* Department of OtolaryngologyHead and Neck Surgery, University of Regensburg, D-93053 Regensburg, Germany; Department of OtolaryngologyHead and Neck Surgery, Ludwig-Maximilians University Munich, D-80337 Munich, Germany;
Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University Munich, D-80337 Munich, Germany
1 To whom correspondence should be addressed at Hals-Nasen-Ohrenklinik und Poliklinik, Universität Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany. Fax: +49-941-944-9431. E-mail: norbert.kleinsasser{at}klinik.uni-regensburg.de.
Received January 27, 2005; accepted April 25, 2005
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ABSTRACT |
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Key Words: genotoxicity; nicotine; Comet assay; human target cells.
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INTRODUCTION |
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This study focuses on a possible direct genotoxic effect of nicotine on cells of the lymphatic tissue of the palatine tonsils (tonsillar cells), a target of tobacco carcinogenesis in the human upper aerodigestive tract. In addition, DNA damage in lymphocytes of the peripheral blood, a well-established surrogate marker of systemic carcinogenic effects, was tested in the same donors (study A). To exclude artifactitious DNA damage, the genotoxic effect of nicotine was tested with two highly pure nicotine batches from two different commercial sources (study B) and under pH-controlled conditions, eliminating alkaline effects at high nicotine concentrations (study C).
The assessment of nicotine as a risk factor for carcinogenesis is of special interest. The health benefit of filter cigarettes and so-called light cigarettes with reduced fractions of tar and nicotine is controversially discussed (Harris et al., 2004; Hoffmann et al., 2001
; Lee and Sanders, 2004
; National Cancer Institute, 2001
). Thus, the cancer risk in smokers of light cigarettes could in fact be much higher than expected because of the undetermined possible genotoxic effects of nicotine.
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MATERIALS AND METHODS |
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Exposure.
Cell suspensions with 5 x 104 lymphocytes or tonsillar cells each were incubated for 60 min in an initial step with (-)-nicotine free base of >99% purity (N 3876, Sigma) and dissolved in PBS at concentrations of 0, 0.125, 0.25, 0.5, 1, 2, and 4 mM (study A). Concentrations of 0.5, 1, 2, and 4 mM of (-)-nicotine from either Sigma or TRC (N 412450, Toronto Research Chemicals, North York, ON, Canada) were used in study B. In study C, concentrations of 1, 2, and 4 mM nicotine (Sigma) were used. In half of the incubation medium the pH was adjusted to neutral (pH 7.2) with hydrochloric acid (Merck, Darmstadt, Germany), whereas in the other half the pH level was only monitored (study C). Nicotine doses were selected according to pilot studies and were held as low as possible to approach concentrations similar to those found in plasma and saliva of smokers (see Discussion). Additional experiments were performed with higher doses, allowing for viability results over 70%.
The directly alkylating mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.02 mM, Sigma) was used as a positive control, and the solvent PBS (1 or 10 %) served as a negative control.
Alkaline single-cell microgel electrophoresis (Comet) assay.
After incubation, cell viability was examined again using trypan blue staining. Once viabilities >80% were obtained, the cells were subjected to the Comet assay (Kleinsasser et al., 2003b). In brief, after resuspending the cells in 0.7% low melting agarose (Cambrex, Rockland, ME) and applying them to coated microscope slides, cell and core membranes were dissolved for at least 90 min in a lysis buffer (10% DMSO, 1% Triton-X in alkaline lysis solution: 2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA; pH 10). The slides were placed into a horizontal gel electrophoresis chamber (Renner, Dannstadt, Germany) and covered with alkaline buffer solution containing NaOH (10 mM) and Na2EDTA (200 mM), pH > 13. A 20-min "unwinding" period was followed by electrophoresis at 25 V and 300 mA for 20 min. Slides were neutralized (Trizma base, pH 7.5, Merck) and stained with ethidium bromide (Sigma). A DMLB microscope (Leica, Heerbrugg, Switzerland) equipped with an adapted CCD camera (Cohu Inc., San Diego, CA) was used to investigate the slides. The software Komet 4.0 (Kinetic Imaging, Liverpool, UK) was applied.
To quantify the induced DNA damage, 100 cells per probe were examined for the Olive tail moment (OTM) reflecting the percentage of DNA in the tail of the comet multiplied by the median migration distance (Olive et al., 1993), the percentage of DNA in the tail (DT), and the tail length (TL) (Lee et al., 2004
).
Statistics.
Evaluation was based on mean OTM values of each individual incubation using Prism 4 (GraphPad Software, Inc., San Diego, CA). Concentration-dependent differences in DNA migration were analyzed by repeated measures analysis of variance (ANOVA) with post test for linear trend and Bonferroni's multiple comparison test for differences between untreated control cells and nicotine-treated cells. After testing for normal distribution, a paired Student's t-test was applied to compare DNA migration in tonsillar cells versus lymphocytes (study A), as well as for evaluation of possible effects with respect to the source of nicotine (study B) and different environmental pH values (study C).
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RESULTS |
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In study A, 1 h of incubation with nicotine induced a significant concentration-dependent increase in DNA migration in the Comet assay in tonsillar cells (up to 3.8-fold, p < 0.0001), as well as in peripheral lymphocytes from the same donors (up to 3.2-fold, p < 0.0001) without affecting cell viability (Fig. 1, Table 2). Compared to the negative control, the effects in tonsillar cells and lymphocytes reached significance at 0.5 mM nicotine (p < 0.05). At all nicotine concentrations there were no significant differences in DNA damage between either cell type as assessed by OTM. The mean values of the positive control for tonsillar cells were 71.8 ± 20.7 (OTM), 67.3 ± 9.1 (DT), and 170.1 ± 33.2 (TL); and for lymphocytes, 76.0 ± 18.1 (OTM), 71.7 ± 11.9 (DT), and 179.5 ± 25.5 (TL).
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DISCUSSION |
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Among the more than 4000 known components of cigarette mainstream smoke, up to 81 compounds are IARC classified carcinogens (Smith et al., 2003). Although both active and passive smoking, as well as use of smokeless tobacco products, have been classified by the IARC as carcinogenic for humans, nicotine as a major component of tobacco and tobacco smoke has never been classified (http://www-cie.iarc.fr/monoeval/grlist.html). Nicotine concentrations in smokers reach up to 100 ng/ml (=0.0006 mM) in plasma and up to 4000 ng/ml (= 0.025 mM) in saliva (Benowitz, 1990
; Schneider et al., 2001
; Teneggi et al., 2002
). In the present study, the lowest concentration of nicotine eliciting significant DNA damage in tonsillar cells was observed after 1 h of incubation with 0.5 mM nicotine (Fig. 1). This concentration is only about 20-fold higher than saliva concentrations, to which a smoker is normally exposed for a much longer time. Therefore, nicotine could express significant direct genotoxic effects of carcinogenesis in human target cells. This possibility is consistent with recent data for human gingival fibroblasts (Argentin and Cicchetti, 2004
) and spermatozoa (Arabi, 2004
). A similar effect was shown previously for myosmine, a minor tobacco alkaloid that also occurs in a variety of foods, e.g., cereals, nuts, cocoa, and dairy products (Kleinsasser et al., 2003b
; Tyroller et al., 2002
; Zwickenpflug et al., 1998
). This effect suggests a possible additional mechanism of induced carcinogenesis by these alkaloids in the upper aerodigestive tract. Other pathways of carcinogenesis for nicotine are non-neuronal acetylcholine receptor (nAchR)-mediated cell growth and suppression of apoptosis (Minna, 2003
; Schuller et al., 2003
), nAchR-mediated angioneogenesis in tumors (Natori et al., 2003
; Cooke and Bitterman, 2004
), epidermal growth factormediated tumor enhancement (Ye et al., 2004
), and cyclooxygenase 2dependent stimulation of gastric tumor growth (Wetscher et al., 1995
). The mechanisms above all belong to the tumor promotion of carcinogenesis, whereas genotoxicity is a possible step in tumor initiation. Other steps of initiation include enhanced oxidative stress and free radical production (Crowley-Weber et al., 2003
; Wetscher et al., 1995
). Controversial results have been published in other classical in vitro tests for genotoxicity, showing either slight positive effects of nicotine e.g., on sister chromatid exchange in mammalian cells (Trivedi et al., 1990
)no nicotine effects (Doolittle et al., 1995
), or even protective effects of nicotine against tobacco smoke exposure and established tobacco carcinogens (Lee et al., 1996
).
Nicotine was equally genotoxic to lymphocytes and tonsillar cells. This finding is in contrast to results obtained for other genotoxic compounds such as vanadium and, to a lesser extent, phthalates and myosmine, which were more genotoxic to lymphocytes than to mucosal cells (Kleinsasser et al., 2000a, 2003a
, 2003b). One reason for this discrepancy could be the closer similarity of lymphocytes to tonsillar cells from lymphatic tissue as compared to cells from mucosal epithelium. Further studies with a higher number of probes will show more clearly whether a lack of difference in mutagen sensitivity between tonsillar cells and lymphocytes will prevail.
Nicotine impurities such as nornicotine could have a significant influence on nicotine effects (Carmella et al., 1997). However, DNA migration in the assay did not differ significantly with nicotine of high purity from two different suppliers (Fig. 2). Therefore the possibility that the nicotine effect could be due to impurities is minimal.
An analysis of a possible influence of smoking status on DNA migration in the negative controls showed no significant differences. This finding is in line with results of Hoffmann and Speit (2005), showing no differences in peripheral blood cells from heavy smokers and nonsmokers, either in the comet assay or in the micronucleus test. The apparent discrepancy between in vitro genotoxic effects of nicotine and no observable effects on DNA damage in vivo could have several sources. The comet assay as well as the micronucleus test might just be not sensitive enough to show a slight but still toxicologically relevant DNA damage by nicotine in vivo. Rapid in vivo repair of DNA damage by nicotine could also lower the chances of detecting genotoxic effects. In this context, the principle established by Druckrey and others (Druckrey et al., 1963
; Peto et al., 1991
), that no threshold values exist for genotoxic compounds, should be taken into account. Another possibility is that nicotine effects might be attenuated by other smoke constituents. This would not be the case in nicotine replacement therapy. To our knowledge, this aspect of nicotine safety has not been addressed in the literature so far.
The increasingly alkaline conditions resulting from higher nicotine concentrations did not have any significant effect on DNA damage by the alkaloid nicotine (Fig. 3). This was proven by adjusting the pH to about 7.2 at the start of the incubation. It will be interesting to look at the effect of pH levels well below neutral, which are commonly found in the gastric mucosa and with gastroesophageal reflux in the esophagus, pharynx, and larynx, taking into account the nitrosative stress from dietary nitrate (Iijima et al., 2003) and the excretion of high concentrations of nicotine with saliva (Boswell et al., 2000
). These investigations will be performed with the aid of mini-organ cultures of epithelia of the upper aerodigestive tract combined with the Comet assay (Kleinsasser et al., 2001
, 2004
). This technique allows repetitive/chronic exposure under varying pH levels, simulating the in vivo situation in active and passive smokers. Further studies should be performed using saliva of smokers and nonsmokers (Reznick et al., 2004
) and by co-incubation of nicotine with cigarette smoke condensate, established tobacco carcinogens as well as ethanol, a commonly found co-carcinogen in the pharynx. Finally, the possible role of metabolic activation for the ability of nicotine to induce DNA damage should be investigated.
In conclusion, nicotine expresses significant genotoxic effects in vitro in human target cells of upper aerodigestive tract carcinogenesis as well as in lymphocytes. The results suggest a direct tumor-initiating potency of this alkaloid.
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NOTES |
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ACKNOWLEDGMENTS |
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