The Tobacco Alkaloid Nicotine Demonstrates Genotoxicity in Human Tonsillar Tissue and Lymphocytes

TOXICOLOGICAL SCIENCES 86(2), 309–317 (2005) doi:10.1093/toxsci/kfi186 Advance Access publication April 27, 2005 The Tobacco Alkaloid Nicotine Demonstrates Genotoxicity in Human Tonsillar Tissue and Lymphocytes Norbert H. Kleinsasser,*,1 Andrea W. Sassen,* Marzell P. Semmler,* Ulrich A. Harréus,† Anna-Katharina Licht,* and Elmar Richter‡ *Department of Otolaryngology–Head and Neck Surgery, University of Regensburg, D-93053 Regensburg, Germany; †Department of Otolaryngology–Head 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 Received January 27, 2005; accepted April 25, 2005 Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNAdamaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with nonadjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis. Key Words: genotoxicity; nicotine; Comet assay; human target cells. The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration. 1 To whom correspondence should be addressed at Hals-NasenOhrenklinik und Poliklinik, Universität Regensburg, Franz-Josef-Straub-Allee 11, D-93053 Regensburg, Germany. Fax: þ49-941-944-9431. E-mail: . Tobacco smoke is composed of a great variety of constituents. Some of these, including polycyclic hydrocarbons, nitrosamines, and aromatic amines, are known to contribute to the carcinogenic risk of tobacco smoke (Hoffmann and Hoffmann, 1997; Smith et al., 2003). Nicotine, the major tobacco alkaloid, however, is accused of the addictive potential of smoking, being mediated by neuronal nicotinic acetylcholine receptors in the central nervous system (Dajas-Bailador and Wonnacott, 2004). With regard to possible carcinogenic risk, nicotine and its demethylation product nornicotine have been discussed mainly for reasons of their nitrosation products 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or N#nitrosonornicotine (NNN) (Hecht, 1998). Recently, direct genotoxic effects of nicotine have been shown in human gingival fibroblasts (Argentin and Cicchetti, 2004) and spermatozoa (Arabi, 2004). Furthermore, nicotine may also stimulate tumor development by non-genotoxic mechanisms such as angioneogenesis (Heeschen et al., 2001; Cooke and Bitterman, 2004), growth stimulation (Shin et al., 2004), antiapoptotic effects (Argentin and Cicchetti, 2004), and receptor-regulated cellular growth (Schuller, 1994). 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 Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: 310 KLEINSASSER ET AL. light cigarettes could in fact be much higher than expected because of the undetermined possible genotoxic effects of nicotine. MATERIALS AND METHODS Cell sources and study groups. In surgery of the oropharynx, tonsillar tissue was harvested from 15 patients (22–47 years, 35.3 on average; 6 male, 9 female; 10 smokers, 5 nonsmokers; for further details see Table 1). This type of surgery is done after recurrent or chronic inflammation of the tonsils in an interval without signs of acute disease. Ten patients each were part of studies A and B, and cells from 5 patients were taken for study C. Study A represents the investigation of dose-dependent effects of nicotine on DNA migration. Study B delineates the comparison between nicotine of two different suppliers. Study C involves the evaluation of a possible impact of the pH of the incubation medium. For the benefit of the patient, only as much tissue as necessary was resected. Whole blood samples were obtained by venous puncture from each patient. The study was approved by the ethics commission of the University of Regensburg Medical Faculty, and all patients signed a written informed consent form. Cell separation. Heparinized blood samples and biopsies stored in MEMJoklik (without L-glutamine and NaHCO3; Linaris, Bettingen, Germany), were transferred to the laboratory immediately after collection. Lymphocytes from whole blood and tonsillar cells were isolated according to the procedure of Kleinsasser et al. (2003a). In bri (...truncated)