Genotoxicity of Nicotine in Mini-Organ Cultures of Human Upper Aerodigestive Tract Epithelia

TOXICOLOGICAL SCIENCES 88(1), 134–141 (2005) doi:10.1093/toxsci/kfi297 Advance Access publication August 24, 2005 Genotoxicity of Nicotine in Mini-Organ Cultures of Human Upper Aerodigestive Tract Epithelia Andrea W. Sassen,* Elmar Richter,† Marzell P. Semmler,* Ulrich A. Harréus,‡ Fernando Gamarra,§ and Norbert H. Kleinsasser*,1 *Otolaryngology-Head and Neck Surgery, University of Regensburg, Germany; †Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University Munich, Germany; ‡Otolaryngology-Head and Neck Surgery, Ludwig-Maximilians University Munich, Germany; and §Internal Medicine-Pneumology, Ludwig-Maximilians University Munich, Germany Received May 2, 2005; accepted August 18, 2005 The direct role of nicotine in tobacco carcinogenesis is still controversial. Recently, DNA damage by nicotine has been demonstrated in isolated human tonsillar tissue cells. Presently, these effects were investigated using mini-organ cultures (MOC) of human nasal epithelia. Intact MOC were repeatedly exposed to 2 and 4 mM nicotine for 1 h on culture days 7, 9, and 11. N-Methyl-N#-nitro-N-nitrosoguanidine (MNNG) served as a positive control. DNA damage was examined by Comet assay either directly after exposure or following a 24-h recovery period. Cell viability was not reduced by any treatment. On day 7, 1 h exposure to 2 and 4 mM nicotine caused a significant dose-dependent 3.3and 5.6-fold increase in DNA damage compared to solvent controls. Although there was no evidence of significant repair within 24 h recovery, DNA damage was not further increased by nicotine on days 9 and 11. After double and triple exposure to 4 mM nicotine a significant reduction in DNA damage following 24 h recovery was observed. In contrast, treatment with MNNG resulted in a highly significant and cumulative increase in DNA migration up to 110-fold compared to controls. During recovery periods, MNNG-induced DNA damage was significantly repaired, leading to a 1.5- to 1.8-fold reduction in DNA migration within 24 h. These results confirm genotoxic effects of nicotine on human nasal epithelia. Further studies are needed to explain the lack of cumulative DNA-damaging effects of nicotine and the absence of significant DNA repair. These studies should include a battery of assays with multiple end points. Key Words: genotoxicity; nicotine; Comet assay; mini-organ cultures; human epithelia. Tobacco smoking is the single most important risk factor for cancer and is responsible for about one third of all cancer deaths (John and Hanke, 2002). In Germany, lung cancer is by 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-Nasen-Ohrenklinik und Poliklinik, Universität Regensburg, Franz-Josef-Straub-Allee 11, D-93053 Regensburg, Germany. Fax: þþ49–941–944–9431. E-mail: norbert.kleinsasser @klinik.uni-regensburg.de. far the most common cause of cancer death in men and ranks third in women (Levi et al., 2004). It is estimated that up to 90% and 60% of male and female lung cancer, respectively, could be prevented by avoiding cigarette smoking (Becker, 2001). Involuntary smoking, e.g., by exposure to secondhand or environmental tobacco smoke, is a risk factor in lung cancer (Boffetta et al., 1998) as well and has been classified as carcinogenic to humans by the IARC (2004). These data emphasize the importance of prevention of smoking initiation and the need for new methods to aid in smoking cessation. However, smoking is a neuronal nicotinic acetylcholine (nACh) receptor-mediated addiction (Dajas-Bailador and Wonnacott, 2004), with nicotine being responsible for the addictive potential of tobacco smoke. The toxicity of nicotine is no longer controversial (Chang et al., 2002; Chen et al., 2004; Cooke and Bitterman, 2004), but its possible contribution to tobaccorelated cancers is less well established. Metabolism of nicotine produces reactive intermediates capable of binding to proteins and DNA (Hukkanen et al., 2005). Up to 1% of 5-3H-nicotine metabolized by human liver microsomes in vitro was covalently bound to proteins (Shigenaga et al., 1987). Prolonged binding of nicotine-derived radioactivity was observed in bronchial mucosa and the urinary bladder wall of rats (Szüts et al., 1978). Using highly sensitive accelerator mass spectrometry, protein and DNA binding was demonstrated in mouse liver both in vivo and in vitro (Li et al., 1996; Sun et al., 2000; Wu et al., 1997). Pretreatment of mice with ascorbic acid, curcuma, grape seed, green tea, vitamin E, or garlic reduced this binding by up to 50% (Cheng et al., 2003). On the other hand, nicotine inhibits metabolic activation of the strongly carcinogenic tobacco-specific nitrosamines (TSNA), N#-nitrosonornicotine (NNN), 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL) arising from nicotine nitrosation (Charest et al., 1989; Lee et al., 1996; Murphy and Heiblum, 1990; Richter and Tricker, 1994, 2002; Schuller et al., 1991; Schulze et al., 1998). Therefore, the net effect of cigarette Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: 135 NICOTINE GENOTOXICITY IN MINI-ORGAN CULTURES smoke on mutagenicity might be quite different from that of nicotine alone. Results concerning the mutagenicity of nicotine in several test systems are controversial. Using the Ames test with several strains of Salmonella typhimurium and the sister chromatid exchange (SCE) test in Chinese hamster ovary cells (CHO), Doolittle et al. (1995) concluded that neither nicotine nor its major metabolites cotinine, nicotine-N#-oxide, cotinine-Noxide, or trans-3#-hydroxycotinine caused genotoxic effects with or without metabolic activation. In contrast, other groups found a modest increase in SCE in CHO (Riebe and Westphal, 1983; Trivedi et al., 1990) and repairable DNA damage using a test system with Escherichia coli polAþ/polA
(Riebe et al., 1982) at high concentrations of nicotine. Recently, nicotine has been shown to increase the frequency of micronuclei in human gingival fibroblasts (Argentin and Cicchetti, 2004) and DNA strand breaks in human spermatozoa (Arabi, 2004). Nicotine may also enhance tumor development by nongenotoxic mechanisms. Nicotine stimulates angioneogenesis in atheromatous plaques and in tumors by an endogenous nicotinic cholinergic pathway in endothelial cells (Cooke and Bitterman, 2004; Heeschen et al., 2001). The growth of human cancer cell lines with biochemical features of neuroendocrine lung cells is strongly stimulated by nicotine (Schuller, 1994). A direct role of nicotine in the growth of gastric tumors and revascularization was shown by Shin et al. (2004). Argentin and Cicchetti (2004) demonstrated genotoxic and antiapoptot (...truncated)