long-term retention of bound substances may in turn lead to degeneration of melanocytes, that takes place when the detoxifying capacities of melanin are exhausted [33,34]. This can be also caused by UVA radiation that may disturb the capacity of melanin pigment. Melanin has ability to absorb UV photons and act as a scavenger of free radicals generated in response to UV irradiation [35]. On the other hand, higher UV doses may cause oxidation of melanin leading to the generation of reactive oxygen species (ROS) [36]. Thereby, melanogenesis can be considered as a prooxidative process increasing the risk of cell’s damage [37]. A phototoxic reaction begins when a substance absorbs photons of energy, usually in the UVA spectrum, and causes excitation of the molecule. When these excited state molecules return to their ground state, they transfer energy to surrounding oxygen, thereby inducing the generation of reactive oxygen species. The ROS cause cellular damage through the oxidation of lipids, nucleic acids and proteins [27,38]. ROS may react with both cellular and extracellular components, leading to induction of cytotoxicity, apoptosis, mutations and carcinogenesis [35]. Moreover, one of the main protective mechanism against oxidative damages involves the NF-E2 – related factor 2 (Nrf2), which induces transcription of genes encoding antioxidant enzymes that build cellular antioxidant system [39]. The antioxidant system, which major role is protection against harmful effects of ROS, consists mainly of superoxide dismutase, catalase and glutathione peroxidase enzymes. Melanin may also act as a free radicals scavenger and possess the superoxide dismutase activity [40]. In the present study it has been observed that tetracycline alone only in the highest analyzed concentration causes alterations in the activities of all tested antioxidant enzymes in melanocytes, causing increase in activities of SOD and GPx, on the contrary to decrease in CAT activity. Exposure of cells to UVA radiation is associated with the concentration-dependent increase in SOD activity
Fig. 7. The glutathione peroxidase (GPx) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
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(Fig. 5) which may be due to overproduction of the superoxide anion, as a result of phototoxic properties of the antibiotic and the augmented antioxidant action of the enzyme. On the other hand, the elevated activity of SOD may enhance the formation of hydroxyl radicals [41]. The observed decrease of CAT activity after exposure of melanocytes to both tested concentrations of tetracycline and UVA irradiation (Fig. 6) may be caused by the sensitivity of catalase to free radicals produced in response to phototoxic action of the antibiotic or damage of the enzyme caused by UVA radiation [35]. Results obtained for the control samples suggest, that CAT deficiency may develop after UVA irradiation without a presence of phototoxic drug. This is consistent with the findings, that catalase may absorb UV radiation and become inactivated [42]. Taking into account, that the main enzyme responsible for inactivation of the proradical hydrogen peroxide in melanocytes is catalase [43,44], we admit the increase in GPx activity observed in our study (Fig. 7) to a compensation mechanism in response to the overproduction of the hydrogen peroxide that cannot be eliminated. Summarizing, we can indicate the phototoxic reactions as a major causing factor for changes in the biochemistry of melanocytes observed in our culture. Tetracycline alone has no influence on melanin content, tyrosinase activity and only in the highest tested concentration may change the cellular antioxidant status in normal human epidermal melanocytes. Simultaneous exposure of the cells to UVA radiation causes alterations of biochemical processes including induction of melanization and occurence of oxidative stress inside melanocytes which is expressed by significant changes in SOD, CAT and GPx activities. This indicates that both factors may be responsible for the changes occurring in vivo. In this study we observed for the first time that tetracycline and UVA evoke a phototoxic action in relation to the biochemistry of normal human epidermal melanocytes. The described effects may explain the mechanisms of the phototoxic impact of the drug on melanin containing tissues. Acknowledgment This study was supported by Medical University of Silesia, Katowice (KNW-2-038/D/3/K). References [1] M.L. Nelson, S.B. Levy, The history of the tetracyclines, Ann. NY. Acad. Sci. 1241 (2011) 17–32. [2] F. Bahrami, D.L. Morris, M.H. Pourgholami, Tetracyclines: drugs with huge therapeutic potential, Mini Rev. Med. Chem. 12 (2012) 44–52. [3] D.E. Brodersen, W.M. Clemons Jr., A.P. Carter, R.J. Morgan-Warren, B.T. Wimberly, V.D.D. Ramakrishnan, The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit, Cell 103 (2000) 1143–1154. [4] B. Zakeri, G.D. Wright, Chemical biology of tetracycline antibiotics, Biochem. Cell Biol. 86 (2008) 124–136. [5] S.G. Vassileva, G. Mateev, L.C. Parish, Antimicrobial photosensitive reactions, Arch. Intern. Med. 158 (1998) 1993–2000. [6] L.B. Valeyrie-Allanore, B. Sassolas, J.C. Roujeau, Drug-induced skin, nail and hair disorders, Drug Saf. 30 (2007) 1011–1030. [7] A.M. Drucker, C.F. Rosen, Drug-induced photosensitivity: culprit drugs, management and prevention, Drug Saf. 34 (2011) 821–837. [8] L.M. Yap, P.A. Foley, R.B. Crouch, C.S. Baker, Drug-induced solar urticaria due to tetracycline, Australas. J. Dermatol. 41 (2000) 181–184. [9] D.E. Moore, Drug-induced cutaneous photosensitivity: incidence, mechanism, prevention and management, Drug Saf. 25 (2002) 345–372. [10] R. Dubakiene, M. Kupriene, Scientific problems of photosensitivity, Medicina (Kaunas) 42 (2006) 619–624. [11] M. Abu Tahir, K. Pramod, S.H. Ansari, J. Ali, Current remedies for vitiligo, Autoimmun. Rev. 9 (2010) 516–520. [12] F. Almutawa, N. Alnomair, Y. Wang, I. Hamzavi, H.W. Lim, Systematic review of UV-based therapy for psoriasis, Am. J. Clin. Dermatol. 14 (2013) 87–109. [13] D. Humme, A. Nast, R. Erdmann, S. Vandersee, M. Beyer, Systematic review of combination therapies for mycosis fungoides, Cancer Treat. Rev. 40 (2014) 927–933.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effect of tetracycline and UV radiation on melanization and antioxidant status of melanocytes Jakub Rok, Ewa Buszman ⇑, Marcin Delijewski, Michał Otre˛ba, Artur Beberok, Dorota Wrzes´niok ´ ska 4, PL 41-200 Sosnowiec, Poland Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellon
a r t i c l e
i n f o
Article history: Received 18 January 2015 Received in revised form 13 April 2015 Accepted 20 April 2015 Available online 27 April 2015
a b s t r a c t Tetracycline is a semisynthetic antibiotic and is used in several types of infections against both grampositive 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. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Tetracycline is one of the first generation tetracycline antibiotics, still in use today. The drug was synthesized by catalytic hydrogenation of chlortetracycline. The obtained C7-dechloro derivative had a better solubility profile and favorable pharmacological activity. This compound was approved by the FDA for clinical use in 1954, as the first novel tetracycline by modification of a natural product, and it was one of the first commercially successful semisynthetic antibiotics used in medicine [1]. Tetracyclines, despite the development of resistance by some bacterial species, are still effectively used against both gram-positive and gram-negative bacteria. These compounds are particularly useful in several types of infections, such as atypical pneumonias, community-acquired pneumonia, rickettsial and chlamydial infections, Lyme disease, cholera, syphilis and periodontal infections [2]. Tetracyclines bind primarily to the 30S subunit of bacterial ribosome where they inhibit protein synthesis by blocking the binding of aminoacylated tRNA (aa-tRNA) to the A site of the ribosome [3]. In addition to their antimicrobial activity, tetracyclines have a number of non-antibacterial effects, including the inhibition
⇑ Corresponding author. Tel.: +48 32 364 16 11. E-mail address: [email protected] (E. Buszman). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.009 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
of inflammation, proteolysis and angiogenesis, as well as anticancer and antimetastatic activity [2,4]. It has been reported that tetracycline causes phototoxic reactions. Although tetracycline is less photoactive than chlortetracycline and doxycycline, it may cause among others photoonycholysis, pseudoporphyria, solar urticaria and the fixed drug eruption [5–8]. In correlation with the clinical observations, it was found that tetracyclines cause singlet oxygen mediated oxidation, which leads to the damage of living cells [9]. Photosensitivity is an adverse cutaneous reaction that results when a certain chemical or drug is applied topically or taken systemically at the same time when a person is exposed to UV radiation or visible light [10]. This phenomenon has been recognized for hundreds of years. In the 13th century, Ibn El-Bitar – the Arab scholar noted that certain plant extracts could be applied in combination in exposure to sunlight to treat vitiligo. These herbal medicines were rediscovered in the 1940s and identified as furocoumarins (psoralens) – phototoxic substances, which have been used in modern photochemotherapy to treat chronic dermatoses, such as vitiligo, psoriasis and fungoides [5,11–13]. It was suggested, that phototoxic reactions may correspond to the binding of drug to melanin biopolymers and drug accumulation in pigmented tissues [14]. Melanins are produced by melanocytes – highly specialized cells residing primarily in the hair follicle, epidermis and eye.
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The melanogenesis process takes place in membrane-bound organelles called melanosomes. The active export of melanosomes from melanocytes to surrounding keratinocytes in the skin or to newly synthesized hair is the basis of skin and hair pigmentation [15]. The most well-known agent that enhances melanogenesis is UV radiation. UV-induced skin pigmentation plays a photoprotective role by preventing from DNA damages and mutations. The shielding effect of melanin, is achieved by its ability to serve as a physical barrier that scatters UV radiation, and as an absorbing filter that reduces the penetration of UV through the epidermis. Melanin, besides functioning as a broadband UV absorbent, has also antioxidant properties, acting as a free radical scavenger and having a superoxide dismutase properties [16]. Previously, we documented that aminoglycoside antibiotics: amikacin [17], kanamycin [18], netilmicin [19] and streptomycin [20] as well as fluoroquinolones: ciprofloxacin [21] and lomefloxacin [22], suppressed melanin biosynthesis and affected antioxidant enzymes activities in human light pigmented melanocytes. We also documented that nicotine modulated melanin biosynthesis and antioxidant enzymes activities in human dark pigmented melanocytes [23]. The purpose of this work was to estimate the effect of tetracycline and UV radiation on viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes, dark pigmented (HEMn-DP).
2. Materials and methods
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2.4. 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 °C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with tetracycline and exposed to UVA irradiation. After 21-h incubation since irradiation, 10 ll of WST-1 were added to 100 ll of culture medium in each well, and the incubation was continued for another 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 a drug) using a microplate reader UVM 340 (Biogenet). The controls were normalized to 100% for each assay and treatments were expressed as the percentage of the controls. 2.5. Melanin content Cell pellets were placed into Eppendorf tubes, dissolved in 100 ll of 1 M NaOH at 80 °C for 1 h, and then centrifuged for 20 min at 16,000g. The supernatants were placed into a 96-well microplate, and absorbance was measured using microplate reader at 405 nm – a wavelength at which melanin absorbs light [24]. Melanin content in tetracycline treated cells was expressed as the percentage of the controls (untreated melanocytes).
2.1. Materials 2.6. Tyrosinase activity Tetracycline hydrochloride, amphotericin B solution (250 lg/ ml), L-3,4-dihydroxyphenylalanine (L-DOPA) and phosphate buffered saline (PBS) were purchased from Sigma–Aldrich Inc. (USA). A growth medium M-254 and a human melanocyte growth supplement-2 (HMGS-2) were acquired from Cascade Biologics (UK). Neomycin sulfate was obtained from Amara (Poland). Penicillin was acquired from Polfa Tarchomin (Poland). Trypsin/EDTA was obtained from Cytogen (Poland). Cell Proliferation Reagent WST1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH SA (Poland).
2.2. Cell culture Human epidermal melanocytes, neonatal, dark pigmented (HEMn-DP, Cascade Biologics) were grown according to the manufacturer’s instruction. The cells were cultured in a M-254 medium supplemented with HMGS-2, penicillin (100 U/ml), neomycin (10 lg/ml) and amphotericin B (0.25 lg/ml) at 37 °C in 5% CO2. All experiments were performed using cells from the passages 5 to 10.
Tyrosinase activity in HEMn-DP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome according to the method described previously [25,26]. Cell lysates were clarified by centrifugation at 10,000g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the phosphate buffer. 100 ll of each lysate were put in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 ll of L-DOPA solution at 37 °C. Control wells contained 100 ll of lysis buffer and 40 ll of L-DOPA solution. Absorbance of dopachrome was measured every 10 min for at least 1.5 h at 475 nm using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls. 2.7. 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.
2.3. UVA irradiation procedure 2.8. Catalase (CAT) assay The ultraviolet light source used in this study was a filtered lamp BVL-8.LM (Vilber Lourmat, France). The intensity of UVA (kmax = 365 nm) radiation was 720 mW/cm2 at 15 cm. The cells, after 24-h incubation with a drug, were irradiated uncovered in petri dishes. Before irradiation the medium had been replaced by PBS. Time of UV exposure was 15 or 30 min. Simultaneously, the nonirradiated cell cultures (control samples) were kept in the dark at 37 °C and 5% CO2. After irradiation PBS was removed from the cells and melanocytes were incubated in the growth medium for 24 h. Then the cells were lysed.
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 of H2O2. The formaldehyde produced is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. One unit of CAT was defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde
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per minute at 25 °C. CAT activity was expressed in nmol/min/mg protein. 2.9. 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 (GSH) 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 °C. GPx activity was expressed in nmol/min/mg protein. 2.10. Statistical analysis In all experiments, mean values of at least three separate experiments (n = 3) performed in triplicate ± standard error of the mean (S.E.M.) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software by means of one-way ANOVA (the influence of UVA radiation or tetracycline) and twoway ANOVA (the influence of UVA radiation and tetracycline), as well as Dunnett’s multiple comparison test in both cases. In all cases the statistical significance was found at least at p < 0.05. 3. Results 3.1. The effect of tetracycline and UVA radiation on cell viability The impact of tetracycline on the viability of melanocytes, was evaluated using antibiotic in concentrations 0.5 lM, 5.0 lM and 50 lM for 24 h (Fig. 1). The concentration of tetracycline 0.5 lM did not cause statistically significant changes in the viability of cells. The exposure of melanocytes to tetracycline in concentrations 5.0 and 50 lM was associated with loss in cell viability by 13.6% and 49.2%, respectively. The effect of simultaneous exposure of melanocytes to tetracycline in concentrations 0.5 lM, 5.0 lM and 50 lM and UVA radiation for 15 min, resulted in cell loss by 6.4%, 14.2% and 47.8%,
Fig. 1. The effect of tetracycline and UVA radiation (15 or 30 min) on viability of melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM, 5.0 lM or 50.0 lM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values ± S.E.M. from three independent experiments (n = 3) performed in triplicate are presented. ⁄⁄ p < 0.005 vs. the control samples.
respectively. After prolongation of the UVA exposure time to 30 min, the changes in cell viability observed for these concentrations of tetracycline were expressed by the cell loss by 20.1%, 36.6% and 70.9%, respectively. The changes of the amount of tested melanocytes and their morphology caused by treatment with 50.0 lM of tetracycline and exposure to UVA radiation for 30 min are presented in Fig. 2. Observed changes show a similar pattern as the results of the WST-1 cell proliferation test. These changes involve mainly cell loss and a decrease of number and the length of dendrites. Although alterations caused by UVA radiation are minor (Fig. 2B) in comparison to the control, there was observed a significant melanocytes loss and changes in their morphology after exposure of cells to tetracycline (Fig. 2C) as well as simultaneous tetracycline and UVA radiation (Fig. 2D). 3.2. The effect of tetracycline and UVA radiation on melanization process The melanization process in cells incubated for 24 h with tetracycline in concentrations 0.5 lM and 5.0 lM was described by melanin content and activity of the main melanotic enzyme, tyrosinase. Tetracycline in these concentrations had no effect on both melanin content (Fig. 3) and the activity of tyrosinase (Fig. 4). In case of cells treated with the same tetracycline concentrations and exposed to UVA radiation for 15 min, melanin production was increased to 108.7% and 115.7%, respectively, when compared with the controls. Prolongation of the UVA exposure time to 30 min resulted in elevation of melanin content to 110.6% and 123.2%, respectively, when compared with the controls. The tyrosinase activity after 24-h incubation of melanocytes with tetracycline in concentrations 0.5 lM and 5.0 lM with simultaneous exposition to UVA radiation for 15 min resulted in the increase of tyrosinase activity to 103.9% (the value not statistically significant) and 114.6%, respectively, when compared with the controls. Prolongation of the UVA exposure time to 30 min resulted in rise of tyrosinase activity to 112.5% and 122.3%, respectively, when compared with the controls. 3.3. The effect of tetracycline and UVA radiation on antioxidant enzymes activities In order to estimate the impact of tetracycline and UVA radiation on antioxidant defense system in normal human melanocytes HEMn-DP, evaluation of the activities of superoxide dismutase, catalase and glutathione peroxidase was made. Melanocytes were exposed to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h. Only in the highest tested concentration, tetracycline alone increased the SOD activity by 20.2% (Fig. 5), when compared with the controls. The simultaneous exposure of cells to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 15 min resulted in increase of the activity of SOD by 13.5% and 32.1%, respectively, while prolongation of the irradiation time to 30 min produced increase in SOD activity by 22.8% and 35.2%, respectively, when compared with the controls. Tetracycline alone only in the highest tested concentration reduced the CAT activity by 8.1% (Fig. 6), when compared with the controls. The simultaneous exposure of melanocytes to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 15 min resulted in the decrease of the activity of CAT by 21.7% and 35.1%, respectively, while prolongation of the irradiation time to 30 min led to decrease in CAT activity by 31.7% and 38.2%, respectively, when compared with the controls. Tetracycline alone in the highest tested concentration (5.0 lM) increased the activity of GPx by 25.6% (Fig. 7), when compared
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Fig. 2. Morphological changes of human epidermal melanocytes HEMn-DP: untreated (A), exposed to UVA radiation for 30 min (B), treated with 50.0 lM of tetracycline (C), treated with 50.0 lM of tetracycline and exposed to UVA radiation for 30 min (D).
5.0 lM and 50 lM decreased the cell viability in a dose-dependent manner (Fig. 1). The value of EC50 was 50 lM. For all tested concentrations, the effect of simultaneous exposure of melanocytes to tetracycline and UVA radiation for 30 min, was associated with heightened cell loss in the examined concentrations, indicating the phototoxic effect of tetracycline. Melanins are the end-products of multistep L-tyrosine oxidation. The key enzyme involved in the synthesis of all types of melanins is tyrosinase (EC 1.14.18.1), which catalyses the rate-limiting initial step in melanogenesis – the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine
(DOPA) and its immediate subsequent oxidation to DOPAquinone [28]. Tetracycline in tested concentrations had no influence on the melanization process in cultured melanocytes. After exposure of melanocytes to UVA radiation, the induction of melanogenesis expressed by consistent increase in melanin content and Fig. 3. The effect of tetracycline and UVA radiation (15 or 30 min) on melanin content in melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM or 5.0 lM for 24 h, and melanin content was measured as described in Section 2. Results are expressed as percentages of the controls. Data are mean ± S.E.M. 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.
with the controls. In case of cells exposed to UVA radiation for 15 min, only tetracycline in concentration 0.5 lM caused significant increase of GPx activity by 15.6%. The exposure of cells to tetracycline in concentrations 0.5 lM and 5.0 lM for 24 h and UVA radiation for 30 min increased the activity of GPx by 41.3% and 22.9%, respectively, when compared with the controls. 4. Discussion More than 300 types of topical and systemic medications are responsible for photosensitivity. Among these agents, one of the most commonly used is group of tetracycline antibiotics [27]. In the present study the simultaneous effect of tetracycline and UVA irradiation on cell viability, melanin content and tyrosinase activity, as well as antioxidant defense system in dark pigmented melanocytes was examined. We used the culture of normal human epidermal melanocytes HEMn-DP. Tetracycline in concentrations
Fig. 4. The effect of tetracycline and UVA radiation (15 or 30 min) on tyrosinase activity in melanocytes. Cells were treated with tetracycline in concentrations: 0.5 lM or 5.0 lM for 24 h, and tyrosinase activity was measured as described in Section 2. Results are expressed as percentages of the controls. Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
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Fig. 5. The superoxide dismutase (SOD) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
tyrosinase activity (Figs. 3 and 4) was observed. The increase was especially seen for the concentration of tetracycline 5.0 lM and UVA exposure for 30 min, reaching a rise by 27.4% in melanin content, corresponding well with increase by 31.1% in tyrosinase activity. Skin pigmentation is determined by over 100 genes including those that encode transcription factors, enzymes, hormones, autocrine and paracrine factors and their receptors [29]. Besides these biochemical factors, the induction of melanogenesis might be a result of the external and internal stress [30], also in form of phototoxicity. Clinically, a phototoxic reaction resembles a sunburn and is characterized by erythema and edema. The erythema usually starts a few hours after exposure, reaches a peak in several hours to a few days, and is followed by desquamation and hyperpigmentation [31]. Hyperpigmentation results from the induction of melanocyte proliferation and migration, enhanced melanin production, deposition of the drug or its photoproducts in the skin or from post-inflammatory changes secondary to subliminal phototoxicity. Other less common manifestations of phototoxicity that
Fig. 6. The catalase (CAT) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. 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.
have been described include slate-gray pigmentation, lichenoid eruptions, photo-onycholysis, and pseudoporphyria [6,32]. The observed specific increase in melanin content may occur in response to the phototoxic effect of the antibiotic which leads to induction of defense mechanisms in the cell, including production of melanin. This is consistent with the increasing activity of tyrosinase with the rising concentration of tetracycline. As melanin may interact with various chemical substances leading to the formation of drug-melanin complexes, thus protecting cells from exposure to harmful compounds, the long-term retention of bound substances may in turn lead to degeneration of melanocytes, that takes place when the detoxifying capacities of melanin are exhausted [33,34]. This can be also caused by UVA radiation that may disturb the capacity of melanin pigment. Melanin has ability to absorb UV photons and act as a scavenger of free radicals generated in response to UV irradiation [35]. On the other hand, higher UV doses may cause oxidation of melanin leading to the generation of reactive oxygen species (ROS) [36]. Thereby, melanogenesis can be considered as a prooxidative process increasing the risk of cell’s damage [37]. A phototoxic reaction begins when a substance absorbs photons of energy, usually in the UVA spectrum, and causes excitation of the molecule. When these excited state molecules return to their ground state, they transfer energy to surrounding oxygen, thereby inducing the generation of reactive oxygen species. The ROS cause cellular damage through the oxidation of lipids, nucleic acids and proteins [27,38]. ROS may react with both cellular and extracellular components, leading to induction of cytotoxicity, apoptosis, mutations and carcinogenesis [35]. Moreover, one of the main protective mechanism against oxidative damages involves the NF-E2 – related factor 2 (Nrf2), which induces transcription of genes encoding antioxidant enzymes that build cellular antioxidant system [39]. The antioxidant system, which major role is protection against harmful effects of ROS, consists mainly of superoxide dismutase, catalase and glutathione peroxidase enzymes. Melanin may also act as a free radicals scavenger and possess the superoxide dismutase activity [40]. In the present study it has been observed that tetracycline alone only in the highest analyzed concentration causes alterations in the activities of all tested antioxidant enzymes in melanocytes, causing increase in activities of SOD and GPx, on the contrary to decrease in CAT activity. Exposure of cells to UVA radiation is associated with the concentration-dependent increase in SOD activity
Fig. 7. The glutathione peroxidase (GPx) activity in HEMn-DP cells after 24 h incubation with 0.5 lM or 5.0 lM of tetracycline and UVA radiation (15 or 30 min). Data are mean ± S.E.M. of at least three independent experiments (n = 3) performed in triplicate. ⁄⁄ p < 0.005 vs. the control samples.
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(Fig. 5) which may be due to overproduction of the superoxide anion, as a result of phototoxic properties of the antibiotic and the augmented antioxidant action of the enzyme. On the other hand, the elevated activity of SOD may enhance the formation of hydroxyl radicals [41]. The observed decrease of CAT activity after exposure of melanocytes to both tested concentrations of tetracycline and UVA irradiation (Fig. 6) may be caused by the sensitivity of catalase to free radicals produced in response to phototoxic action of the antibiotic or damage of the enzyme caused by UVA radiation [35]. Results obtained for the control samples suggest, that CAT deficiency may develop after UVA irradiation without a presence of phototoxic drug. This is consistent with the findings, that catalase may absorb UV radiation and become inactivated [42]. Taking into account, that the main enzyme responsible for inactivation of the proradical hydrogen peroxide in melanocytes is catalase [43,44], we admit the increase in GPx activity observed in our study (Fig. 7) to a compensation mechanism in response to the overproduction of the hydrogen peroxide that cannot be eliminated. Summarizing, we can indicate the phototoxic reactions as a major causing factor for changes in the biochemistry of melanocytes observed in our culture. Tetracycline alone has no influence on melanin content, tyrosinase activity and only in the highest tested concentration may change the cellular antioxidant status in normal human epidermal melanocytes. Simultaneous exposure of the cells to UVA radiation causes alterations of biochemical processes including induction of melanization and occurence of oxidative stress inside melanocytes which is expressed by significant changes in SOD, CAT and GPx activities. This indicates that both factors may be responsible for the changes occurring in vivo. In this study we observed for the first time that tetracycline and UVA evoke a phototoxic action in relation to the biochemistry of normal human epidermal melanocytes. The described effects may explain the mechanisms of the phototoxic impact of the drug on melanin containing tissues. Acknowledgment This study was supported by Medical University of Silesia, Katowice (KNW-2-038/D/3/K). References [1] M.L. Nelson, S.B. Levy, The history of the tetracyclines, Ann. NY. Acad. Sci. 1241 (2011) 17–32. [2] F. Bahrami, D.L. Morris, M.H. Pourgholami, Tetracyclines: drugs with huge therapeutic potential, Mini Rev. Med. Chem. 12 (2012) 44–52. [3] D.E. Brodersen, W.M. Clemons Jr., A.P. Carter, R.J. Morgan-Warren, B.T. Wimberly, V.D.D. Ramakrishnan, The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit, Cell 103 (2000) 1143–1154. [4] B. Zakeri, G.D. Wright, Chemical biology of tetracycline antibiotics, Biochem. Cell Biol. 86 (2008) 124–136. [5] S.G. Vassileva, G. Mateev, L.C. Parish, Antimicrobial photosensitive reactions, Arch. Intern. Med. 158 (1998) 1993–2000. [6] L.B. Valeyrie-Allanore, B. Sassolas, J.C. Roujeau, Drug-induced skin, nail and hair disorders, Drug Saf. 30 (2007) 1011–1030. [7] A.M. Drucker, C.F. Rosen, Drug-induced photosensitivity: culprit drugs, management and prevention, Drug Saf. 34 (2011) 821–837. [8] L.M. Yap, P.A. Foley, R.B. Crouch, C.S. Baker, Drug-induced solar urticaria due to tetracycline, Australas. J. Dermatol. 41 (2000) 181–184. [9] D.E. Moore, Drug-induced cutaneous photosensitivity: incidence, mechanism, prevention and management, Drug Saf. 25 (2002) 345–372. [10] R. Dubakiene, M. Kupriene, Scientific problems of photosensitivity, Medicina (Kaunas) 42 (2006) 619–624. [11] M. Abu Tahir, K. Pramod, S.H. Ansari, J. Ali, Current remedies for vitiligo, Autoimmun. Rev. 9 (2010) 516–520. [12] F. Almutawa, N. Alnomair, Y. Wang, I. Hamzavi, H.W. Lim, Systematic review of UV-based therapy for psoriasis, Am. J. Clin. Dermatol. 14 (2013) 87–109. [13] D. Humme, A. Nast, R. Erdmann, S. Vandersee, M. Beyer, Systematic review of combination therapies for mycosis fungoides, Cancer Treat. Rev. 40 (2014) 927–933.
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