The ability of melanin to interact with many drugs and other chemical substances has both beneficial and adverse effects on the body. Binding of potentially dangerous compounds to this biopolymer protects cells from exposure to excessive concentrations of harmful substances through previous accumulation and further elimination in non-toxic concentrations. However, a long-term exposition to xenobiotics with high affinity for melanin may lead to degeneration of pigmented cells. It is believed that the process of drug-induced damages of melanin-containing tissues takes place when the detoxifying capacities of melanin are exhausted [8, 32]. Nicotine forms complexes with melanin and the amounts of nicotine bound to melanin increase with rising initial drug concentrations and prolongation of incubation time. These complexes were characterized by two classes of independent binding sites with the association constants K 1 = 2.44 × 104 M−1 and K 2 = 7.72 × 102 M−1. The total number of binding sites was estimated to be 1,748 μmol nicotine/mg melanin [33]. It has been demonstrated that the laboratory synthesis of melanin, involving oxidation of tyrosine under the influence of tyrosinase in the presence of 3H-nicotine, results in a polymer with incorporated radionuclide [19]. Data from the literature indicate that concentration of nicotine in some organs is related to the pigmentation [14]. Deposition of nicotine in hair containing melanin takes place both during development of hair and after this process [18, 21]. Melanin-containing tissues can store nicotine up to 30 days after a single injection, what was observed in mice [22, 34]. Interactions of nicotine with melanin are still discussed in the context of possible role of the accumulated nicotine in melanin rich tissues, according to nicotine dependence as a topic of health risk significance. That would surely be important, because melanin could act as a non indifferent factor in absorption of nicotine from cigarette smoke and NRT.

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. We used the culture of normal human melanocytes HEMn-DP as an in vitro experimental model system. We have found that nicotine in concentrations from 0.0001 to 10 mM decreases the cell viability in a dose-dependent manner (Fig. 1). The value of EC 50 was determined to be 2.52 mM.

The obtained results show that nicotine affects the melanization process in tested cell line. For concentrations of nicotine 0.01 and 0.05 mM, we observed an activation of melanogenesis expressed by increase in melanin content and tyrosinase activity (Figs. 2, 3). The specific increases in the melanin content for the concentration of nicotine 0.01 and 0.05 mM are probably due to induction of tyrosinase activity by nicotine in these concentrations. The multistep process of melanogenesis is strongly influenced by the key enzyme, tyrosinase. In our study, tyrosinase has increased which correlates with the increasing melanin content. Taking into account the possibility of melanin dispersion occurrence in smokers and in people exposed directly to the effects of nicotine, it can be assumed that the existence of skin coloration may be, among other things, related to the dispersion of melanin-filled melanosomes to the dendrites. It has been shown that smokers’ melanocytes contain melanosomes found in the third or fourth stage of development, entirely filled with melanin, while among non-smokers, melanosomes in the second stage prevail [16]. Nicotine would also activate melanization of melanocytes in an indirect way. It happens through beta-adrenergic effects of epinephrine, which release from the adrenal medulla is augmented while smoking a cigarette. It may cause an increase in the level of cyclic adenosine monophosphate (cAMP), which is an important factor stimulating the biosynthesis of melanin [35]. In humans smoking cigarettes, levels of cAMP in plasma and urine are increased [36]. There might be also a possibility that the high content of melanin can lead to higher absorption of nicotine and lower tobacco cessation rates [14, 23]. A positive correlation between the degree of melanization and the number of cigarettes smoked per day, the level of cotinine—the main metabolite of nicotine in the urine, and test results on the degree of nicotine dependence based on a questionnaire (Fagerström test) was found [23, 37].

For the concentrations from 0.0001 to 0.005 mM as well as for 0.1 mM and 0.5 mM, we observed that the melanin content and tyrosinase activity were similar to control. The lack of increase in the melanin content for the concentrations of nicotine 0.1 and 0.5 mM may be caused by emerging induction of oxidative stress by nicotine that presumably affects the function of tyrosinase enzyme. High H 2 O 2 content stated for these concentrations may also have inhibitory effect on melanogenic enzymes. For the highest tested concentration of nicotine (1.0 mM) inhibition of melanization process, expressed by reduction of tyrosinase activity and melanin content in melanocytes by about 14 and 16 %, respectively, was demonstrated. This indicates that an inhibitory effect of nicotine in concentration of 1.0 mM on melanogenesis is probably due to its direct inhibition of tyrosinase activity.

In the present study, it has been observed for the first time that nicotine causes significant alterations in the activities of antioxidant enzymes: SOD, CAT, and GPx in melanocytes. The concentration-dependent increase in SOD activity, after exposure of melanocytes to nicotine in concentrations from 0.1 to 1.0 mM (Fig. 4), is probably associated with overproduction of the superoxide anion and subsequent formation of H 2 O 2 (Fig. 7), which leads to the increase in CAT activity (Fig. 5). We observed decreased activities of GPx for these nicotine concentrations (Fig. 6), what may be explained by the redundant H 2 O 2 level that cannot be eliminated. After treatment of cells with nicotine in lower concentrations (0.01 and 0.05 mM), the activities of antioxidant enzymes, as well as the H 2 O 2 content were similar to the controls. Increase of oxidative stress in melanocytes can be observed after exposition to nicotine in concentrations higher than 0.1 mM. The overproduction of ROS may cause damages in basic cellular components of cells resulting in dysfunctions or leading to cell death [38]. Harmful effects of oxidative stress should be overcome by three main enzymes: SOD, CAT, and GPx. Additionally, the scavenger properties of melanin may complement the effectiveness of the antioxidant system of melanocytes. CAT is the main enzyme responsible for degradation of hydrogen peroxide in melanocytes [39]. Thus, it may be possible that CAT and melanin act synergistically to protect cells from oxidative stress, while lower activity of GPx.

To summarize, we have demonstrated that nicotine at lower concentrations induces melanogenesis in normal human melanocytes, having no influence on cellular antioxidant status. At higher nicotine concentrations (above 0.1 mM), the occurence of oxidative stress inside melanocytes (expressed by significant changes of antioxidant enzymes activity) was stated.