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Effect of nicotine on melanogenesis and antioxidant status in HEMn-LP melanocytes
Environmental Research 134 (2014) 309–314
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Effect of nicotine on melanogenesis and antioxidant status in HEMn-LP melanocytes Marcin Delijewski, Artur Beberok, Michał Otręba, Dorota Wrześniok, Jakub Rok, Ewa Buszman n Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellońska 4, 41-200 Sosnowiec, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 June 2014 Received in revised form 5 August 2014 Accepted 14 August 2014 Available online 7 September 2014
Nicotine is a natural ingredient of tobacco plants and is responsible for the addictive properties of tobacco. Nowadays nicotine is also commonly used as a form of smoking cessation therapy. It is suggested that nicotine may be accumulated in human tissues containing melanin. This may in turn affect biochemical processes in human cells producing melanin. The aim of this study was to examine the effect of nicotine on melanogenesis and antioxidant status in cultured normal human melanocytes HEMn-LP. Nicotine induced concentration-dependent loss in melanocytes viability. The value of EC50 was determined to be 7.43 mM. Nicotine inhibited a melanization process in human light pigmented melanocytes and caused alterations of antioxidant defense system. Significant changes in cellular antioxidant enzymes: superoxide dismutase and catalase activities and in hydrogen peroxide content were stated. The obtained results may explain a potential influence of nicotine on biochemical processes in melanocytes in vivo during long term exposition to nicotine. & 2014 Elsevier Inc. All rights reserved.
Keywords: Nicotine Melanin Melanocytes Tyrosinase Antioxidant enzymes
1. Introduction Nicotine is one of the tobacco alkaloids and due to its addictive properties is responsible for the high smoking prevalence in the world. It was estimated by World Health Organization (2013) that tobacco is used by about one billion smokers and tobacco smoke contributes to premature deaths of 6 million people each year. Nicotine is also commonly applied in a form of smoking cessation therapy, like nicotine replacement therapy (NRT) which consists of nicotine gums, lozenges, sublingual tablets, inhalers, nasal sprays and transdermal patches. After reaching nicotinic acetylcholine receptors in brain, it causes the reinforcing effect or softens withdrawal symptoms (Benowitz, 2010). The distribution of nicotine in the body has been investigated since 1851 but about 1972, after conducting autoradiographic studies on pigmented animals, the evidence for nicotine accumulation in melanin containing tissues was provided (Yerger and Malone, 2006). Moreover, studies on mice revealed that pigmented tissues can store nicotine up to 30 days after a single injection (Lindquist and Ullberg, 1974; Szüts et al., 1978). Melanin is a polymeric pigment and the major determinant of the color of skin and hair. It is also present in eye, inner ear, heart, lungs,
n
Corresponding author. E-mail address: [email protected] (E. Buszman).
http://dx.doi.org/10.1016/j.envres.2014.08.015 0013-9351/& 2014 Elsevier Inc. All rights reserved.
liver, lymphocytes and brain. Melanin biopolymer is produced, stored and transported in melanosomes which are the specialized membrane-bound organelles of melanocytes. Melanin is biosynthesized in a complex process called menalogenesis, in which three main melanogenic enzymes are involved: tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). Tyrosinase is the crucial enzyme for the whole process (Sulaimon and Kitchell, 2003; Park et al., 2009). The role of melanin is to protect cells from UV radiation by creating a supranuclear cap in a cell, absorbing energy and working as an antioxidant agent and free radicals scavenger (Różanowska et al., 1999; Park et al., 2009). The oxidative/antioxidative balance in melanocytes depends on the activity of the main antioxidative enzymes: superoxide dismutase, catalase and glutathione peroxidase. The additional unique contribution of melanin in this aspect has to be considered. As reactive oxygen species pose a risk for the biochemical processes in melanocytes, like production of melanin, protective action of unaffected antioxidative enzymes appears to be crucial. The activity of the main enzyme responsible for the production of melanin, tyrosinase, is also sensitive to oxidative stress (Schallreuter et al., 2008). Thereby the reduced melanin content may also indicate the oxidative damage in cells. On the other hand, it can also be a reason for higher susceptibility of cells to oxidative stress, as melanin seems to work as a radical scavenger and supporting agent in the oxidative/antioxidative balance in melanocytes (Tolleson, 2005).
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Melanins can act as free radical scavengers for oxidizing and reducing radicals, what may be important in the protection against free radical damage. This is due to the content of many reduced groups that may react with the radicals. These interactions can be realized through one-electron transfer processes or through radical addition (Różanowska et al., 1999). On the other hand, synthesis of melanin is also by itself burdened with the risk of production of reactive oxygen species, due to formation of compounds undergoing redox reactions and polymerizations. As observed by Smit et al. (2008), cells containing more melanin, showed at least 60% increase of ROS generation, when compared with less pigmented cells, under the same conditions. This may be in turn associated with oxidative DNA damages. Melanin is capable of binding many chemical substances, including drugs (Larsson, 1993), like aminoglycoside antibiotics (Buszman et al., 2007), fluoroquinolones (Beberok et al., 2011), anticancer agents (Surażyński et al., 2001), psychotropic drugs (Buszman et al., 2008) and also nicotine (Delijewski et al., 2013). Previously, we documented that aminoglycoside antibiotics: amikacin (Wrześniok et al., 2013b), kanamycin (Wrześniok et al., 2013d), netilmicin (Wrześniok et al., 2013c) and streptomycin (Wrześniok et al., 2013a) as well as fluoroquinolones: ciprofloxacin (Beberok et al., 2011) and lomefloxacin (Beberok et al., 2013) suppressed biosynthesis of melanin in human light pigmented melanocytes. We also demonstrated that the analyzed antibiotics modified antioxidant status in the analyzed cells. Literature suggests that retention of nicotine in melanincontaining tissues may influence melanin synthesis (Yerger and Malone, 2006). The purpose of this work was to estimate the effect of nicotine on viability, melanogenesis and an antioxidant defense system in cultured normal human melanocytes HEMn-LP.
2.4. Measurement of melanin content The melanocytes were seeded in T-25 flasks at a density of 1 105 cells per flask. Nicotine treatment in a concentration range from 0.0001 to 1.0 mM began 48 h after seeding. After 24 h of incubation, the cells were detached with trypsin/ EDTA. Cell pellets were placed into Eppendorf tubes, dissolved in 100 μl of 1 M NaOH at 80 1C 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 at 405 nm – a wavelength at which melanin absorbs light (Ozeki et al., 1996). Melanin content in nicotine treated cells was expressed as the percentage of the controls (untreated melanocytes). 2.5. Tyrosinase activity assay Tyrosinase activity in HEMn-LP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome, according to the method described by Kim et al. (2005) and Buscá et al. (1996), with a slight modification. The cells were cultured at a density of 1 105 cells in T-25 flasks for 48 h. After 24-h incubation with nicotine (concentration range from 0.0001 to 1.0 mM) cells were lysed and clarified by centrifugation at 10,000g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the same lysis phosphate buffer. 100 μl of each lysate were put in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 μl of L-DOPA solution at 37 1C. Absorbance 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 (untreated melanocytes). 2.6. Hydrogen peroxide assay Hydrogen peroxide (H2O2) content was measured using an assay kit (Cell Biolabs, Inc., USA) according to the manufacturer's instruction. This method is based on the ability of sorbitol to convert peroxide to a peroxyl radical, which oxidizes Fe2 þ into Fe3 þ . Then Fe3 þ reacts with an equimolar amount of xylenol orange in the presence of acid to create a purple product that absorbs light at maximal wavelength 595 nm. The antioxidant – butylated hydroxytoluene (BHT) is provided to prevent further undesirable chain peroxidation. Hydrogen peroxide content in the samples was expressed in mmol/mg protein. 2.7. Superoxide dismutase assay
2. Materials and methods 2.1. Materials Nicotine, phosphated-buffered saline (PBS), 3,4-dihydroxy-L-phenylalanine (LDOPA) and amphotericin B were purchased from Sigma-Aldrich Inc. (USA). Neomycin sulfate was obtained from Amara (Poland). Penicillin was acquired from Polfa Tarchomin (Poland). Growth medium M-254 and 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). 2.2. Cell culture The normal human epidermal melanocytes (HEMn-LP, Cascade Biologics) were grown according to the manufacturer's instruction. The cells were cultured in M-254 basal medium supplemented with HMGS-2, penicillin (100 U/ml), neomycin (10 μg/ml) and amphotericin B (0.25 μg/ml) at 37 1C in 5% CO2. All experiments were performed using cells in the passages 5–8. 2.3. 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 1C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with nicotine solutions in a concentration range from 0.0001 to 10 mM. After 21-h incubation, 10 μl of WST-1 were added to 100 μl 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 nicotine) 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.
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. 2.8. Catalase 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 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 per minute at 25 1C. CAT activity was expressed in nmol/ min/mg protein. 2.9. Glutathione peroxidase 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 nicotinamide-adenine dinucleotide phosphate (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 1C. 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 ¼3) performed in triplicate7standard error of the mean (S.E.M.) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software. A value of
M. Delijewski et al. / Environmental Research 134 (2014) 309–314 p o 0.05 (n) or p o 0.005 (nn), obtained with a Student's t-test by comparing the data with those for control (cells without nicotine), was considered statistically significant.
3. Results To assess the influence of nicotine on the viability of melanocytes, cells were treated with nicotine in a range of concentrations from 0.0001 mM to 10 mM for 24 h (Fig. 1). For nicotine concentrations from 0.0001 mM to 2.5 mM, changes were not statistically significant. After treatment of cells with 5.0, 7.5 and 10 mM of nicotine, the loss in cell viability by 35.4%, 51.2% and 73.7%, respectively, was observed. The concentration of a drug that produces loss in cell viability by 50% (EC50) was determined to be 7.43 mM. To study the effect of nicotine on the effectiveness of melanization process in cells, we measured the melanin content and the activity of the main melanogenic enzyme, tyrosinase. Cells were incubated for 24 h with nicotine in a range of concentrations from 0.0001 mM to 1.0 mM. Nicotine in concentrations from 0.0001 mM to 0.01 mM had
Fig. 1. The impact of nicotine on viability of HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–10 mM) and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values 7S.E.M. from three independent experiments (n ¼3) performed in triplicate are presented. ** Po 0.005 vs. the control samples.
Fig. 2. The impact of nicotine on melanin content in HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–1.0 mM) for 24 h, and melanin content was measured as described in Materials and Methods. Results are expressed as percentages of the controls. Data are mean7 S.E.M. of at least three independent experiments (n¼3) performed in triplicate. *Po 0.05 vs. the control samples; **P o 0.005 vs. the control samples.
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no effect on melanin content (Fig. 2). In cells treated with nicotine at concentrations of 0.05, 0.1, 0.5 and 1.0 mM for 24 h, melanin production was suppressed to 95.2%, 91.7%, 85.1% and 76.1%, respectively, when compared with the controls. Changes of the tyrosinase activity in melanocytes treated with nicotine corresponded with alterations in melanin formation (Fig. 3). Nicotine in the range of concentrations from 0.0001 mM to 0.01 mM had no effect on the cellular tyrosinase activity. After 24-h incubation with nicotine at concentrations of 0.05, 0.1, 0.5 and 1.0 mM, tyrosinase activity was decreased to 94.8%, 90.6%, 86.7% and 77.1%, respectively, when compared with the controls (Fig. 3). To characterize the effect of nicotine on antioxidant defense system in melanocytes, the activities of superoxide dismutase, catalase and glutathione peroxidase were determined, as well as the H2O2 content was estimated. Human melanocytes HEMn-LP were exposed to nicotine in concentrations of 0.01, 0.05, 0.1, 0.5 or 1.0 mM for 24 h. It has been demonstrated that the activity of SOD increases with rising concentration of nicotine (Fig. 5). The treatment of cells with 0.1, 0.5 or 1.0 mM of nicotine significantly increased the SOD activity by 10.7%, 13.1%, or 18.3%, respectively, as
Fig. 3. The impact of nicotine on tyrosinase activity in HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–1.0 mM) for 24 h, and tyrosinase activity was measured as described in Materials and Methods. Data are mean7 S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *P o0.05 vs. the control samples; **P o0.005 vs. the control samples.
Fig. 4. The hydrogen peroxide (H2O2) content in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *Po 0.05 vs. the control samples; **P o0.005 vs. the control samples.
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Fig. 5. The superoxide dismutase (SOD) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7 S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. *P o0.05 vs. the control samples.
Fig. 6. The catalase (CAT) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. *Po 0.05 vs. the control samples.
compared with the controls. The intracellular CAT activity was also increased by 10.3%, 18.0% or 25.2% for cells treated with nicotine in concentration of 0.1 mM, 0.5 mM or 1.0 mM, respectively (Fig. 6). Treatment of melanocytes with 0.1, 0.5 and 1.0 mM of nicotine enhanced the H2O2 content by 13.8%, 25.5% and 37.0%, respectively, as compared with the controls (Fig. 4). Nicotine in the concentration of 0.01 and 0.05 mM had no effect on cellular SOD and CAT activities as well as on H2O2 content. In contrast to SOD and CAT, nicotine had no statistically significant influence on the activity of GPx (Fig. 7).
4. Discussion Melanocytes have a unique feature that distinguishes them from other human cells, that is ability to biosynthesis of diversified and complex biopolymers called melanins. These pigments may bind chemical substances, metal ions and many drugs (Larsson, 1993). This is significant when we take into account detoxifying role of melanin and its properties to serve as a drug reservoir.
Fig. 7. The glutathione peroxidase (GPx) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean7 S.E.M. of at least three independent experiments (n¼3) performed in triplicate.
Nicotine can also form complexes with melanin and the amounts of this alkaloid bound to melanin increase with rising initial nicotine concentration and prolongation of incubation time. Complexes of nicotine with melanin were characterized by two classes of independent binding sites with the association constants K1 ¼ 2.44 104 M 1 and K2 ¼7.72 102 M 1, while the total number of binding sites was estimated to be 1.748 μmol nicotine/mg melanin (Delijewski et al., 2013). The accumulation of nicotine in hair allows to use it in toxicology as a popular biomarker of exposition to nicotine, both in smokers as well as in people exposed to second-hand smoke (Gerstenberg et al., 1995; Koszowski et al., 2008). The contribution of nicotine–melanin interaction may be seen in Black smokers that have substantially higher hair nicotine levels than White smokers (Apelberg et al., 2012). Substances with affinity to hair melanin should also have affinity for the melanin in other tissues. The retention of nicotine in melanin-containing cells could involve nicotine as a false precursor in the formation of new melanin, because of a structural analogy between nicotine and indole-5,6quinone, as one of the precursors of melanin (Larsson et al., 1979). There are still remaining questions if melanin-rich cells protect surrounding tissues by promoting detoxification of nicotine, or if accumulated nicotine affects its metabolism, addiction and results in health risk (Hébert, 2006). In the present study the effect of nicotine on melanocytes viability, as well as on melanization process and antioxidant defense system in pigmented cells was analyzed. As an in vitro experimental model system, we used the culture of normal human melanocytes HEMn-LP. We have found that nicotine in concentrations from 0.0001 mM to 10 mM decreases the cell viability (Fig.1). The value of EC50 was determined to be 7.43 mM, which indicates that nicotine is less toxic to melanocytes than lomefloxacin, streptomycin and kanamycin, for which EC50 values were determined to be 0.75 mM, 5.0 mM and 6.0 mM, respectively (Beberok et al., 2013; Wrześniok et al., 2013a, 2013d). EC50 value obtained for amikacin (7.5 mM) (Wrześniok et al., 2013b) was similar to that obtained for nicotine. Results that we have obtained show that nicotine affects the melanization process in tested cell line. For concentrations of nicotine from 0.05 mM to 1.0 mM we observed inhibition of melanogenesis expressed by decrease in melanin content and tyrosinase activity (Figs. 2 and 3). The inhibitory effect of nicotine in concentrations starting from 0.05 mM on melanogenesis in HEMn-LP cells is probably due to its direct inhibition of tyrosinase
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activity. For the concentrations from 0.0001 mM to 0.01 mM we observed that the melanin content and tyrosinase activity were similar to the control. Reactive oxygen species (ROS) are generated in aerobic processes in cellular interior and in limited quantities are needed in signaling pathways in cells. However, the overproduction of ROS may cause damages in basic cellular components like DNA, proteins and lipids, resulting in cell dysfunction or death (Mari et al., 2010). The complex antioxidant system of cells consists mainly of superoxide dismutase, catalase and glutathione peroxidase enzymes and is responsible for protection against harmful effects of ROS. Melanin can also act as a free radical, including ROS, scavenger and it has been suggested that this biopolymer possesses the superoxide dismutase activity (Hoogduijn et al., 2004). Catalase and melanin may act synergistically to counteract the deleterious effects, especially oxidative stress (Maresca et al., 2008). SOD is the enzyme which catalyzes the formation of hydrogen peroxide from superoxide anion (Gardner et al., 2002). CAT and GPx catalyze the breakdown of hydrogen peroxide, produced by SOD, to water (Mari et al., 2010). Lightly pigmented melanocytes, that in general express lower melanogenic and catalase activity, are probably more susceptible to the harmful effects of free radicals (Maresca et al., 2008). The influence of nicotine on antioxidant status in melanocytes seems to be complex. It was demonstrated that nicotine forms weak complexes with Fe2 þ (Bridge et al., 2004). Moreover, melanin binds Fe2 þ as far as nicotine. It has been reported, that Fenton reaction may be strongly inhibited by nicotine in the concentrations from 0.1 mM to 0.5 mM (Williams and Linert, 2004). It was also observed, that nicotine may interact with the complex I of mitochondrial respiratory chain, thereby decreasing the formation of ROS, by antagonizing the NADH binding onto complex I (Cormier et al., 2001). Therefore, it may be possible that nicotine supports melanin in modulating the antioxidant status of melanocytes and the ability of melanin to work as a free radicals scavenger. In the present study it has been observed that nicotine causes significant alterations in the activities of antioxidant enzymes: superoxide dismutase and catalase in melanocytes. The exposure of melanocytes to nicotine in concentrations from 0.1 mM to 1.0 mM probably causes overproduction of the superoxide anion. The resulting increase in SOD activity (Fig. 5) is associated with subsequent formation of H2O2 (Fig. 4), that leads to the increase in CAT activity (Fig. 6). After treatment of cells with nicotine in lower concentrations (0.01 mM and 0.05 mM), the activities of SOD and CAT, and the H2O2 content were similar to the controls. Simultaneously, no significant changes in cellular GPx activity were observed (Fig. 7), what indicates that in melanocytes catalase is the main enzyme responsible for inactivation of the proradical hydrogen peroxide, what was suggested by Maresca et al. (2008). It may be also possible, that while catalase acts synergistically with melanin to protect cells from oxidative stress in effective manner, the activity of GPx may remain unaltered. Moreover, in case of light pigmented melanocytes, which contain less melanin than dark pigmented cells, the amount of nicotine bound to melanin is relatively low and therefore the antioxidative enzymes response is sufficient to compensate the increase in ROS formation. It may be worth to consider the contribution of tyrosinase to the oxidative/antioxidative balance in melanocytes, as it seems that this enzyme represents a protective mechanism against ROSgenerating compounds. (Perluigi et al., 2003). However, the induction of oxidative stress presumably affects the function of tyrosinase enzyme, what may have a shift in the decrease of melanin content, what was observed for the concentrations of nicotine from 0.05 mM. The increase in H2O2 content observed for
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the concentrations of nicotine from 0.1 mM, may also lead to the inhibition of melanogenic enzymes. Altogether, our results demonstrate that nicotine in noncytotoxic concentrations causes significant alterations of biochemical processes in melanocytes, like inhibition of melanogenesis (reduction in melanin content and tyrosinase activity) and induction of oxidative stress (increase in SOD, CAT activity and H2O2 content). The results presented in this work concerning the effect of nicotine on viability and biochemical processes in normal human melanocytes have to be taken into consideration in case of people with low melanin content and smoking cigarettes or using nicotine replacement therapy.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by the Medical University of Silesia (Grant no. KNW-1-094/K/3/0).
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Surażyński, A., Pałka, J., Wrześniok, D., Buszman, E., Kaczmarczyk, P., 2001. Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts. Eur. J. Pharmacol. 419, 139–145. Szüts, T., Olsson, S., Lindquist, N.G., Ullberg, S., Pilotti, A., Enzell, C., 1978. Long-term fate of [14C]nicotine in the mouse: retention in the bronchi, melanincontaining tissues and urinary bladder wall. Toxicology 10, 207–220. Tolleson, W.H., 2005. Human melanocyte biology, toxicology, and pathology. J. Environ. Sci. Health 23, 105–161. Williams, E., Linert, W., 2004. In vitro evidence supporting the therapeutic role of nicotine against neurodegeneration. In Vivo 18, 391–399. World Health Organization. Tobacco Fact sheet No 339, updated 2013, World Health Organization, Geneva. 〈http://www.who.int/mediacentre/factsheets/fs339/en/〉 (accessed: 8.02.14.). Wrześniok, D., Beberok, A., Oręba, M., Buszman, E., 2013a. Effect of streptomycin on melanogenesis and antioxidant status in melanocytes. Mol. Cell. Biochem. 383, 77–84. Wrześniok, D., Beberok, A., Otręba, M., Buszman, E., 2013b. Modulation of melanogenesis and antioxidant defense system in melanocytes by amikacin. Toxicol. In Vitro 27, 1102–1108. Wrześniok, D., Beberok, A., Otręba, M., Buszman, E., 2013c. Netilmicin-induced modulation of melanogenesis in HEMa-LP melanocytes. Acta Pol. Pharm. Drug Res. 70, 803–808. Wrześniok, D., Otręba, M., Beberok, A., Buszman, E., 2013d. Impact of kanamycin on melanogenesis and antioxidant enzymes activity in melanocytes–an in vitro study. J. Cell Biochem. 114, 2746–2752. Yerger, V.B., Malone, R.E., 2006. Melanin and nicotine: a review of the literature. Nicotine Tob. Res. 8, 487–498.
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Effect of nicotine on melanogenesis and antioxidant status in HEMn-LP melanocytes Marcin Delijewski, Artur Beberok, Michał Otręba, Dorota Wrześniok, Jakub Rok, Ewa Buszman n Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellońska 4, 41-200 Sosnowiec, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 June 2014 Received in revised form 5 August 2014 Accepted 14 August 2014 Available online 7 September 2014
Nicotine is a natural ingredient of tobacco plants and is responsible for the addictive properties of tobacco. Nowadays nicotine is also commonly used as a form of smoking cessation therapy. It is suggested that nicotine may be accumulated in human tissues containing melanin. This may in turn affect biochemical processes in human cells producing melanin. The aim of this study was to examine the effect of nicotine on melanogenesis and antioxidant status in cultured normal human melanocytes HEMn-LP. Nicotine induced concentration-dependent loss in melanocytes viability. The value of EC50 was determined to be 7.43 mM. Nicotine inhibited a melanization process in human light pigmented melanocytes and caused alterations of antioxidant defense system. Significant changes in cellular antioxidant enzymes: superoxide dismutase and catalase activities and in hydrogen peroxide content were stated. The obtained results may explain a potential influence of nicotine on biochemical processes in melanocytes in vivo during long term exposition to nicotine. & 2014 Elsevier Inc. All rights reserved.
Keywords: Nicotine Melanin Melanocytes Tyrosinase Antioxidant enzymes
1. Introduction Nicotine is one of the tobacco alkaloids and due to its addictive properties is responsible for the high smoking prevalence in the world. It was estimated by World Health Organization (2013) that tobacco is used by about one billion smokers and tobacco smoke contributes to premature deaths of 6 million people each year. Nicotine is also commonly applied in a form of smoking cessation therapy, like nicotine replacement therapy (NRT) which consists of nicotine gums, lozenges, sublingual tablets, inhalers, nasal sprays and transdermal patches. After reaching nicotinic acetylcholine receptors in brain, it causes the reinforcing effect or softens withdrawal symptoms (Benowitz, 2010). The distribution of nicotine in the body has been investigated since 1851 but about 1972, after conducting autoradiographic studies on pigmented animals, the evidence for nicotine accumulation in melanin containing tissues was provided (Yerger and Malone, 2006). Moreover, studies on mice revealed that pigmented tissues can store nicotine up to 30 days after a single injection (Lindquist and Ullberg, 1974; Szüts et al., 1978). Melanin is a polymeric pigment and the major determinant of the color of skin and hair. It is also present in eye, inner ear, heart, lungs,
n
Corresponding author. E-mail address: [email protected] (E. Buszman).
http://dx.doi.org/10.1016/j.envres.2014.08.015 0013-9351/& 2014 Elsevier Inc. All rights reserved.
liver, lymphocytes and brain. Melanin biopolymer is produced, stored and transported in melanosomes which are the specialized membrane-bound organelles of melanocytes. Melanin is biosynthesized in a complex process called menalogenesis, in which three main melanogenic enzymes are involved: tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). Tyrosinase is the crucial enzyme for the whole process (Sulaimon and Kitchell, 2003; Park et al., 2009). The role of melanin is to protect cells from UV radiation by creating a supranuclear cap in a cell, absorbing energy and working as an antioxidant agent and free radicals scavenger (Różanowska et al., 1999; Park et al., 2009). The oxidative/antioxidative balance in melanocytes depends on the activity of the main antioxidative enzymes: superoxide dismutase, catalase and glutathione peroxidase. The additional unique contribution of melanin in this aspect has to be considered. As reactive oxygen species pose a risk for the biochemical processes in melanocytes, like production of melanin, protective action of unaffected antioxidative enzymes appears to be crucial. The activity of the main enzyme responsible for the production of melanin, tyrosinase, is also sensitive to oxidative stress (Schallreuter et al., 2008). Thereby the reduced melanin content may also indicate the oxidative damage in cells. On the other hand, it can also be a reason for higher susceptibility of cells to oxidative stress, as melanin seems to work as a radical scavenger and supporting agent in the oxidative/antioxidative balance in melanocytes (Tolleson, 2005).
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Melanins can act as free radical scavengers for oxidizing and reducing radicals, what may be important in the protection against free radical damage. This is due to the content of many reduced groups that may react with the radicals. These interactions can be realized through one-electron transfer processes or through radical addition (Różanowska et al., 1999). On the other hand, synthesis of melanin is also by itself burdened with the risk of production of reactive oxygen species, due to formation of compounds undergoing redox reactions and polymerizations. As observed by Smit et al. (2008), cells containing more melanin, showed at least 60% increase of ROS generation, when compared with less pigmented cells, under the same conditions. This may be in turn associated with oxidative DNA damages. Melanin is capable of binding many chemical substances, including drugs (Larsson, 1993), like aminoglycoside antibiotics (Buszman et al., 2007), fluoroquinolones (Beberok et al., 2011), anticancer agents (Surażyński et al., 2001), psychotropic drugs (Buszman et al., 2008) and also nicotine (Delijewski et al., 2013). Previously, we documented that aminoglycoside antibiotics: amikacin (Wrześniok et al., 2013b), kanamycin (Wrześniok et al., 2013d), netilmicin (Wrześniok et al., 2013c) and streptomycin (Wrześniok et al., 2013a) as well as fluoroquinolones: ciprofloxacin (Beberok et al., 2011) and lomefloxacin (Beberok et al., 2013) suppressed biosynthesis of melanin in human light pigmented melanocytes. We also demonstrated that the analyzed antibiotics modified antioxidant status in the analyzed cells. Literature suggests that retention of nicotine in melanincontaining tissues may influence melanin synthesis (Yerger and Malone, 2006). The purpose of this work was to estimate the effect of nicotine on viability, melanogenesis and an antioxidant defense system in cultured normal human melanocytes HEMn-LP.
2.4. Measurement of melanin content The melanocytes were seeded in T-25 flasks at a density of 1 105 cells per flask. Nicotine treatment in a concentration range from 0.0001 to 1.0 mM began 48 h after seeding. After 24 h of incubation, the cells were detached with trypsin/ EDTA. Cell pellets were placed into Eppendorf tubes, dissolved in 100 μl of 1 M NaOH at 80 1C 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 at 405 nm – a wavelength at which melanin absorbs light (Ozeki et al., 1996). Melanin content in nicotine treated cells was expressed as the percentage of the controls (untreated melanocytes). 2.5. Tyrosinase activity assay Tyrosinase activity in HEMn-LP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome, according to the method described by Kim et al. (2005) and Buscá et al. (1996), with a slight modification. The cells were cultured at a density of 1 105 cells in T-25 flasks for 48 h. After 24-h incubation with nicotine (concentration range from 0.0001 to 1.0 mM) cells were lysed and clarified by centrifugation at 10,000g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the same lysis phosphate buffer. 100 μl of each lysate were put in a 96-well plate, and the enzymatic assay was initiated by the addition of 40 μl of L-DOPA solution at 37 1C. Absorbance 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 (untreated melanocytes). 2.6. Hydrogen peroxide assay Hydrogen peroxide (H2O2) content was measured using an assay kit (Cell Biolabs, Inc., USA) according to the manufacturer's instruction. This method is based on the ability of sorbitol to convert peroxide to a peroxyl radical, which oxidizes Fe2 þ into Fe3 þ . Then Fe3 þ reacts with an equimolar amount of xylenol orange in the presence of acid to create a purple product that absorbs light at maximal wavelength 595 nm. The antioxidant – butylated hydroxytoluene (BHT) is provided to prevent further undesirable chain peroxidation. Hydrogen peroxide content in the samples was expressed in mmol/mg protein. 2.7. Superoxide dismutase assay
2. Materials and methods 2.1. Materials Nicotine, phosphated-buffered saline (PBS), 3,4-dihydroxy-L-phenylalanine (LDOPA) and amphotericin B were purchased from Sigma-Aldrich Inc. (USA). Neomycin sulfate was obtained from Amara (Poland). Penicillin was acquired from Polfa Tarchomin (Poland). Growth medium M-254 and 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). 2.2. Cell culture The normal human epidermal melanocytes (HEMn-LP, Cascade Biologics) were grown according to the manufacturer's instruction. The cells were cultured in M-254 basal medium supplemented with HMGS-2, penicillin (100 U/ml), neomycin (10 μg/ml) and amphotericin B (0.25 μg/ml) at 37 1C in 5% CO2. All experiments were performed using cells in the passages 5–8. 2.3. 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 1C and 5% CO2 for 48 h. Then the medium was removed and cells were treated with nicotine solutions in a concentration range from 0.0001 to 10 mM. After 21-h incubation, 10 μl of WST-1 were added to 100 μl 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 nicotine) 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.
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. 2.8. Catalase 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 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 per minute at 25 1C. CAT activity was expressed in nmol/ min/mg protein. 2.9. Glutathione peroxidase 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 nicotinamide-adenine dinucleotide phosphate (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 1C. 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 ¼3) performed in triplicate7standard error of the mean (S.E.M.) were calculated. The results were analyzed statistically using GraphPad Prism 6.01 Software. A value of
M. Delijewski et al. / Environmental Research 134 (2014) 309–314 p o 0.05 (n) or p o 0.005 (nn), obtained with a Student's t-test by comparing the data with those for control (cells without nicotine), was considered statistically significant.
3. Results To assess the influence of nicotine on the viability of melanocytes, cells were treated with nicotine in a range of concentrations from 0.0001 mM to 10 mM for 24 h (Fig. 1). For nicotine concentrations from 0.0001 mM to 2.5 mM, changes were not statistically significant. After treatment of cells with 5.0, 7.5 and 10 mM of nicotine, the loss in cell viability by 35.4%, 51.2% and 73.7%, respectively, was observed. The concentration of a drug that produces loss in cell viability by 50% (EC50) was determined to be 7.43 mM. To study the effect of nicotine on the effectiveness of melanization process in cells, we measured the melanin content and the activity of the main melanogenic enzyme, tyrosinase. Cells were incubated for 24 h with nicotine in a range of concentrations from 0.0001 mM to 1.0 mM. Nicotine in concentrations from 0.0001 mM to 0.01 mM had
Fig. 1. The impact of nicotine on viability of HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–10 mM) and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values 7S.E.M. from three independent experiments (n ¼3) performed in triplicate are presented. ** Po 0.005 vs. the control samples.
Fig. 2. The impact of nicotine on melanin content in HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–1.0 mM) for 24 h, and melanin content was measured as described in Materials and Methods. Results are expressed as percentages of the controls. Data are mean7 S.E.M. of at least three independent experiments (n¼3) performed in triplicate. *Po 0.05 vs. the control samples; **P o 0.005 vs. the control samples.
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no effect on melanin content (Fig. 2). In cells treated with nicotine at concentrations of 0.05, 0.1, 0.5 and 1.0 mM for 24 h, melanin production was suppressed to 95.2%, 91.7%, 85.1% and 76.1%, respectively, when compared with the controls. Changes of the tyrosinase activity in melanocytes treated with nicotine corresponded with alterations in melanin formation (Fig. 3). Nicotine in the range of concentrations from 0.0001 mM to 0.01 mM had no effect on the cellular tyrosinase activity. After 24-h incubation with nicotine at concentrations of 0.05, 0.1, 0.5 and 1.0 mM, tyrosinase activity was decreased to 94.8%, 90.6%, 86.7% and 77.1%, respectively, when compared with the controls (Fig. 3). To characterize the effect of nicotine on antioxidant defense system in melanocytes, the activities of superoxide dismutase, catalase and glutathione peroxidase were determined, as well as the H2O2 content was estimated. Human melanocytes HEMn-LP were exposed to nicotine in concentrations of 0.01, 0.05, 0.1, 0.5 or 1.0 mM for 24 h. It has been demonstrated that the activity of SOD increases with rising concentration of nicotine (Fig. 5). The treatment of cells with 0.1, 0.5 or 1.0 mM of nicotine significantly increased the SOD activity by 10.7%, 13.1%, or 18.3%, respectively, as
Fig. 3. The impact of nicotine on tyrosinase activity in HEMn-LP cells. Melanocytes were treated with various nicotine concentrations (0.0001–1.0 mM) for 24 h, and tyrosinase activity was measured as described in Materials and Methods. Data are mean7 S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *P o0.05 vs. the control samples; **P o0.005 vs. the control samples.
Fig. 4. The hydrogen peroxide (H2O2) content in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *Po 0.05 vs. the control samples; **P o0.005 vs. the control samples.
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Fig. 5. The superoxide dismutase (SOD) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7 S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. *P o0.05 vs. the control samples.
Fig. 6. The catalase (CAT) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean 7S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. *Po 0.05 vs. the control samples.
compared with the controls. The intracellular CAT activity was also increased by 10.3%, 18.0% or 25.2% for cells treated with nicotine in concentration of 0.1 mM, 0.5 mM or 1.0 mM, respectively (Fig. 6). Treatment of melanocytes with 0.1, 0.5 and 1.0 mM of nicotine enhanced the H2O2 content by 13.8%, 25.5% and 37.0%, respectively, as compared with the controls (Fig. 4). Nicotine in the concentration of 0.01 and 0.05 mM had no effect on cellular SOD and CAT activities as well as on H2O2 content. In contrast to SOD and CAT, nicotine had no statistically significant influence on the activity of GPx (Fig. 7).
4. Discussion Melanocytes have a unique feature that distinguishes them from other human cells, that is ability to biosynthesis of diversified and complex biopolymers called melanins. These pigments may bind chemical substances, metal ions and many drugs (Larsson, 1993). This is significant when we take into account detoxifying role of melanin and its properties to serve as a drug reservoir.
Fig. 7. The glutathione peroxidase (GPx) activity in HEMn-LP cells after 24 h incubation with 0.01, 0.05, 0.1, 0.5 or 1.0 mM of nicotine. Data are mean7 S.E.M. of at least three independent experiments (n¼3) performed in triplicate.
Nicotine can also form complexes with melanin and the amounts of this alkaloid bound to melanin increase with rising initial nicotine concentration and prolongation of incubation time. Complexes of nicotine with melanin were characterized by two classes of independent binding sites with the association constants K1 ¼ 2.44 104 M 1 and K2 ¼7.72 102 M 1, while the total number of binding sites was estimated to be 1.748 μmol nicotine/mg melanin (Delijewski et al., 2013). The accumulation of nicotine in hair allows to use it in toxicology as a popular biomarker of exposition to nicotine, both in smokers as well as in people exposed to second-hand smoke (Gerstenberg et al., 1995; Koszowski et al., 2008). The contribution of nicotine–melanin interaction may be seen in Black smokers that have substantially higher hair nicotine levels than White smokers (Apelberg et al., 2012). Substances with affinity to hair melanin should also have affinity for the melanin in other tissues. The retention of nicotine in melanin-containing cells could involve nicotine as a false precursor in the formation of new melanin, because of a structural analogy between nicotine and indole-5,6quinone, as one of the precursors of melanin (Larsson et al., 1979). There are still remaining questions if melanin-rich cells protect surrounding tissues by promoting detoxification of nicotine, or if accumulated nicotine affects its metabolism, addiction and results in health risk (Hébert, 2006). In the present study the effect of nicotine on melanocytes viability, as well as on melanization process and antioxidant defense system in pigmented cells was analyzed. As an in vitro experimental model system, we used the culture of normal human melanocytes HEMn-LP. We have found that nicotine in concentrations from 0.0001 mM to 10 mM decreases the cell viability (Fig.1). The value of EC50 was determined to be 7.43 mM, which indicates that nicotine is less toxic to melanocytes than lomefloxacin, streptomycin and kanamycin, for which EC50 values were determined to be 0.75 mM, 5.0 mM and 6.0 mM, respectively (Beberok et al., 2013; Wrześniok et al., 2013a, 2013d). EC50 value obtained for amikacin (7.5 mM) (Wrześniok et al., 2013b) was similar to that obtained for nicotine. Results that we have obtained show that nicotine affects the melanization process in tested cell line. For concentrations of nicotine from 0.05 mM to 1.0 mM we observed inhibition of melanogenesis expressed by decrease in melanin content and tyrosinase activity (Figs. 2 and 3). The inhibitory effect of nicotine in concentrations starting from 0.05 mM on melanogenesis in HEMn-LP cells is probably due to its direct inhibition of tyrosinase
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activity. For the concentrations from 0.0001 mM to 0.01 mM we observed that the melanin content and tyrosinase activity were similar to the control. Reactive oxygen species (ROS) are generated in aerobic processes in cellular interior and in limited quantities are needed in signaling pathways in cells. However, the overproduction of ROS may cause damages in basic cellular components like DNA, proteins and lipids, resulting in cell dysfunction or death (Mari et al., 2010). The complex antioxidant system of cells consists mainly of superoxide dismutase, catalase and glutathione peroxidase enzymes and is responsible for protection against harmful effects of ROS. Melanin can also act as a free radical, including ROS, scavenger and it has been suggested that this biopolymer possesses the superoxide dismutase activity (Hoogduijn et al., 2004). Catalase and melanin may act synergistically to counteract the deleterious effects, especially oxidative stress (Maresca et al., 2008). SOD is the enzyme which catalyzes the formation of hydrogen peroxide from superoxide anion (Gardner et al., 2002). CAT and GPx catalyze the breakdown of hydrogen peroxide, produced by SOD, to water (Mari et al., 2010). Lightly pigmented melanocytes, that in general express lower melanogenic and catalase activity, are probably more susceptible to the harmful effects of free radicals (Maresca et al., 2008). The influence of nicotine on antioxidant status in melanocytes seems to be complex. It was demonstrated that nicotine forms weak complexes with Fe2 þ (Bridge et al., 2004). Moreover, melanin binds Fe2 þ as far as nicotine. It has been reported, that Fenton reaction may be strongly inhibited by nicotine in the concentrations from 0.1 mM to 0.5 mM (Williams and Linert, 2004). It was also observed, that nicotine may interact with the complex I of mitochondrial respiratory chain, thereby decreasing the formation of ROS, by antagonizing the NADH binding onto complex I (Cormier et al., 2001). Therefore, it may be possible that nicotine supports melanin in modulating the antioxidant status of melanocytes and the ability of melanin to work as a free radicals scavenger. In the present study it has been observed that nicotine causes significant alterations in the activities of antioxidant enzymes: superoxide dismutase and catalase in melanocytes. The exposure of melanocytes to nicotine in concentrations from 0.1 mM to 1.0 mM probably causes overproduction of the superoxide anion. The resulting increase in SOD activity (Fig. 5) is associated with subsequent formation of H2O2 (Fig. 4), that leads to the increase in CAT activity (Fig. 6). After treatment of cells with nicotine in lower concentrations (0.01 mM and 0.05 mM), the activities of SOD and CAT, and the H2O2 content were similar to the controls. Simultaneously, no significant changes in cellular GPx activity were observed (Fig. 7), what indicates that in melanocytes catalase is the main enzyme responsible for inactivation of the proradical hydrogen peroxide, what was suggested by Maresca et al. (2008). It may be also possible, that while catalase acts synergistically with melanin to protect cells from oxidative stress in effective manner, the activity of GPx may remain unaltered. Moreover, in case of light pigmented melanocytes, which contain less melanin than dark pigmented cells, the amount of nicotine bound to melanin is relatively low and therefore the antioxidative enzymes response is sufficient to compensate the increase in ROS formation. It may be worth to consider the contribution of tyrosinase to the oxidative/antioxidative balance in melanocytes, as it seems that this enzyme represents a protective mechanism against ROSgenerating compounds. (Perluigi et al., 2003). However, the induction of oxidative stress presumably affects the function of tyrosinase enzyme, what may have a shift in the decrease of melanin content, what was observed for the concentrations of nicotine from 0.05 mM. The increase in H2O2 content observed for
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the concentrations of nicotine from 0.1 mM, may also lead to the inhibition of melanogenic enzymes. Altogether, our results demonstrate that nicotine in noncytotoxic concentrations causes significant alterations of biochemical processes in melanocytes, like inhibition of melanogenesis (reduction in melanin content and tyrosinase activity) and induction of oxidative stress (increase in SOD, CAT activity and H2O2 content). The results presented in this work concerning the effect of nicotine on viability and biochemical processes in normal human melanocytes have to be taken into consideration in case of people with low melanin content and smoking cigarettes or using nicotine replacement therapy.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by the Medical University of Silesia (Grant no. KNW-1-094/K/3/0).
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