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The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis
Environmental Research 151 (2016) 44–49
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The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis Marcin Delijewski, Dorota Wrześniok, Artur Beberok, Jakub Rok, Michał Otręba, Ewa Buszman n Department of Pharmaceutical Chemistry, School of Pharmacy with the Division of Laboratory Medicine, 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 26 February 2016 Received in revised form 6 July 2016 Accepted 7 July 2016
Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMnLP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells. & 2016 Elsevier Inc. All rights reserved.
Keywords: Nicotine UV radiation Melanocytes Melanin Tyrosinase
1. Introduction Nicotine is a main compound of tobacco plants which due to its addictive potential contributes to smoking-related deaths and diseases as a result of exposure to toxins in tobacco smoke (Benowitz, 2010). Nicotine is also one of the most often used agents for smoking cessation therapies, like nicotine replacement therapy (NRT). It is also a promising substance in pharmacology, because of its presumed neuroprotective and antioxidant properties (Linert et al., 1999; Williams and Linert, 2004; Takeuchi et al., 2009). More than a billion of people all over the world are exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. Tobacco smoke is the most effective way of nicotine absorption, however it contains many cancerogenic compounds, like polycyclic aromatic n
Corresponding author. E-mail address: [email protected] (E. Buszman).
http://dx.doi.org/10.1016/j.envres.2016.07.009 0013-9351/& 2016 Elsevier Inc. All rights reserved.
hydrocarbons. The safer way of nicotine absorption is NRT in form of gums, sublingual tablets, inhalers, and transdermal patches (Benowitz et al., 2009). Melanin is a natural pigment present in the skin, eyes, hair, inner ear, heart, lungs, liver, lymphocytes and brain. It is produced in melanocytes, in a multistep process called melanogenesis, where the key enzyme is tyrosinase (Larsson, 1993; Sulaimon and Kitchell, 2003). Melanin is known to work as UV radiation absorbent, antioxidant agent and free radicals scavenger (Brenner and Hearing, 2008). It is the main determinant of skin colour and may absorb UV radiation (Meredith and Sarna, 2006; Brenner and Hearing, 2008), which is a powerful factor enhancing skin pigmentation that protects DNA from damages (Yamaguchi and Hearing, 2009). Moreover, melanin may bind many substances, including 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).
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It has been suggested that nicotine may be accumulated in human tissues containing melanin and retention in these tissues may increase melanin biosynthesis. This may influence viability of cells, as well as safety and efficacy of smoking cessation therapies (Yerger and Malone, 2006). UV radiation is a particular inductor of melanogenesis, what may further influence changes in melanin containing cells. It is known that nicotine may lead to melanin pigmentations in oral mucosa of smokers and children exposed to cigarette smoke (Hedin and Larsson, 1984; Haresaku et al., 2007; Sridharan et al., 2011). When we consider binding of nicotine to melanin (Claffey et al., 2000; 2001), long-term retention of nicotine in melanin containing tissues in laboratory animals (Szüts et al.. 1978) and incorporation of nicotine to the pigmented hair (Stout and Ruth, 1999), interactions between nicotine and melanin seem to be significant especially in case of dark pigmented individuals or those exposed to UV radiation. Taking into account that the accumulation of nicotine may be associated with increased time of exposure to nicotine (King et al., 2009), the accompanying exposure to UV radiation makes the problem more complex. We have already documented that aminoglycoside antibiotics: amikacin (Wrześniok et al., 2013a), kanamycin (Wrześniok et al., 2013b), netilmicin (Wrześniok et al., 2013c) and streptomycin (Wrześniok et al., 2013d) as well as fluoroquinolones: ciprofloxacin (Beberok et al., 2011), lomefloxacin (Beberok et al., 2013), norfloxacin and moxifloxacin (Beberok et al., 2015) suppressed melanin biosynthesis in human light pigmented melanocytes. On the other hand, we observed that chlorpromazine (Otręba et al., 2015) and nicotine (Delijewski et al., 2014a) have potential to induce melanogenesis is dark pigmented melanocytes. Our studies also demonstrated that simultaneous exposure of dark pigmented melanocytes to tetracycline and UVA radiation (Rok et al., 2015) caused induction of melanogenesis and that this induction was increasing with rising concentration of the drug. In order to estimate the effect of nicotine in UV-irradiated melanin-containing human cells, we performed a comparative investigation of the simultaneous effect of nicotine and UVA radiation on viability, melanin content and tyrosinase activity in cultured normal human melanocytes dark – (HEMn-DP) and light – (HEMn-LP) pigmented.
2. Materials and methods
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experiments were performed using cells from the passages 5–10. 2.3. UVA irradiation procedure The ultraviolet light source used in this study was a filtered lamp BVL-8. LM (Vilber Lourmat, France). The intensity of UVA (λmax ¼365 nm) radiation was 720 mW/cm2 at 15 cm. The cells, after 24-h incubation with nicotine, 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. 2.4. Cell lysis procedure After incubation the medium was removed, cells were washed twice with PBS, detached using trypsin/EDTA solution and centrifuged. Then, cells were counted and suspended in lysis buffer (PBS supplemented with protease and phosphatase inhibitors) to reach the density of 1 000 000 cells/ml. Afterwards cells were kept in liquid nitrogen ( 196 °C) for 30 min. The obtained cell lysates were stored at 86 °C until analysis. 2.5. 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 disulfonate) 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 nicotine and exposed to UVA irradiation. After 21-h incubation since irradiation, 10 μl of WST-1 were added to 100 μl 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 nicotine) 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.
2.1. Materials 2.6. Melanin content Nicotine, phosphate-buffered saline (PBS), 3,4-dihydroxy-Lphenylalanine (L-DOPA), amphotericin B, SIGMAFAST Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 3 were purchased from Sigma-Aldrich Inc. (USA). Neomycin sulphate 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 Human epidermal melanocytes, neonatal, dark pigmented (HEMn-DP, Cascade Biologics) and light pigmented (HEMn-LP, 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 μg/ml) and amphotericin B (0.25 μg/ml) at 37 °C in 5% CO2. All
Cell pellets were placed into Eppendorf tubes, dissolved in 100 μl of 1 M NaOH at 80 °C for 1 h, and then centrifuged for 20 min at 16,000 g. 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. Melanin content in nicotine treated cells was expressed as the percentage of the controls. 2.7. Tyrosinase activity Tyrosinase activity in HEMn-DP and HEMn-LP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome. Cell lysates were clarified by centrifugation at 10,000 g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the 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 °C. Control wells contained 100 μl of lysis buffer and 40 μl of L-DOPA solution. Absorbance of DOPAchrome was measured every 10 min for at least 1.5 h at 475 nm
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using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls. 2.8. 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 by means of one-way ANOVA (the influence of UVA radiation or nicotine) and two-way ANOVA (the influence of UVA radiation and nicotine), as well as Dunnett's multiple comparison test in both cases. In all cases the statistical significance was found at least at p o0.05, vs. the control samples (cells non-treated with nicotine and non-irradiated).
3. Results In order to assess the simultaneous influence of nicotine and UVA radiation on the viability of melanocytes, cells were treated with nicotine in concentrations 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM for 24 h (Figs. 1 and 2) and exposed to UVA radiation for 15 or 30 min The effect of simultaneous exposure of HEMn-DP melanocytes to nicotine in concentrations 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM and UVA radiation for 30 min was the cell loss by 7.6% (p ¼0.0005), 11.6% (p o0.0001), 15.5% (p o0.0001) and 24.4% (p o0.0001), respectively, when compared with the controls (cells non-treated with nicotine and non-irradiated) (Fig. 1). After simultaneous exposure of HEMn-DP melanocytes to UVA radiation for 15 min, the significant decrease in cell viability was only observed for the highest tested concentration of nicotine (1.0 mM) and was 9.3% (p o0.0001). The simultaneous exposure of HEMn-LP melanocytes to nicotine in concentrations 0.1 mM, 0.5 mM and 1.0 mM and UVA radiation for 30 min, resulted in cell loss by 5.4% (p ¼0.0016), 6.6% (p o0.0001) and 14.2% (p o0.0001), respectively, when compared with the controls (Fig. 2). After exposure of melanocytes to UVA radiation for 15 min, the significant decrease in cell viability was observed only for the highest tested concentration of nicotine
Fig. 1. The effect of nicotine and UVA radiation (15 or 30 min) on viability of HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values 7 SEM. from three independent experiments (n ¼ 3) performed in triplicate are presented. * P o 0.05 vs. the control samples; ** P o 0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
Fig. 2. The effect of nicotine and UVA radiation (15 or 30 min) on viability of HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values7 S.E.M. from three independent experiments (n ¼3) performed in triplicate are presented. * P o0.05 vs. the control samples; ** P o0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
(1.0 mM) and was 10.3% (p o0.0001). The simultaneous effect of nicotine and UVA radiation on the effectiveness of melanization process in HEMn-DP and HEMn-LP melanocytes was evaluated on the basis of melanin content and tyrosinase activity after incubation of cells for 24 h with nicotine in concentrations 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM (Figs. 3–6). After incubation of dark pigmented melanocytes with nicotine in concentrations 0.01 mM, 0.05 mM and 0.1 mM for 24 h and simultaneous exposure to UVA radiation for 30 min, melanin content was increased by 32.1% (p o0.0001), 19.6% (p o0.0001) and 7.0% (p ¼0.0171), respectively, when compared with the controls (Fig. 3). The simultaneous exposure of cells to UVA radiation for 15 min and nicotine in concentration 0.01 mM and 0.05 mM,
Fig. 3. The effect of nicotine and UVA radiation (15 or 30 min) on melanin content in HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 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 (n ¼ 3) performed in triplicate. * P o 0.05 vs. the control samples; **Po 0.005 vs. the control samples (cells non-treated with nicotine and nonirradiated).
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Fig. 4. The effect of nicotine and UVA radiation (15 or 30 min) on melanin content in HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 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 mean 7 S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. ** P o 0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
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Fig. 6. The effect of nicotine and UVA radiation (15 or 30 min) on tyrosinase activity in HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM for 24 h, and tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean 7S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *P o 0.05 vs. the control samples; ** P o 0.005 vs. the control samples (cells non-treated with nicotine and nonirradiated).
(Fig. 5). Tyrosinase activity for the same concentrations of nicotine and exposure to UVA radiation for 15 min, was increased by 28.2% (p o0.0001) and 17.7% (p o0.0001) respectively, when compared with the controls. Exposure of HEMn-LP melanocytes to UVA radiation for 15 and 30 min, only for the highest tested concentration of nicotine (1.0 mM) resulted in decrease in tyrosinase activity by 18.6% (p o0.0001) and 11.8% (p ¼ 0.003), respectively (Fig. 6). For other tested concentrations of nicotine no significant changes in tyrosinase activity were stated.
4. Discussion
Fig. 5. The effect of nicotine and UVA radiation (15 or 30 min) on tyrosinase activity in HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM for 24 h, and tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean 7 S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. **P o0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
caused increase in melanin content by 26.6% (p o0.0001) and 16.5% (p o0.0001), respectively. Exposure of light pigmented melanocytes to UVA radiation for 30 min, only for the highest tested concentration of nicotine (1.0 mM) caused decrease in melanin content by 11.1% (p o0.0001), when compared with the controls (Fig. 4). Simultaneous exposure of cells to UVA radiation for 15 min and nicotine in concentration 0.5 mM and 1.0 mM caused decrease in melanin content by 8.8% (p ¼0.0007) and 20.0% (p o 0.0001) respectively. Changes of the tyrosinase activity in cells treated with nicotine and exposed to UVA radiation correspond with alterations in melanin formation. The simultaneous exposure of HEMn-DP melanocytes to UVA radiation for 30 min and nicotine in concentrations 0.01 mM and 0.05 mM caused increase in tyrosinase activity by 34.5% (p o 0.0001) and 17.3% (p o0.0001), respectively
Nicotine known for years as a main component of tobacco draws attention because of the growing popularity of electronic cigarettes and reports about potential therapeutic action of this alkaloid (Williams and Linert, 2004; Shimohama, 2009; Quik et al., 2014; Smith et al., 2015). The former evidence about nicotine is being currently revised due to contradictive reports about the addictive properties, toxicity or effects of the pure substance on living organisms (Talhout et al., 2007; Mayer, 2014). Following the nicotine's pathway in human body we can discover the potential signifinance of the link between nicotine and melanin (Benowitz et al., 2009; Yerger and Malone, 2006; Uematsu et al., 1995). In the present study the simultaneous effect of nicotine and UVA radiation on cells viability, melanin content and tyrosinase activity in dark- and light- pigmented melanocytes was examined. We used the culture of normal human epidermal melanocytes HEMn-DP and HEMn-LP. In case of melanocytes HEMn-DP, for all tested concentrations beginning from 0.05 mM, the effect of simultaneous exposure of cells to nicotine and UVA radiation for 30 min, was associated with heightened cell loss, what suggests intensification of the toxic effect of nicotine by UVA radiation. The similar effect for HEMn-LP cells, was observed when melanocytes were exposed to nicotine in concentrations from 0.1 mM, suggesting higher cytotoxicity of nicotine and UVA radiation towards dark pigmented cells. This is in agreement with our previous studies (Delijewski et al., 2014a, 2014b) which indicate the higher cytotoxicity of the nicotine alone
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against cells containing more melanin, probably as an effect of the accumulation of the alkaloid in the pigment. Nicotine may form complexes with melanin and the amount of the alkaloid that is bound to melanin increases with rising initial nicotine concentration and prolongation of the incubation time. The formed nicotine-melanin complexes were characterized by two classes of independent binding sites with the association constants K1 ¼2.44 104 M 1 and K2 ¼7.72 102 M 1 and the total number of binding sites was estimated to be 1,748 μmol nicotine/mg melanin (Delijewski et al., 2013). This indicates, that nicotine binds to melanin in a higher amount than substances whose affinity for melanin was traditionally defined as high, for example: chloroquine – approx. 0.9 mmol drug/mg melanin (Buszman et al., 1992) or ciprofloxacin – about 1.2 mmol drug/mg melanin (Beberok et al., 2011). The nature of the interaction of nicotine and other xenobiotics with melanin is still not well established but the existence of ionic bonds, non-electrostatic van der Waals interactions, hydrophobic forces or charge transfer reactions have been proposed (Delijewski et al., 2013). The possibility of nicotine-melanin complex formation may explain higher toxicity of the alkaloid towards HEMn-DP in comparison to HEMn-LP cells. Melanin is synthesized in a multistep process – melanogenesis, in which the key enzyme is tyrosinase that catalyses the transformation of L-tyrosine to DOPA and DOPAquinone (Hearing, 2011). Melanin may interact with various chemical substances what leads to the formation of complexes (Larsson, 1993). Melanin has also ability to absorb UV photons what affects the capacity of the pigment. Due to its ability to absorb UV radiation and accumulate different compounds, including drugs, melanin protects cells from exposure to harmful environmental factors. Moreover, melanin may act as a scavenger of free radicals generated in response to UV radiation (Maresca et al., 2006) and possess superoxide dismutase activity, converting superoxide radical to hydrogen peroxide (Hoogduijn et al., 2004). Melanin may also act synergistically with catalase, to counteract the oxidative stress, by catalyzing the breakdown of hydrogen peroxide to water (Maresca et al., 2008; Mari et al., 2010). On the other hand, the long-term exposition of cells to various substances as well as exposure to UV radiation may lead to degeneration of melanin or melanocytes, what takes place when the detoxifying capacities of melanin are exhausted (Larsson, 1993; Mårs and Larsson, 1999). UV radiation may cause oxidation of melanin (Wood et al., 2006) and melanogenesis alone can be considered as a prooxidative process increasing the risk of cell death, due to generation of reactive orthodihydroxyindoles, – phenols and semiquinones. The redox-cycling of the ortho-dihydroxy compounds is known to result in the production of reactive oxygen species (ROS), like hydrogen peroxide or superoxide radical (Denat et al., 2014; Huang et al., 2014; Smit et al., 2008). According to Jenkins and Grossman (2013), melanocytes exhibit significantly higher basal levels of ROS than other epidermal cell types. It is also known that all types of UVinduced tanning may result in DNA and cellular damage (Miyamura et al., 2011). The dual role of melanin, in where melanin protects against mitochondria superoxide generation and DNA damage but may also act as a direct photosensitizer of mitochondrial DNA damage during UVA irradiation of human melanoma cells, has been shown by Swalwell et al. (2012). This seems to be consistent with the results obtained for dark pigmented melanocytes exposed to nicotine and UVA radiation. These cells contain more melanin in comparison to light pigmented melanocytes and the reduction in their viability was noticeably higher. After exposure of HEMn-DP melanocytes to UVA radiation, the induction of melanogenesis expressed by increase in melanin content and tyrosinase activity was observed. This effect was especially seen for the concentration of nicotine 0.01 mM and 0.05 mM and UVA exposure for 30 min, reaching a rise by 32.1%
and 19.6% respectively, in melanin content, corresponding well with increase by 34.5% and 17.3% respectively, in tyrosinase activity. The specific increases in the melanin content for the concentration of nicotine 0.01 mM and 0.05 mM, additionally intensified by exposure to UVA radiation, are probably due to induction of tyrosinase activity by both nicotine and UVA radiation. In contrary, such an effect has not been seen for HEMn-LP cells, where the only changes in melanogenesis were observed for higher nicotine concentrations (from 0.5 mM) and referred to inhibition of melanogenesis, presumably as a result of the toxic effects of nicotine in these concentrations. Pigmentation is regulated by many genes which encode enzymes, transcription factors, hormones, autocrine and paracrine factors and their receptors, as well as by external or internal stress (Costin and Hearing, 2007; Maddodi et al., 2012). It is possible that UVA radiation as well as together with nicotine evokes greater stress in HEMn-DP melanocytes, compared to HEMn-LP melanocytes what results in induction of melanin biosynthesis. Nevertheless, the process of melanogenesis is strongly influenced by the key enzyme, tyrosinase. In our study, in case of dark pigmented cells, activity of tyrosinase has increased what correlates well with the increasing melanin content. Summarizing, we have observed that UVA radiation strengthens toxic effects of nicotine in HEMn-DP melanocytes and attenuates some biochemical alterations caused by nicotine in HEMn-LP melanocytes. However, in both tested cell lines, it is nicotine that remains a major factor determining changes in the biochemistry of cells. Nicotine alone has influence on viability, melanin content and tyrosinase activity in melanocytes and together with UV radiation may be responsible for changes occurring often in vivo. Our results demonstrate that simultaneous exposure of cells to UVA radiation and nicotine causes important alterations of viability and biochemical processes in melanocytes, what may be valuable especially in case of people with high melanin content during long-term exposure to nicotine and UV radiation.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgment This work was supported by the Medical University of Silesia (Grant No. KNW-1-050/K/6/0).
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human skin. Pigment Cell Melanoma Res. 24, 136–147. Otręba, M., Wrześniok, D., Beberok, A., Rok, J., Buszman, E., 2015. Melanogenesis and antioxidant defense system in normal human melanocytes cultured in the presence of chlorpromazine. Toxicol. In Vitro 29, 221–227. Quik, M., Zhang, D., Perez, X.A., Bordia, T., 2014. Role for the nicotinic cholinergic system in movement disorders; therapeutic implications. Pharmacol. Ther. 144, 50–59. Rok, J., Buszman, E., Delijewski, M., Otręba, M., Beberok, A., Wrześniok, D., 2015. Effect of tetracycline and UV radiation on melanization and antioxidant status of melanocytes. J. Photochem. Photobiol. B Biol. 148, 168–173. Shimohama, S., 2009. Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Biol. Pharm. Bull. 32, 332–336. Smit, N.P., van Nieuwpoort, F.A., Marrot, L., Out, C., Poorthuis, B., van Pelt, H., Meunier, J.R., Pavel, S., 2008. Increased melanogenesis is a risk factor for oxidative DNA damage–study on cultured melanocytes and atypical nevus cells. Photochem. Photobiol. 84, 550–555. Smith, D.M., Bansal-Travers, M., O’Connor, R.J., Goniewicz, M.L., Hyland, A., 2015. Associations between perceptions of e-cigarette advertising and interest in product trial amongst US adult smokers and non-smokers: results from an internet-based pilot survey. Tob. Induc. Dis. 13, 14. Sridharan, S., Ganiger, K., Satyanarayana, A., Rahul, A., Shetty, S., 2011. Effect of environmental tobacco smoke from smoker parents on gingival pigmentation in children and young adults: a cross-sectional study. J. Periodontol. 82, 956–962. Stout, P.R., Ruth, J.A., 1999. Deposition of [3H]cocaine, [3H]nicotine, and [3H]flunitrazepam in mouse hair melanosomes after systemic administration. Drug. Metab. Dispos. 27, 731–735. Sulaimon, S.S., Kitchell, B.E., 2003. The biology of melanocytes. Vet. Dermatol. 14, 57–65. 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. Swalwell, H., Latimer, J., Haywood, R.M., Birch-Machin, M.A., 2012. Investigating the role of melanin in UVA/UVB – and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells. Free Radic. Biol. Med. 52, 626–634. 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, melanin-containing tissues and urinary bladder wall. Toxicology 10, 207–220. Takeuchi, H., Yanagida, T., Inden, M., Takata, K., Kitamura, Y., Yamakawa, K., Sawada, H., Izumi, Y., Yamamoto, N., Kihara, T., Uemura, K., Inoue, H., Taniguchi, T., Akaike, A., Takahashi, R., Shimohama, S., 2009. Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinson’s disease models. J. Neurosci. Res. 87, 576–585. Talhout, R., Opperhuizen, A., van Amsterdam, J.G., 2007. Role of acetaldehyde in tobacco smoke addiction. Eur. Neuropsychopharmacol. 17, 627–636. Uematsu, T., Mizuno, A., Nagashima, S., Oshima, A., Nakamura, M., 1995. The Axial Distribution of Nicotine Content along Hair Shaft as an Indicator of Changes in smoking Behaviour: Evaluation in a smoking-Cessation programme with or without the AID of Nicotine chewing GUM. BR. J. Clin. Pharmacol. 39, 665–669. Williams, E., Linert, W., 2004. In vitro evidence supporting the therapeutic role of nicotine against neurodegeneration. In Vivo 18, 391–399. Wood, S.R., Berwick, M., Ley, R.D., Walter, R.B., Setlow, R.B., Timmins, G.S., 2006. UV causation of melanoma in Xiphophorus is dominated by melanin photosensitized oxidant production. Proc. Natl. Acad. Sci. USA 103, 4111–4115. Wrześniok, D., Beberok, A., Otręba, M., Buszman, E., 2013a. Modulation of melanogenesis and antioxidant defense system in melanocytes by amikacin. Toxicol. In Vitro 27, 1102–1108. Wrześniok, D., Otręba, M., Beberok, A., Buszman, E., 2013a. Impact of kanamycin on melanogenesis and antioxidant enzymes activity in melanocytes – an in vitro study. J. Cell Biochem. 114, 2746–2752. Wrześniok, D., Beberok, A., Otręba, M., Buszman, E., 2013a. Netilmicin-induced modulation of melanogenesis in HEMa-LP melanocytes. Acta Pol. Pharm. Drug Res. 70, 803–808. Wrześniok, D., Beberok, A., Otręba, M., Buszman, E., 2013b. Effect of streptomycin on melanogenesis and antioxidant status in melanocytes. Mol. Cell Biochem. 383, 77–84. Yerger, V.B., Malone, R.E., 2006. Melanin and nicotine: a review of the literature. Nicot. Tob. Res. 8, 487–498. Yamaguchi, Y., Hearing, V.J., 2009. Physiological factors that regulate skin pigmentation. Biofactors 35, 193–199.
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Environmental Research journal homepage: www.elsevier.com/locate/envres
The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis Marcin Delijewski, Dorota Wrześniok, Artur Beberok, Jakub Rok, Michał Otręba, Ewa Buszman n Department of Pharmaceutical Chemistry, School of Pharmacy with the Division of Laboratory Medicine, 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 26 February 2016 Received in revised form 6 July 2016 Accepted 7 July 2016
Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMnLP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells. & 2016 Elsevier Inc. All rights reserved.
Keywords: Nicotine UV radiation Melanocytes Melanin Tyrosinase
1. Introduction Nicotine is a main compound of tobacco plants which due to its addictive potential contributes to smoking-related deaths and diseases as a result of exposure to toxins in tobacco smoke (Benowitz, 2010). Nicotine is also one of the most often used agents for smoking cessation therapies, like nicotine replacement therapy (NRT). It is also a promising substance in pharmacology, because of its presumed neuroprotective and antioxidant properties (Linert et al., 1999; Williams and Linert, 2004; Takeuchi et al., 2009). More than a billion of people all over the world are exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. Tobacco smoke is the most effective way of nicotine absorption, however it contains many cancerogenic compounds, like polycyclic aromatic n
Corresponding author. E-mail address: [email protected] (E. Buszman).
http://dx.doi.org/10.1016/j.envres.2016.07.009 0013-9351/& 2016 Elsevier Inc. All rights reserved.
hydrocarbons. The safer way of nicotine absorption is NRT in form of gums, sublingual tablets, inhalers, and transdermal patches (Benowitz et al., 2009). Melanin is a natural pigment present in the skin, eyes, hair, inner ear, heart, lungs, liver, lymphocytes and brain. It is produced in melanocytes, in a multistep process called melanogenesis, where the key enzyme is tyrosinase (Larsson, 1993; Sulaimon and Kitchell, 2003). Melanin is known to work as UV radiation absorbent, antioxidant agent and free radicals scavenger (Brenner and Hearing, 2008). It is the main determinant of skin colour and may absorb UV radiation (Meredith and Sarna, 2006; Brenner and Hearing, 2008), which is a powerful factor enhancing skin pigmentation that protects DNA from damages (Yamaguchi and Hearing, 2009). Moreover, melanin may bind many substances, including 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).
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It has been suggested that nicotine may be accumulated in human tissues containing melanin and retention in these tissues may increase melanin biosynthesis. This may influence viability of cells, as well as safety and efficacy of smoking cessation therapies (Yerger and Malone, 2006). UV radiation is a particular inductor of melanogenesis, what may further influence changes in melanin containing cells. It is known that nicotine may lead to melanin pigmentations in oral mucosa of smokers and children exposed to cigarette smoke (Hedin and Larsson, 1984; Haresaku et al., 2007; Sridharan et al., 2011). When we consider binding of nicotine to melanin (Claffey et al., 2000; 2001), long-term retention of nicotine in melanin containing tissues in laboratory animals (Szüts et al.. 1978) and incorporation of nicotine to the pigmented hair (Stout and Ruth, 1999), interactions between nicotine and melanin seem to be significant especially in case of dark pigmented individuals or those exposed to UV radiation. Taking into account that the accumulation of nicotine may be associated with increased time of exposure to nicotine (King et al., 2009), the accompanying exposure to UV radiation makes the problem more complex. We have already documented that aminoglycoside antibiotics: amikacin (Wrześniok et al., 2013a), kanamycin (Wrześniok et al., 2013b), netilmicin (Wrześniok et al., 2013c) and streptomycin (Wrześniok et al., 2013d) as well as fluoroquinolones: ciprofloxacin (Beberok et al., 2011), lomefloxacin (Beberok et al., 2013), norfloxacin and moxifloxacin (Beberok et al., 2015) suppressed melanin biosynthesis in human light pigmented melanocytes. On the other hand, we observed that chlorpromazine (Otręba et al., 2015) and nicotine (Delijewski et al., 2014a) have potential to induce melanogenesis is dark pigmented melanocytes. Our studies also demonstrated that simultaneous exposure of dark pigmented melanocytes to tetracycline and UVA radiation (Rok et al., 2015) caused induction of melanogenesis and that this induction was increasing with rising concentration of the drug. In order to estimate the effect of nicotine in UV-irradiated melanin-containing human cells, we performed a comparative investigation of the simultaneous effect of nicotine and UVA radiation on viability, melanin content and tyrosinase activity in cultured normal human melanocytes dark – (HEMn-DP) and light – (HEMn-LP) pigmented.
2. Materials and methods
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experiments were performed using cells from the passages 5–10. 2.3. UVA irradiation procedure The ultraviolet light source used in this study was a filtered lamp BVL-8. LM (Vilber Lourmat, France). The intensity of UVA (λmax ¼365 nm) radiation was 720 mW/cm2 at 15 cm. The cells, after 24-h incubation with nicotine, 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. 2.4. Cell lysis procedure After incubation the medium was removed, cells were washed twice with PBS, detached using trypsin/EDTA solution and centrifuged. Then, cells were counted and suspended in lysis buffer (PBS supplemented with protease and phosphatase inhibitors) to reach the density of 1 000 000 cells/ml. Afterwards cells were kept in liquid nitrogen ( 196 °C) for 30 min. The obtained cell lysates were stored at 86 °C until analysis. 2.5. 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 disulfonate) 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 nicotine and exposed to UVA irradiation. After 21-h incubation since irradiation, 10 μl of WST-1 were added to 100 μl 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 nicotine) 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.
2.1. Materials 2.6. Melanin content Nicotine, phosphate-buffered saline (PBS), 3,4-dihydroxy-Lphenylalanine (L-DOPA), amphotericin B, SIGMAFAST Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 3 were purchased from Sigma-Aldrich Inc. (USA). Neomycin sulphate 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 Human epidermal melanocytes, neonatal, dark pigmented (HEMn-DP, Cascade Biologics) and light pigmented (HEMn-LP, 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 μg/ml) and amphotericin B (0.25 μg/ml) at 37 °C in 5% CO2. All
Cell pellets were placed into Eppendorf tubes, dissolved in 100 μl of 1 M NaOH at 80 °C for 1 h, and then centrifuged for 20 min at 16,000 g. 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. Melanin content in nicotine treated cells was expressed as the percentage of the controls. 2.7. Tyrosinase activity Tyrosinase activity in HEMn-DP and HEMn-LP cells was determined by measuring the rate of oxidation of L-DOPA to DOPAchrome. Cell lysates were clarified by centrifugation at 10,000 g for 5 min. A tyrosinase substrate L-DOPA (2 mg/ml) was prepared in the 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 °C. Control wells contained 100 μl of lysis buffer and 40 μl of L-DOPA solution. Absorbance of DOPAchrome was measured every 10 min for at least 1.5 h at 475 nm
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using a microplate reader. Tyrosinase activity was expressed as the percentage of the controls. 2.8. 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 by means of one-way ANOVA (the influence of UVA radiation or nicotine) and two-way ANOVA (the influence of UVA radiation and nicotine), as well as Dunnett's multiple comparison test in both cases. In all cases the statistical significance was found at least at p o0.05, vs. the control samples (cells non-treated with nicotine and non-irradiated).
3. Results In order to assess the simultaneous influence of nicotine and UVA radiation on the viability of melanocytes, cells were treated with nicotine in concentrations 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM for 24 h (Figs. 1 and 2) and exposed to UVA radiation for 15 or 30 min The effect of simultaneous exposure of HEMn-DP melanocytes to nicotine in concentrations 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM and UVA radiation for 30 min was the cell loss by 7.6% (p ¼0.0005), 11.6% (p o0.0001), 15.5% (p o0.0001) and 24.4% (p o0.0001), respectively, when compared with the controls (cells non-treated with nicotine and non-irradiated) (Fig. 1). After simultaneous exposure of HEMn-DP melanocytes to UVA radiation for 15 min, the significant decrease in cell viability was only observed for the highest tested concentration of nicotine (1.0 mM) and was 9.3% (p o0.0001). The simultaneous exposure of HEMn-LP melanocytes to nicotine in concentrations 0.1 mM, 0.5 mM and 1.0 mM and UVA radiation for 30 min, resulted in cell loss by 5.4% (p ¼0.0016), 6.6% (p o0.0001) and 14.2% (p o0.0001), respectively, when compared with the controls (Fig. 2). After exposure of melanocytes to UVA radiation for 15 min, the significant decrease in cell viability was observed only for the highest tested concentration of nicotine
Fig. 1. The effect of nicotine and UVA radiation (15 or 30 min) on viability of HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values 7 SEM. from three independent experiments (n ¼ 3) performed in triplicate are presented. * P o 0.05 vs. the control samples; ** P o 0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
Fig. 2. The effect of nicotine and UVA radiation (15 or 30 min) on viability of HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM and examined by the WST-1 assay. Data are expressed as % of cell viability. Mean values7 S.E.M. from three independent experiments (n ¼3) performed in triplicate are presented. * P o0.05 vs. the control samples; ** P o0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
(1.0 mM) and was 10.3% (p o0.0001). The simultaneous effect of nicotine and UVA radiation on the effectiveness of melanization process in HEMn-DP and HEMn-LP melanocytes was evaluated on the basis of melanin content and tyrosinase activity after incubation of cells for 24 h with nicotine in concentrations 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM and 1.0 mM (Figs. 3–6). After incubation of dark pigmented melanocytes with nicotine in concentrations 0.01 mM, 0.05 mM and 0.1 mM for 24 h and simultaneous exposure to UVA radiation for 30 min, melanin content was increased by 32.1% (p o0.0001), 19.6% (p o0.0001) and 7.0% (p ¼0.0171), respectively, when compared with the controls (Fig. 3). The simultaneous exposure of cells to UVA radiation for 15 min and nicotine in concentration 0.01 mM and 0.05 mM,
Fig. 3. The effect of nicotine and UVA radiation (15 or 30 min) on melanin content in HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 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 (n ¼ 3) performed in triplicate. * P o 0.05 vs. the control samples; **Po 0.005 vs. the control samples (cells non-treated with nicotine and nonirradiated).
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Fig. 4. The effect of nicotine and UVA radiation (15 or 30 min) on melanin content in HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 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 mean 7 S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. ** P o 0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
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Fig. 6. The effect of nicotine and UVA radiation (15 or 30 min) on tyrosinase activity in HEMn-LP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM for 24 h, and tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean 7S.E.M. of at least three independent experiments (n¼ 3) performed in triplicate. *P o 0.05 vs. the control samples; ** P o 0.005 vs. the control samples (cells non-treated with nicotine and nonirradiated).
(Fig. 5). Tyrosinase activity for the same concentrations of nicotine and exposure to UVA radiation for 15 min, was increased by 28.2% (p o0.0001) and 17.7% (p o0.0001) respectively, when compared with the controls. Exposure of HEMn-LP melanocytes to UVA radiation for 15 and 30 min, only for the highest tested concentration of nicotine (1.0 mM) resulted in decrease in tyrosinase activity by 18.6% (p o0.0001) and 11.8% (p ¼ 0.003), respectively (Fig. 6). For other tested concentrations of nicotine no significant changes in tyrosinase activity were stated.
4. Discussion
Fig. 5. The effect of nicotine and UVA radiation (15 or 30 min) on tyrosinase activity in HEMn-DP melanocytes. Cells were treated with nicotine in concentrations: 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 1.0 mM for 24 h, and tyrosinase activity was measured as described in Materials and methods. Results are expressed as percentages of the controls. Data are mean 7 S.E.M. of at least three independent experiments (n ¼3) performed in triplicate. **P o0.005 vs. the control samples (cells non-treated with nicotine and non-irradiated).
caused increase in melanin content by 26.6% (p o0.0001) and 16.5% (p o0.0001), respectively. Exposure of light pigmented melanocytes to UVA radiation for 30 min, only for the highest tested concentration of nicotine (1.0 mM) caused decrease in melanin content by 11.1% (p o0.0001), when compared with the controls (Fig. 4). Simultaneous exposure of cells to UVA radiation for 15 min and nicotine in concentration 0.5 mM and 1.0 mM caused decrease in melanin content by 8.8% (p ¼0.0007) and 20.0% (p o 0.0001) respectively. Changes of the tyrosinase activity in cells treated with nicotine and exposed to UVA radiation correspond with alterations in melanin formation. The simultaneous exposure of HEMn-DP melanocytes to UVA radiation for 30 min and nicotine in concentrations 0.01 mM and 0.05 mM caused increase in tyrosinase activity by 34.5% (p o 0.0001) and 17.3% (p o0.0001), respectively
Nicotine known for years as a main component of tobacco draws attention because of the growing popularity of electronic cigarettes and reports about potential therapeutic action of this alkaloid (Williams and Linert, 2004; Shimohama, 2009; Quik et al., 2014; Smith et al., 2015). The former evidence about nicotine is being currently revised due to contradictive reports about the addictive properties, toxicity or effects of the pure substance on living organisms (Talhout et al., 2007; Mayer, 2014). Following the nicotine's pathway in human body we can discover the potential signifinance of the link between nicotine and melanin (Benowitz et al., 2009; Yerger and Malone, 2006; Uematsu et al., 1995). In the present study the simultaneous effect of nicotine and UVA radiation on cells viability, melanin content and tyrosinase activity in dark- and light- pigmented melanocytes was examined. We used the culture of normal human epidermal melanocytes HEMn-DP and HEMn-LP. In case of melanocytes HEMn-DP, for all tested concentrations beginning from 0.05 mM, the effect of simultaneous exposure of cells to nicotine and UVA radiation for 30 min, was associated with heightened cell loss, what suggests intensification of the toxic effect of nicotine by UVA radiation. The similar effect for HEMn-LP cells, was observed when melanocytes were exposed to nicotine in concentrations from 0.1 mM, suggesting higher cytotoxicity of nicotine and UVA radiation towards dark pigmented cells. This is in agreement with our previous studies (Delijewski et al., 2014a, 2014b) which indicate the higher cytotoxicity of the nicotine alone
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against cells containing more melanin, probably as an effect of the accumulation of the alkaloid in the pigment. Nicotine may form complexes with melanin and the amount of the alkaloid that is bound to melanin increases with rising initial nicotine concentration and prolongation of the incubation time. The formed nicotine-melanin complexes were characterized by two classes of independent binding sites with the association constants K1 ¼2.44 104 M 1 and K2 ¼7.72 102 M 1 and the total number of binding sites was estimated to be 1,748 μmol nicotine/mg melanin (Delijewski et al., 2013). This indicates, that nicotine binds to melanin in a higher amount than substances whose affinity for melanin was traditionally defined as high, for example: chloroquine – approx. 0.9 mmol drug/mg melanin (Buszman et al., 1992) or ciprofloxacin – about 1.2 mmol drug/mg melanin (Beberok et al., 2011). The nature of the interaction of nicotine and other xenobiotics with melanin is still not well established but the existence of ionic bonds, non-electrostatic van der Waals interactions, hydrophobic forces or charge transfer reactions have been proposed (Delijewski et al., 2013). The possibility of nicotine-melanin complex formation may explain higher toxicity of the alkaloid towards HEMn-DP in comparison to HEMn-LP cells. Melanin is synthesized in a multistep process – melanogenesis, in which the key enzyme is tyrosinase that catalyses the transformation of L-tyrosine to DOPA and DOPAquinone (Hearing, 2011). Melanin may interact with various chemical substances what leads to the formation of complexes (Larsson, 1993). Melanin has also ability to absorb UV photons what affects the capacity of the pigment. Due to its ability to absorb UV radiation and accumulate different compounds, including drugs, melanin protects cells from exposure to harmful environmental factors. Moreover, melanin may act as a scavenger of free radicals generated in response to UV radiation (Maresca et al., 2006) and possess superoxide dismutase activity, converting superoxide radical to hydrogen peroxide (Hoogduijn et al., 2004). Melanin may also act synergistically with catalase, to counteract the oxidative stress, by catalyzing the breakdown of hydrogen peroxide to water (Maresca et al., 2008; Mari et al., 2010). On the other hand, the long-term exposition of cells to various substances as well as exposure to UV radiation may lead to degeneration of melanin or melanocytes, what takes place when the detoxifying capacities of melanin are exhausted (Larsson, 1993; Mårs and Larsson, 1999). UV radiation may cause oxidation of melanin (Wood et al., 2006) and melanogenesis alone can be considered as a prooxidative process increasing the risk of cell death, due to generation of reactive orthodihydroxyindoles, – phenols and semiquinones. The redox-cycling of the ortho-dihydroxy compounds is known to result in the production of reactive oxygen species (ROS), like hydrogen peroxide or superoxide radical (Denat et al., 2014; Huang et al., 2014; Smit et al., 2008). According to Jenkins and Grossman (2013), melanocytes exhibit significantly higher basal levels of ROS than other epidermal cell types. It is also known that all types of UVinduced tanning may result in DNA and cellular damage (Miyamura et al., 2011). The dual role of melanin, in where melanin protects against mitochondria superoxide generation and DNA damage but may also act as a direct photosensitizer of mitochondrial DNA damage during UVA irradiation of human melanoma cells, has been shown by Swalwell et al. (2012). This seems to be consistent with the results obtained for dark pigmented melanocytes exposed to nicotine and UVA radiation. These cells contain more melanin in comparison to light pigmented melanocytes and the reduction in their viability was noticeably higher. After exposure of HEMn-DP melanocytes to UVA radiation, the induction of melanogenesis expressed by increase in melanin content and tyrosinase activity was observed. This effect was especially seen for the concentration of nicotine 0.01 mM and 0.05 mM and UVA exposure for 30 min, reaching a rise by 32.1%
and 19.6% respectively, in melanin content, corresponding well with increase by 34.5% and 17.3% respectively, in tyrosinase activity. The specific increases in the melanin content for the concentration of nicotine 0.01 mM and 0.05 mM, additionally intensified by exposure to UVA radiation, are probably due to induction of tyrosinase activity by both nicotine and UVA radiation. In contrary, such an effect has not been seen for HEMn-LP cells, where the only changes in melanogenesis were observed for higher nicotine concentrations (from 0.5 mM) and referred to inhibition of melanogenesis, presumably as a result of the toxic effects of nicotine in these concentrations. Pigmentation is regulated by many genes which encode enzymes, transcription factors, hormones, autocrine and paracrine factors and their receptors, as well as by external or internal stress (Costin and Hearing, 2007; Maddodi et al., 2012). It is possible that UVA radiation as well as together with nicotine evokes greater stress in HEMn-DP melanocytes, compared to HEMn-LP melanocytes what results in induction of melanin biosynthesis. Nevertheless, the process of melanogenesis is strongly influenced by the key enzyme, tyrosinase. In our study, in case of dark pigmented cells, activity of tyrosinase has increased what correlates well with the increasing melanin content. Summarizing, we have observed that UVA radiation strengthens toxic effects of nicotine in HEMn-DP melanocytes and attenuates some biochemical alterations caused by nicotine in HEMn-LP melanocytes. However, in both tested cell lines, it is nicotine that remains a major factor determining changes in the biochemistry of cells. Nicotine alone has influence on viability, melanin content and tyrosinase activity in melanocytes and together with UV radiation may be responsible for changes occurring often in vivo. Our results demonstrate that simultaneous exposure of cells to UVA radiation and nicotine causes important alterations of viability and biochemical processes in melanocytes, what may be valuable especially in case of people with high melanin content during long-term exposure to nicotine and UV radiation.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgment This work was supported by the Medical University of Silesia (Grant No. KNW-1-050/K/6/0).
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