http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2014; 37(3): 322–328 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.851693

RESEARCH ARTICLE

In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay Robert Sobkowiak, Jakub Musidlak, and Andrzej Lesicki Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University, Poznan´, Poland

Abstract

Keywords

The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head area, tail length, percent DNA in the tail, and Olive tail moment. Samples of whole blood were collected and blood cells were challenged with acute doses of 0.1, 1 and 10 mM of ()-nicotine for 60 minutes. We found that nicotine treatment had dose-dependent effects on the level of DNA damage. At 1 and 10 mM of nicotine, both Olive tail moment and percent DNA in the tail significantly increased (p50.001), compared to the control. In the presence of 10 mM of nicotine, the shortest tail length and the smallest head area were detected. At a concentration of 0.1 mM, surprisingly, DNA damage detected by the comet assay was lower than in the control, which was proved by the observed significantly (p50.001) lower Olive tail moment and percent DNA in the tail as well as larger head area. The results suggest that nicotine, at a reasonably low concentration (0.1 mM), comparable to those found in the blood of habitual smokers, may have a protective effect, whereas higher doses of nicotine (1 and 10 mM) are genotoxic. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine is discussed.

Blood cells, DNA damage, DNA protection, drug

Introduction Nicotine is part of the regular human diet, being present in a number of food plants. The content of this alkaloid was estimated as, for example, 5–43 and 16.8 mg/kg of wet weight in tomatoes and cauliflower, respectively, whereas green pepper and eggplant may contain even up to 100 mg/kg (Koleva et al., 2012). The largest amount of nicotine is found in leaves of tobacco plants Nicotiana tabacum and N. rustica, from 8 to 100 mg/g of dry weight of plants (Idris et al., 1998). However, the main source of nicotine exposure for people is, in general, inhaling tobacco smoke from either active or passive smoking. A cigarette contains approximately 0.3– 2.0 mg of nicotine, and plasma nicotine levels in heavy smokers reach up to 100 ng/mL (&0.6 mM) (Kleinsasser et al., 2005; Matta et al., 2007). For many smokers, nicotine is delivered in sufficient quantities to produce physiological and behavioral effects that comprise addiction (Swan & Lessov-Schlaggar, 2009). Although it can be treated as addictive, nicotine alone does not fully explain all aspects of symptoms, clinical course or need for treatment that relate to addiction (IARC, 2007). Since the late 1990s, there have been several commercial

Address for correspondence: Robert Sobkowiak, Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan´, Poland. Fax: +48 61 8295829. E-mail: [email protected]

History Received 30 April 2013 Revised 29 July 2013 Accepted 25 August 2013 Published online 14 November 2013

products containing nicotine offered on the market (Pogocki et al., 2007). Surprisingly, nicotine as medication is widely used in nicotine replacement therapies to assist smoking cessation as transdermal nicotine patches and nicotinecontaining gum and, more recently, have been proposed for use concurrently with smoking as part of a risk reduction strategy (Herman & Sofuoglu, 2010). Moreover, despite popular prejudice against this drug, nicotine treatment has been studied as an experimental therapy for Parkinson’s disease, Alzheimer’s disease, ulcerative colitis, and others (Baron, 1996; Jarvik, 1991; Pogocki et al., 2007). Summing up, almost every one of us is exposed to this alkaloid, although the source of nicotine may vary (e.g. food, smoke or medicines). Nicotine has been identified as a noncarcinogenic component of cigarette smoke (IARC, 2004), and DNA alteration induced by tobacco is not related to nicotine content (Mizusaki et al., 1977). However, its high doses are toxic. The genotoxicity and mutagenicity of drugs in animals were evaluated with the use of several experimental methods, such as alkaline elution (Turner et al., 1982), nucleoid sedimentation (Harris et al., 1986), sister chromatid exchange (SCE) (Doolittle et al., 1995), chromosome aberration (DeMarini et al., 2008), DNA adduct measurements (Hall et al., 1993) and micronuclei counting (Attia, 2007a,b). However, the alkaline comet assay (Singh et al., 1988), increasingly used for in vitro testing of genotoxicity of pharmaceuticals (Hartmann et al., 2001, 2004), is also very useful for

Effect of nicotine on human leukocytes

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

DOI: 10.3109/01480545.2013.851693

examination of the DNA-damaging effects of nicotine (Ginzkey et al., 2009; Kleinsasser et al., 2005; Muthukumaran et al., 2008; Sassen et al., 2005; Sudheer et al., 2007a,b). The method has several advantages over other in vitro genotoxicity test methods on leukocytes, especially over those applicable to proliferating cells only (Collins et al., 2008; Lee et al., 2004), whereas almost all leukocytes are in the same phase of the cell cycle (G0) (Collins et al., 2008). The comet assay is also highly sensitive (Leroy et al., 1996; Singh, 2000). Therefore, the method is ideally suited for human investigations because it requires no prelabeling with radioactivity or other harmful procedures and can be applied to easily obtainable cells. Normally, white blood cells are used, because they are obtained in a relatively noninvasive way, do not require tissue disaggregation and behave well in the comet assay (Collins, 2004). A lethal dosage of nicotine for adult humans has been estimated to be 30–60 mg (0.5–1.0 mg/kg or 6 mM) (Gosselin et al., 1988). Many investigators (e.g. Arabi, 2004; Argentin & Cicchetti, 2004; Doolittle et al., 1995; Ginzkey et al., 2009; Kleinsasser et al., 2005; Muthukumaran et al., 2008; Sassen et al., 2005; Sudheer et al., 2007a,b) reported toxic effects of nicotine on different human cells or tissues. However, those investigators have not taken into account the real nicotine blood concentrations in smokers. Peak arterial nicotine concentrations can be as high as 0.6 mM, depending on how the cigarette is smoked, whereas peak venous blood levels are typically 0.06–0.31 mM (Matta et al., 2007). Research on effects of low nicotine concentration in a physiological range with the use of the comet assay is limited (Sobkowiak & Lesicki, 2009, with experiments on Caenorhabditis elegans with nicotine concentrations ranged from 1 to 100 mM; Ginzkey et al., 2012, with experiments on cells of human nasal mucosa and human bronchial epithelial cells with nicotine concentrations ranging from 1 to 4000 mM). Keeping in view the above-cited reports, and the scarcity of the lowconcentration in vitro studies on effects of nicotine on DNA damage, we initiated a study on in vitro genotoxic effects of low nicotine concentrations on human leukocytes. To our knowledge, this is the first study demonstrating that nicotine, at concentrations similar to those generally found in the blood of habitual smokers (Benowitz et al., 2009; Hukkanen et al., 2005), performs a possible genoprotective role in leukocytes.

Methods Blood collection, preservation and exposure to nicotine Just before blood collection, ()-nicotine (CAS no. 54-11-5, free base; purity, 499%; Sigma-Aldrich, St. Louis, MO) was diluted in phosphate buffered saline (PBS; 137 mM of NaCl, 2.7 mM of KCl, 1.4 mM of KH2PO4 and 10 mM of Na2HPO4) to prepare separate tubes with three nicotine stock solutions. To avoid potential effects produced by chronic exposure to nicotine, both volunteers (authors R.S. and J.M.) were nonsmokers. To prevent DNA damage, cell samples were handled in dimmed light. A 100-mL blood sample from each volunteer was collected by a finger prick into a tube. Immediately, the blood was mixed with a one-fourth volume of the anticoagulant (23 mM of citric acid monohydrate,

323

45 mM of trisodium citrate and 45 mM of glucose) and divided into 24-mL aliquots in four Eppendorf test tubes. To prepare the assay sample, 1 mL of appropriate nicotine stock solution was added – then, the final concentrations of nicotine were reached (0.1, 1 and 10 mM). In the control variant, we added 1 mL of PBS instead of 1 mL of nicotine stock solution. Exposure to nicotine was carried out at 37  C in a water bath for 60 minutes. We tested nicotine at concentrations simulating physiological blood-nicotine level (or slightly higher) in smokers (Matta et al., 2007). To prove that, in this concentration, the range of nicotine is not cytotoxic, cell viability was examined by the trypan blue test (Sudheer et al., 2007a,b). These experiments were carried out in accord with the ethics standards as formulated in the Helsinki Declaration of 1975 (revised, 2008). Alkaline single-cell microgel electrophoresis (comet) assay After incubation with nicotine, without undue delay, the alkaline comet assay was conducted according to the method described by Olive & Banath (2006). In brief, 3 mL of the assay sample were resuspended in 100 mL of 0.5% lowmelting-point agarose (Prona; ABO, Gdan´sk, Poland) and applied to slides coated with 1% normal-melting-point agarose (Prona, ABO). Slides were covered with a 24  60 mm coverslip and left on a cold plate for 3 minutes (to solidify agarose), and next the coverslip was removed. After cell lysis for at least 12 hours at 4  C in alkaline lysis buffer (0.03 M of NaOH, 1 M of NaCl and 0.5% sodium dodecyl sulfate; pH 10), slides were placed in a horizontal gel electrophoresis chamber and covered with alkaline buffer (300 mM of NaOH and 1 mM of Na2 ethylenediaminetetraacetic acid) at pH413. After a 50-minute DNA ‘‘unwinding’’ period, the electrophoresis was performed under standard conditions (25 V and 300 mA) for 20 minutes in a chamber cooled on ice. The duration of electrophoresis was designed so that DNA of control cells showed a significant migration. It was necessary to reveal the presence of DNA damages in cells (treated with 0.1 mM of nicotine), which exhibited lower DNA migration, as compared to the control sample. Electrophoresis was followed by neutralization at pH 7.5 (0.4 M of Tris; pH 7.5) and rinsing with water and cold 70% ethanol. Slides were air-dried at room temperature and stored until analysis in a desiccator. Just before image acquisition, DNA was stained with 300 mL of SybrGreen I (dilution, 1:10 000; Molecular Probes, Invitrogen, Eugene, OR) of a sensitivity at least 25 times greater than standard ethidium bromide. Comets were observed at 300 magnification, using a confocal fluorescence microscope (LSM 510; Carl Zeiss, Jena, Germany) equipped with an excitation filter of 500– 550 nm and a barrier filter of 590 nm. Image analysis Quantification of the DNA strand breaks in the stored images was done using CASP software (CASP image-analysis system; University of Wroclaw, Wroclaw, Poland) (Konca et al., 2003). The head corresponds to the amount of DNA that still remains in the nuclear matrix, whereas the tail

324

R. Sobkowiak et al.

Drug Chem Toxicol, 2014; 37(3): 322–328

visualizes the fragments of DNA migrating from nuclei. At least 150 cells for every test sample were counted and analyzed for the following parameters to quantify the induced DNA damage: head area; tail length; percent DNA in the tail and Olive tail moment (Olive et al., 1993). Olive tail moment is the product of tail length and percent DNA in the tail. The percent DNA in the tail is a measure of relative fluorescent intensity in the head and tail, measured from the head center to the tail end.

greater head area than in the control (Figure 1; p50.001). On the other hand, at 1 and 10 mM, increased DNA damage was detected. For both concentrations, Olive tail moment and percent DNA in the tail were significantly higher than in the control (p50.001). Besides, in the presence of 10 mM of nicotine, a significantly lower head area than in the control was observed. At all examined nicotine concentrations, tail length was lower than in the control, but, significantly, this was confirmed only at 10 mM of nicotine (Figure 1; p50.001).

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

Statistical analysis

Discussion

Data from four experiments were pooled and median values were calculated. Kruskal–Wallis’ analysis of variance test was used to determine statistical differences with nonparametric distribution. Statistical significance was considered at p50.001. Calculations and graphs were done using Statistica software (StatSoft, Inc., Tulsa, OK).

In this study, we used the comet assay to investigate in vitro response of human leukocytes to nicotine at concentrations similar to those found in the blood of habitual smokers (Hukkanen et al., 2005). In this concentration range, nicotine is not cytotoxic for many kinds of cells, including blood cells (Ginzkey et al., 2012; Kleinsasser et al., 2005; Sassen et al., 2005). This is confirmed by the trypan blue test for nicotinetreated lymphocytes. Nicotine did not exert any cytotoxic effects up to the 1000-mM concentration based on the trypan blue test (Sudheer et al., 2007a,b). Cell viability was between 75 and 85% in the 2000- and 3000-mM concentrations of nicotine-treated lymphocytes, respectively, but cell viability was dropped to 60% in 4000-mM nicotine-treated lymphocytes (Sudheer et al., 2007a,b). Cell viability was 495% in all our tested samples. Moreover, nicotine (1, 10 and 100 mM) did not produce any cytotoxic effects in our previous studies of C. elegans cells (Sobkowiak & Lesicki, 2009). Therefore, we can conclude that the nicotine concentrations used (at the tested duration of treatment) do not have cytotoxic effects. Genotoxic in vitro effects of nicotine have been reported in human gingival fibroblast (Argentin & Cicchetti, 2004), tonsillar cells (Kleinsasser et al., 2005), lymphocytes

Results After treatment of human leukocytes with nicotine for 60 minutes, even at 10 mM, which was the highest concentration, cell viability (evaluated in the trypan blue test) did not change significantly. Nicotine did not have cytotoxic effects in any of the four experiments, because cell viability was above 95% after exposure to 0.1, 1 and 10 mM of nicotine (data not presented). Table 1 shows the individual data sets from each of the four experiments. The DNA alterations in leukocytes are expressed as head area, tail length, percent DNA in the tail and Olive tail moment. Distinct differences were recorded between the experimental variants (Figure 1). At 0.1 mM, we observed lower values of percent DNA in the tail and Olive tail moment than in control experiments (Figure 1; p50.001). Also, in the presence of 0.1 mM of nicotine, we observed

Table 1. DNA damage by nicotine, as measured by the comet assay, in human leukocytes. Nicotine 0 mM N

Mean Median

First experiment HA 366 1215.6 TL 103.8 TD% 53.7 OTM 33.8 Second experiment HA 72 1551.9 TL 124.1 TD% 50.6 OTM 41.5 Third experiment HA 82 1647.9 TL 139.5 TD% 52.2 OTM 45.3 Fourth experiment HA 39 1647.1 TL 125.7 TD% 52.9 OTM 44.1

0.1 mM SD

SE

N

Mean Median

1 mM SD

SE

N

Mean Median

10 mM SD

SE

1161.5 375.2 19.6 394 1643.9 1569.0 446.8 22.5 215 1381.6 1322.0 468.7 32.0 104.0 19.6 1.0 111.9 112.0 13.9 0.7 101.3 101.0 14.8 1.0 54.2 13.4 0.7 43.3 43.1 12.3 0.6 56.6 59.7 13.6 0.9 33.3 9.8 0.5 32.0 30.7 9.9 0.5 37.3 38.2 9.2 0.6 1568.5 293.2 34.5 103 2189.5 2175.0 396.7 39.1 68 1035.0 124.5 13.0 1.5 107.3 110.0 16.2 1.6 103.4 54.0 9.2 1.1 39.1 37.5 11.4 1.1 59.6 42.5 8.1 0.9 29.3 30.2 10.0 1.0 35.7

N

SD

SE

520 1177.7 1149.5 319.5 103.2 102.0 12.8 63.7 63.6 9.5 43.2 42.4 8.4

14.0 0.6 0.4 0.4

945.5 347.2 42.14 71 103.5 11.0 1.3 60.2 9.4 1.1 35.1 9.3 1.1

Mean Median

933.5 123.5 71.2 49.9

846.0 376.1 127.0 14.9 73.1 8.3 50.7 9.5

44.6 1.8 1.0 1.1

1671.0 271.1 29.9 109 1991.0 1980.0 519.9 49.8 101 1698.0 1665.0 598.1 59.5 109 1043.9 1056.0 319.9 30.6 140.0 13.0 1.4 120.5 117.0 21.9 2.1 124.9 124.0 13.4 1.3 100.9 101.0 9.3 0.98 54.8 11.4 1.2 45.8 45.9 9.8 0.9 62.0 60.3 15.8 1.6 59.6 58.8 13.2 1.3 46.3 9.3 1.0 35.3 36.0 9.9 0.9 45.5 45.3 11.1 1.18 40.6 40.2 8.53 0.8 1692.0 259.7 41.6 36 1664.3 1671.5 384.7 64.1 102 1685.8 1710.0 465.4 46.1 130.0 20.9 3.3 102.0 102.0 13.5 2.3 120.6 119.5 15.2 1.5 54.7 7.0 1.1 51.2 52.7 9.4 1.6 58.2 58.5 10.5 1.0 44.3 7.1 1.1 32.5 33.3 7.1 1.2 42.0 42.4 8.4 0.8

114 1347.9 1286.0 367.5 114.4 112.0 14.9 64.2 64.4 9.0 43.4 42.1 7.8

34.4 1.4 0.8 0.7

HA, head area; TL, tail length; TD%, tail DNA %; OTM, Olive tail moment; N, number of comets; SD, standard deviation; SE, standard error.

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

DOI: 10.3109/01480545.2013.851693

Effect of nicotine on human leukocytes

325

Figure 1. Head area (A), tail length (B), percent DNA in the tail (C) and Olive tail moment (D) of comets obtained from human control cells (0) or those exposed to 0.1, 1 and 10 mM of ()-nicotine for 60 minutes. Mean values of four experiments are given with standard error and standard deviation values. Asterisks denote significant differences from the control (Kruskal–Wallis’ analysis of variance; p50.001).

(Sudheer et al., 2007a,b), cells of human nasal epithelia (Ginzkey et al., 2012; Sassen et al., 2005), tumor-free human parotid glands (Ginzkey et al., 2009), oral epidermal carcinoma cells (Wu et al., 2005) and spermatozoids (Arabi, 2004), as well as in animal cells: Chinese hamster ovary (CHO) cells (Doolittle et al., 1995); mouse bone marrow cells (Attia, 2007a,b) and culture of C. elegans cells (Sobkowiak & Lesicki, 2009). In most experiments, nicotine tested at concentrations from 1 to 4000 mM induced a dose-dependent increase in DNA migration, with statistical differences from the control, starting at 1 mM (Argentin & Cicchetti, 2004; Wu et al., 2005). However, taking into account a realistic dispersion of nicotine after usage, for example, after cigarette smoking (Matta et al., 2007), a 1000-mM nicotine concentration or higher chosen for experiments is unrealistically high. In this study of nicotine effects on human leukocytes, we observed dual effects of this alkaloid. Similarly to results of other researchers (Argentin & Cicchetti, 2004; Ginzkey et al., 2012; Kleinsasser et al., 2005; Sassen et al., 2005; Sudheer et al., 2007a,b; Wu et al., 2005), we observed genotoxic effects in the presence 1 and 10 mM of nicotine

(Figure 1C and D). In the comet assay, the tail length increases at relatively low damage levels and therefore is treated as the most informative (Collins et al., 2008). In some cases, significant dose-dependent DNA damages may appear, which is manifested by an increased amount of DNA in the tail, with no tail elongation and sometimes even tail contraction. We observed this situation in the presence of 10 mM of nicotine, and the best description of DNA damage level in such cases was provided by percent DNA in the tail and Olive tail moment parameters. Generally, percent DNA in the tail covers the widest range of damage, and it is linearly related to break frequency over most of this range (Collins et al., 2008; Lovell & Omori, 2008). However, in our study, we also found that the percent DNA in the tail, together with the head area and Olive tail moment, were significantly different in cells treated with 0.1 mM than in control cells. These results suggest that DNA in leukocytes was not only undamaged, but also even protected against natural damage detected in control cells. We suppose that relatively high Olive tail moment values in control cells were a result of spontaneous DNA damages (Kohn & Bohr, 2002) caused by, for example, oxidative damage from sudden exposure to the

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

326

R. Sobkowiak et al.

high concentration of O2 in the atmosphere, compared with blood (Collins et al., 2008) or damages generated in every experimental manipulation. However, these types of damages appeared in control, as well in nicotine-treated, samples. Nicotine, at concentrations ranging up to 6000 mM, was not genotoxic to CHO cells tested by SCE assay (Doolittle et al., 1995). DNA damages at nicotine concentrations of 1 and 10 mM were lower than in the control in our study of in vitro response of cell culture of C. elegans to nicotine (Sobkowiak & Lesicki, 2009). This was reflected in significantly lower Olive tail moment and tail length in comet assays for both concentrations. Similarly, Ginzkey et al. (2012), in the comet assay, observed lower DNA damage in isolated single cells of human nasal mucosa of some patients in the presence of 1 mM of nicotine than in the control, although this difference is not significant. It should be stressed that our results indicate a significantly lower value of DNA damage in cells treated with 0.1 mM of nicotine than control cells (Figure 1). Leukocytes are known to express the nicotinic acetylcholine receptor (nAChR) as a possible target for nicotine, and these results underscore the fact that nAChR plays an important role in nicotine-induced genotoxicity (Arias et al., 2009; Sobkowiak & Lesicki, 2011). Nicotinic receptors mediate responses in PC12 cells and probably in C. elegans cell culture, as in most in vitro systems utilizing transformed cells, but these responses require substantially higher concentrations than in vivo (Abreu-Villaca et al., 2005 and references cited therein). Abreu-Villaca et al. (2005) and Sobkowiak & Lesicki (2009) reported on significant effects elicited at nicotine concentrations approximately 10-fold higher than those in venous blood of smokers. People are continuously exposed to a variety of harmful (e.g. genotoxic) and beneficial (e.g. antioxidant) chemicals. The basal level of DNA damage in primary human (diploid) lymphocytes has been estimated to contain approximately 5000 different DNA damages detected by the simple alkaline comet assay (Møller, 2006). Probably immediately after isolation, leukocytes suffer oxidative damage from sudden exposure to the high concentration of O2 in the atmosphere, compared with blood (Collins et al., 2008). This process increases the damage to the DNA and emphasizes the protective effects of nicotine. We suppose that nicotine at low concentrations (approximately 0.1 mM) protects DNA from damage, which is proved by higher comet head area and lower value of percent DNA in the tail and Olive tail moment than in the control (Figure 1). The results suggesting opposite – genotoxic versus genoprotective – roles of nicotine are controversial. The differences between results obtained in different test systems can be attributed to different drug concentrations and treatment duration, as well as different endpoints of genotoxicity, and the repair capacities of the various cell types used can explain the discrepancies. Nicotine may act as an oxidative stressor or as an antioxidant. This suggests biphasic effects of nicotine, with the shift somewhere between 10 and 100 mM (Abreu-Villaca et al., 2005; Guan et al., 2003; Sobkowiak & Lesicki, 2009). Villablanca (1998) described bimodal, dose-dependent activities of nicotine in calf pulmonary endothelial cells (ECs). Lower concentrations (0.1–10 nM; i.e. lower than the

Drug Chem Toxicol, 2014; 37(3): 322–328

concentrations found in the blood of smokers, but at the level of exposure to nicotine from food) stimulated proliferation of ECs (up to 180%), whereas a higher concentration (100 mM) led to a decrease in DNA synthesis and apoptosis (Villablanca, 1998). It is plausible that nicotine treatment may play dual effects on oxidative stress (OS) and cell protection, in which the effects are dependent on differences in dosage of the drug used and its mechanism of action (Guan et al., 2003). Generally, high doses of nicotine may induce toxicity and stimulate OS, whereas reasonably low concentrations may act as an antioxidant and play an important protective role (Guan et al., 2003). The fact that only high doses of nicotine (1000 mM and over), but not a low dose (100 mM), can induce the decrease in glutathione content and increase in the level of lipid peroxidation markers in CHO cells and lymphocytes also provides evidence to show dose-differentiated effects of nicotine (Sudheer et al., 2007a,b; Yildiz et al., 1998). Although the exact mechanism of nicotine influence on DNA is still unknown, OS induced by nicotine seems to play an important role. It has been reported that nicotine disrupts the mitochondrial respiratory chain, leading to increased generation of superoxide anions and hydrogen peroxide (Gvozdjakova et al., 1992). High doses of nicotine (over 1000 mM) stimulate OS and increase the activity of cytochrome P 450 enzymes during intracellular metabolism of nicotine (Yildiz et al., 1998). Interestingly, nicotine in vitro has been found to have radical scavenging properties (Ferger et al., 1998). It has been suggested that nicotine may have a capacity to chelate Fe2þ to block the Fenton reaction (SotoOtero et al., 2002). Overall, smoking harms health and this statement contains the assumption that smoking leads to DNA damage resulting from the presence of a large number of carcinogens in tobacco smoke. However, Hoffmann & Speit (2005), under the experimental conditions of the comet assay used in their study, did not reveal – as it could be expected – any effect of cigarette smoking on the amount of DNA damage in peripheral blood cells. This negative finding is in agreement with their previous study (Speit et al., 2003) and the majority of reviewed human biomonitoring studies (Faust et al., 2004), which also failed to show an effect of smoking on DNA migration in the comet assay (Møller et al., 2000). Several other articles dealing with smoking effects in the comet assay reported discrepant results (for a review, see Faust et al., 2004).

Conclusions Many factors can contribute to the generation of conflicting results, but, in our opinion, a bimodal and partly protective effect of nicotine should be taken into account. The role of nicotine in reducing DNA damage by other components of tobacco smoke in human cells needs to be confirmed in future experiments.

Acknowledgements The authors thank Sylwia Ufnalska, an author’s editor, for help in improving the manuscript. She is not responsible for the final version of the article.

DOI: 10.3109/01480545.2013.851693

Declaration of interest The authors report no declarations of interest. This study was supported by funds from the Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University (Poznan´, Poland).

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

References Abreu-Villaca Y, Seidler FJ, Qiao D, et al. (2005). Modeling the developmental neurotoxicity of nicotine in vitro: cell acquisition, growth and viability in PC12 cells. Brain Res Dev Brain Res 154: 239–246. Arabi M. (2004). Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa. Andrologia 36:305–310. Argentin G, Cicchetti R. (2004). Genotoxic and antiapoptotic effect of nicotine on human gingival fibroblasts. Toxicol Sci 79:75–81. Arias HR, Richards VE, Ng D, et al. (2009). Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis. Int J Biochem Cell Biol 41:1441–1451. Attia SM. (2007a). Chromosomal composition of micronuclei in mouse bone marrow treated with rifampicin and nicotine, analyzed by multicolor fluorescence in situ hybridization with pancentromeric DNA probe. Toxicology 235:112–118. Attia SM. (2007b). The genotoxic and cytotoxic effects of nicotine in the mouse bone marrow. Mutat Res 632:29–36. Baron JA. (1996). Beneficial effects of nicotine and cigarette smoking: the real, the possible and the spurious. Br Med Bull 52:58–73. Benowitz NL, Hukkanen J, Jacob 3rd P. (2009). Nicotine chemistry, metabolism, kinetics and biomarkers. Handb Exp Pharmacol 192: 29–60. Collins AR. (2004). The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249–261. Collins AR, Oscoz AA, Brunborg G, et al. (2008). The comet assay: topical issues. Mutagenesis 23:143–151. DeMarini DM, Gudi R, Szkudlinska A, et al. (2008). Genotoxicity of 10 cigarette smoke condensates in four test systems: comparisons between assays and condensates. Mutat Res 650:15–29. Doolittle DJ, Winegar R, Lee CK, et al. (1995). The genotoxic potential of nicotine and its major metabolites. Mutat Res 344:95–102. Faust F, Kassie F, Knasmu¨ller S, et al. (2004). The use of the alkaline comet assay with lymphocytes in human biomonitoring studies. Mutat Res 566:209–229. Ferger B, Spratt C, Earl CD, et al. (1998). Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-induced neurotoxicity in vivo. Naunyn Schmiedebergs Arch Pharmacol 358:351–359. Ginzkey C, Kampfinger K, Friehs G, et al. (2009). Nicotine induces DNA damage in human salivary glands. Toxicol Lett 184:1–4. Ginzkey C, Stueber T, Friehs G, et al. (2012). Analysis of nicotineinduced DNA damage in cells of the human respiratory tract. Toxicol Lett 208:23–29. Gosselin RE, Smith RP, Hodge HC. (1988). Clinical toxicology of commercial products, 6th ed. Baltimore, MD: Williams & Wilkins. Guan ZZ, Yu WF, Nordberg A. (2003). Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells. Neurochem Int 43: 243–249. Gvozdjakova A, Kucharska J, Gvozdja´k J. (1992). Effect of smoking on the oxidative processes of cardiomyocytes. Cardiology 81:81–84. Hall J, Bre´sil H, Donato F, et al. (1993). Alkylation and oxidative-DNA damage repair activity in blood leukocytes of smokers and nonsmokers. Int J Cancer 54:728–733. Harris G, Holmes A, Sabovljev SA, et al. (1986). Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors. Int J Radiat Biol Relat Stud Phys Chem Med 50:685–694. Hartmann A, Elhajouji A, Kiskinis E, et al. (2001). Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test. Food Chem Toxicol 39: 843–858. Hartmann A, Schumacher M, Plappert-Helbig U, et al. (2004). Use of the alkaline in vivo comet assay for mechanistic genotoxicity investigations. Mutagenesis 19:51–59. Herman AI, Sofuoglu M. (2010). Comparison of available treatments for tobacco addiction. Curr Psychiatry Rep 12:433–440.

Effect of nicotine on human leukocytes

327

Hoffmann H, Speit G. (2005). Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test. Mutat Res 581:105–114. Hukkanen J, Jacob 3rd P, Benowitz NL. (2005). Metabolism and disposition kinetics of nicotine. Pharmacol Rev 57:79–115. IARC (International Agency for Research on Cancer). (2004). IARC monographs on the evaluation of carcinogenic risks to humans: tobacco smoke and involuntary smoking, vol. 83. Lyon, France: IARC Scientific Publications. IARC (International Agency for Research on Cancer). (2007). IARC monographs on the evaluation of carcinogenic risks to humans: smokeless tobacco and some tobacco-specific N-nitrosamines, vol. 89. Lyon, France: IARC Scientific Publications. Idris AM, Ibrahim SO, Vasstrand EN, et al. (1998). The Swedish snus and the Sudanese toombak: are they different? Oral Oncol 34:558–566 Jarvik ME. (1991). Beneficial effects of nicotine. Br J Addict 86: 571–575. Kleinsasser NH, Sassen AW, Semmler MP, et al. (2005). The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes. Toxicol Sci 86:309–317. Kohn KW, Bohr VA. (2002). Genomic instability and DNA repair. In: Alison M, ed. The cancer handbook. London: Nature Publishing Group, 87–106. Koleva II, van Beek TA, Soffers AE, et al. (2012). Alkaloids in the human food chain – natural occurrence and possible adverse effects. Mol Nutr Food Res 56:30–52. Konca K, Lankoff A, Banasik A, et al. (2003). A cross-platform public domain PC image-analysis program for the comet assay. Mutat Res 534:15–20. Lee E, Oh E, Lee J, et al. (2004). Use of the tail moment of the lymphocytes to evaluate DNA damage in human biomonitoring studies. Toxicol Sci 81:121–132. Leroy T, Van Hummelen P, Anard D, et al. (1996). Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis. J Toxicol Environ Health 47:409–422. Lovell DP, Omori T. (2008). Statistical issues in the use of the comet assay. Mutagenesis 23:171–182. Matta SG, Balfour DJ, Benowitz NL, et al. (2007). Guidelines on nicotine dose selection for in vivo research. Psychopharmacology (Berl) 190:269–319. Mizusaki S, Okamoto H, Akiyama A, Fukuhara Y. (1977). Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate. Mutat Res 48:319–325. Møller P. (2006). The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. Basic Clin Pharmacol Toxicol 98:336–345. Møller P, Knudsen LE, Loft S, Wallin H. (2000). The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiol Biomarkers Prev 9:1005–1015. Muthukumaran S, Sudheer AR, Nalini N, Menon VP. (2008). Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes. Redox Rep 13: 217–224. Olive PL, Banath JP. (2006). The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29. Olive PL, Durand RE, Le Riche J, et al. (1993). Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers. Cancer Res 53:733–736. Pogocki D, Ruman T, Danilczuk M, et al. (2007). Application of nicotine enantiomers, derivatives and analogues in therapy of neurodegenerative disorders. Eur J Pharmacol 563:18–39. Sassen AW, Richter E, Semmler MP, et al. (2005). Genotoxicity of nicotine in mini-organ cultures of human upper aerodigestive tract epithelia. Toxicol Sci 88:134–141. Singh NP. (2000). Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res 455:111–127. Singh NP, McCoy MT, Tice RR, Schneider EL. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. Sobkowiak R, Lesicki A. (2009). Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay. Drug Chem Toxicol 32:252–257.

328

R. Sobkowiak et al.

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.

Sobkowiak R, Lesicki A. (2011). Cell signaling pathways activated by nicotine. Adv Cell Biol 38:581–596. Soto-Otero R, Me´ndez-Alvarez E, Hermida-Ameijeiras A, et al. (2002). Effects of ()-nicotine and ()-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson’s disease. Biochem Pharmacol 64:125–135. Speit G, Witton-Davies T, Heepchantree W, et al. (2003). Investigations on the effect of cigarette smoking in the comet assay. Mutat Res 542: 33–42. Sudheer AR, Muthukumaran S, Devipriya N, Menon VP. (2007a). Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: with the comparison of N-acetylcysteine. Toxicology 230: 11–21. Sudheer AR, Muthukumaran S, Kalpana C, et al. (2007b). Protective effect of ferulic acid on nicotine-induced DNA damage and cellular

Drug Chem Toxicol, 2014; 37(3): 322–328

changes in cultured rat peripheral blood lymphocytes: a comparison with N-acetylcysteine. Toxicol In Vitro 21:576–585. Swan GE, Lessov-Schlaggar CN. (2009). Tobacco addiction and pharmacogenetics of nicotine metabolism. J Neurogenet 23: 262–271. Turner DR, Griffith VC, Morley AA. (1982). Ageing in vivo does not alter the kinetics of DNA strand break repair. Mech Ageing Dev 19: 325–331. Villablanca AC. (1998). Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Physiol 84:2089–2098. Wu HJ, Chi CW, Liu TY. (2005). Effects of pH on nicotine-induced DNA damage and oxidative stress. J Toxicol Environ Health A 68: 1511–1523. Yildiz D, Ercal N, Armstrong DW. (1998). Nicotine enantiomers and oxidative stress. Toxicology 130:155–165.

In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay.

The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head a...
338KB Sizes 0 Downloads 0 Views