Genotoxicity of Nicotine in Mini-Organ Cultures of Human Upper Aerodigestive Tract Epithelia
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《毒物学科学杂志》
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
Internal Medicine-Pneumology, Ludwig-Maximilians University Munich, Germany
ABSTRACT
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 3.3- and 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.
INTRODUCTION
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 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 tobacco-related 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 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-N-oxide, 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 polA+/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 antiapoptotic effects of nicotine in human gingival fibroblasts.
In a prior study, the genotoxic potential of nicotine was demonstrated in human peripheral lymphocytes and lymphatic tissue of the palatine tonsils. Starting at 0.5 mM, nicotine showed significant enhancement of DNA migration in the Comet assay (Kleinsasser et al., 2005a). The present study focuses on mini-organ cultures (MOC), which were first described by Steinsvg et al. (1991) and further developed for upper aerodigestive tract epithelia by Kleinsasser et al. (2001, 2004). Being fully covered with partially ciliated epithelium, the MOC are closer to the in vivo situation than isolated cells. Whether or not possible genotoxic effects of nicotine are dose dependent, cumulative, and repaired sufficiently after repetitive exposure should be elucidated in MOC with the aid of the Comet assay, a sensitive short-term genetic toxicity test detecting strand breaks, alkali-labile sites, and incomplete DNA repair in human mucosa cells, thereby indicating a possible step in tumor initiation (Tice et al., 2000).
MATERIALS AND METHODS
Preparation of mini-organ cultures.
During surgery of the nasal air passage, human mucosa specimens from the lower ridge of the inferior nasal turbinate of 14 male patients were harvested (35.3 years on average). All tissue donors consumed less than 60 g alcohol per day and were healthy in other respects (Table 1). The mucosa was resected to improve nasal breathing and to diminish the patient's snoring. The study was approved by the Ethics Commission of the University of Regensburg Medical Faculty, and all participants provided written informed consent.
Fresh specimens were immediately transported to the laboratory and dissected into cubes of about 1 mm3 restricted only to mucosa and excluding deeper layers. Connective tissue and bony structures were resected. This preparation technique ensures that the cube contained respiratory epithelium with cilia, goblet cells, and mucosal gland cells, exclusively. The cubes were washed three times in Airway Epithelial Cell Growth Medium (AECGM; PromoCell, Heidelberg, Germany) and transferred into 24-well plates, one fragment per well. Thus the sample extent decided on the number of possible experimental days. Adhesion to the dish surface was prevented by coating the wells with 0.75% Agar Noble (BD Becton Dickinson, Heidelberg, Germany) dissolved in Dulbecco's modified eagle medium (DMEM; Gibco-BRL, Eggenstein, Germany) containing 10% fetal calf serum (FCS), nonessential amino acids, penicillin, and streptomycin (Biochrom, Berlin, Germany). The mini-organ cultures (MOC) were incubated in 250 μl AECGM at 37°C, 5% CO2, and 95% relative humidity. The medium was exchanged every other day. Until the seventh day of culture the mucosa fragments were completely coated with partially ciliated epithelium.
Exposure.
The experimental protocol is outlined in Figure 1. Epithelization of the surface area of the MOC was allowed to occur without experimental disruption up to the seventh day. On days 7, 9 and 11, the cultured MOC were repeatedly exposed to nicotine (MP Biomedicals, Eschwege, Germany) for 60 min. According to results of preliminary pilot studies on single cells of the inferior nasal turbinate (Sassen et al., 2003), nicotine dissolved in phosphate buffered saline (PBS) (Sigma-Aldrich, Taufkirchen, Germany) was added at 0, 2, and 4 mM. Subsequently, the Comet assay was performed on the exposed MOC. Recovery from genotoxic effects was controlled in cubes placed onto fresh plates after nicotine exposure on days 8, 10, and 12 and incubated for 24 h in AECGM.
The directly alkylating mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.02 mM, 60 min; Sigma-Aldrich) was used as a positive control to point out technical malfunctions, e.g., failures during electrophoresis.
Cell separation.
The samples were digested enzymatically by treatment with collagenase P (1 mg/ml), hyaluronidase from bovine testes (1 mg/ml) (Roche, Mannheim, Germany), and pronase E Type XIV from Streptomyces griseus (5 mg/ml) (Sigma-Aldrich), dissolved in AECGM, for 45 min at 37°C in a shaking water bath (Pool-Zobel et al., 1994). Following digestion, the reaction tubes were vortexed and residual connective tissue was removed. The tubes were centrifuged and the supernatant discarded. The enzymes were neutralized by fetal bovine serum and the cells subsequently washed in PBS.
Alkaline single cell microgel electrophoresis (Comet) assay.
After examining the viability of the cells using trypan blue staining, the cells were subjected to the Comet assay (e.g., Tice et al., 2000; Kleinsasser et al., 2001, 2003, 2005a). 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) with pH > 13. A 20 min "unwinding" period was followed by an 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-Aldrich). A DMLB microscope (Leica, Heerbrugg, Switzerland) equipped with an adapted CCD camera (Cohu Inc., San Diego, CA) was used to examine the slides. The software Komet 4.0 (Kinetic Imaging, Liverpool, UK) was applied for planimetric evaluation of fluorescence intensity.
To quantify the level of induced DNA damage, 80 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 tail (DT), and the tail length (TL) (Lee et al., 2004).
Statistics.
Evaluation was based on mean OTM values of individual MOC from patient 1–14. Missing data was not available because of technical conditions, e.g., insufficient sample material. Statistical analyses using the Wilcoxon Test and ANOVA for repeated measures were performed using the SPSSTM 10.0 program (SPSS Inc., Chicago, IL, USA).
RESULTS
Ciliary beats were not restricted by prolonged culture. Cell viability tested by trypan blue exclusion remained unchanged between 90 and 99%. Neither parameter was affected by single, double, or triple exposure to either 2 and 4 mM nicotine or 0.02 mM MNNG as the positive control.
Individual results for all three parameters of DNA damage, OTM, DT, and TL, together with mean and standard deviation are shown in Tables 2–4. Box plots of OTM values are shown for comparison of DNA damage in controls, nicotine-, and MNNG-treated MOC in Figure 2. In untreated MOC, DNA damage remained low between culture days 7 and 12.
Treatment with the direct mutagen MNNG for 1 h on day 7 (Fig. 2) resulted in a highly significant increase in DNA damage compared to controls (52-fold, p < 0.001). During the following 24 h recovery period, the cells showed significant DNA repair (p < 0.01) resulting in a nearly two-fold reduction in DNA damage. After the second and third treatment with MNNG, OTM increased further (65-fold and 110-fold, p < 0.001) as compared to the controls. ANOVA for repeated analysis for days 7, 9, and 11 confirmed highly significant differences (p < 0.001). On days 10 and 12 MNNG-exposed MOC again showed significant recovery from DNA damage (p < 0.01).
A single 1-h treatment of MOC with 2 and 4 mM nicotine on day 7 resulted in a dose-dependent increase in OTM values (3.9- and 5.6-fold, p < 0.001). A second treatment of the same MOC on days 9 and 11 did not further increase DNA damage at either nicotine concentration. At 2 mM, no significant recovery from DNA damage was observed in MOC 24 h after either single, double, or triple 1-h exposures. After the high dose of 4 mM nicotine, a slight but significant recovery from DNA damage was observed on days 10 and 12, 24 h after the second (1.3-fold reduction, p < 0.05) and third (1.5-fold reduction, p < 0.05) 1-h treatment, respectively. Throughout days 7 to 12, nicotine-treated MOC had significantly higher dose-dependent OTM values compared to the controls (p < 0.01).
Comparing the negative controls of smokers with a history of 4.5–55 package years (n = 7; patient 2, 4, 6, 7, 12–14) and nonsmokers or smokers with a history of less than 2 package years (n = 7; patient 1, 3, 5, 8–11), the Mann-Whitney-U-test showed no differences (p = 0.805).
DISCUSSION
In the present study, genotoxic effects of nicotine were investigated with the aid of a three-dimensional mini-organ culture (MOC) of human upper aerodigestive tract epithelia. The integrity of cells with their intercellular organ-specific formation allows a better simulation of the in vivo situation than assaying single cells (Kleinsasser et al., 2001, 2004; Steinsvg et al., 1991). Repetitive exposures to nicotine, followed by recovery phases, give a more precise depiction of the genotoxic consequences of nicotine. Viability of the cultures did not change within the experimental period and was not affected by nicotine treatment. In the negative control, DNA migration did not increase with prolonged incubation in PBS.
Nicotine showed a dose-dependent increase in DNA fragmentation in MOC, affirming recent results in lymphatic tissue of the palatine tonsils and peripheral lymphocytes (Kleinsasser et al., 2005a). In these cells, nicotine induced a dose-dependent increase in DNA migration by 0.125 to 4 mM, with statistical differences from controls starting at 0.5 mM. Genotoxic effects due to artifactitious conditions, such as increasing pH with increasing nicotine concentrations due to its alkaline properties, and metabolite formation during storage were excluded. There were also no relevant differences in DNA migration when using nicotine from two different commercial sources (Kleinsasser et al., 2005a). Significant direct genotoxic effects have also been shown for human gingival fibroblasts (Argentin and Cicchetti, 2004) and spermatozoa (Arabi, 2004). To what extent this nicotine-derived DNA damage is triggered by enhanced oxidative stress and free radical production (Crowley-Weber et al., 2003; Wetscher et al., 1995) has to be clarified in further experiments.
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 (Hukkanen et al., 2005; Schneider et al.; 2001; Teneggi et al., 2002). The lowest concentration in the present study, 2 mM nicotine, elicited significant DNA damage in mucosa cells of the nose after 1 h incubation (Fig. 2). This concentration is about 80-fold higher than saliva concentrations. However, dose-dependency of DNA damage by nicotine in MOC was not studied in the present experiments. For carcinogenic substances acting through direct genotoxic mechanisms, no threshold exists. Especially after prolonged exposure in habitual tobacco users, lower concentrations of nicotine may lead to irreversible changes in genetic material. The net genotoxic effect of nicotine after exposure to a complex mixture of compounds in cigarette smoke cannot be predicted from single exposure experiments (Lee et al., 1996).
No cumulative effects of 2 and 4 mM nicotine were observed after double or triple exposure (Fig. 2). These findings need further evaluation by additional tests. However, the results proved to be quite constant during the present studies. Within 24 h, recovery from nicotine exposure, as seen by a decrease in DNA migration as an indicator of DNA repair, was very weak, reaching significance only after double and triple exposure to the higher nicotine concentration of 4 mM. This is in sharp contrast to the results achieved with the directly alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine MNNG, which was used as a positive control to detect technical failures (Fig. 2). Repetitive exposure to MNNG led to cumulative DNA damage, which was repaired to a significant extent during the recovery phases. A similar two-fold reduction in DNA damage by MNNG within 24 h was reported to occur in V79 cells (Labaj et al., 2003). The obviously different nature of DNA damage caused by nicotine as compared to MNNG requires further advanced investigations with prolonged exposure mimicking the human in vivo situation. For such studies, the MOC model may offer certain advantages. As with nicotine (Kleinsasser et al., 2005a), genotoxic effects detected with the Comet assay in single cells were confirmed in cells from MOC after 7 days of culture for benzo[a]pyrene-7,8-diol-9,10-epoxide and mono(2-ethylhexyl)phthalate (Kleinsasser et al., 2004). The capacity for repair of DNA damage observed with MNNG in the present study, as well as the metabolism of foreign compounds (Teissier et al., 1998), is an indicator of the retained metabolic competence in MOC. This was also demonstrated with the aid of flow cytometry, detecting no decrease in the cytochrome P450 2A6, the most important enzyme of nicotine metabolism (Kleinsasser et al., manuscript in preparation).
No significant differences were found concerning the possible influence of smoking status on DNA migration in untreated controls. This was not unexpected, in view of the lack of differences in the Comet assay between smokers and nonsmokers in our previous study in isolated lymphocytes and cells from lymphatic tissue of the palatine tonsils (Kleinsasser et al., 2005a). These results are in line with Hoffmann and Speit (2005), showing no differences between heavy and nonsmokers in peripheral blood cells in the Comet assay and in the micronucleus test. However, more studies need to be performed as concern further end points of genotoxicity, e.g., the frequency of micronuclei and sister chromatid exchanges.
Nicotine is known to be activated by the cytochrome P450 2A6 system. This metabolic pathway may be anticipated in this cell system of metabolic competent mini-organ cultures. However, at present we are not able to clearly differentiate possible direct genotoxic effects from effects after metabolic activation. In additional studies (data not shown), an enzyme mix led to a diminished effect which may be discussed as a possible hint for a direct genotoxic effect without the necessity of activation.
The increased DNA fragmentation induced by nicotine in mini-organ cultures of human nasal epithelia reflects direct genotoxic effects and warrants further investigations. In order to exclude possible apoptosis or necrosis reactions induced by nicotine, mini-organ cultures will be examined histologically. Furthermore, dose-dependent effects of long-term exposure of MOC to nicotine, simulating the in vivo situation even more effectively, will be performed as well. Finally, in view of possible antimutagenic effects of nicotine (Lee et al., 1996), co-incubation of nicotine with cigarette smoke condensate and established tobacco carcinogens are warranted. In conclusion, the mechanisms of nicotinic DNA damage and its repair are yet not fully understood and demand future investigations.
However, the present results may add to the controversial discussion on filter cigarettes and so-called light cigarettes with reduced fractions of tar and nicotine concerning their possible health benefit (Harris et al., 2004; Hoffmann et al., 2001; Lee and Sanders, 2004; National Cancer Institute, 2001). In view of nicotine compensation, the cancer risk in smokers of light cigarettes could in fact be far less reduced than expected due to the underestimated genotoxic potential of nicotine.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
The technical expertise of Barbara Goricnik is highly appreciated. We would also like to thank Imperial Tobacco Ltd. for financial support of this study.
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Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University Munich, Germany
Otolaryngology-Head and Neck Surgery, Ludwig-Maximilians University Munich, Germany
Internal Medicine-Pneumology, Ludwig-Maximilians University Munich, Germany
ABSTRACT
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 3.3- and 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.
INTRODUCTION
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 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 tobacco-related 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 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-N-oxide, 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 polA+/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 antiapoptotic effects of nicotine in human gingival fibroblasts.
In a prior study, the genotoxic potential of nicotine was demonstrated in human peripheral lymphocytes and lymphatic tissue of the palatine tonsils. Starting at 0.5 mM, nicotine showed significant enhancement of DNA migration in the Comet assay (Kleinsasser et al., 2005a). The present study focuses on mini-organ cultures (MOC), which were first described by Steinsvg et al. (1991) and further developed for upper aerodigestive tract epithelia by Kleinsasser et al. (2001, 2004). Being fully covered with partially ciliated epithelium, the MOC are closer to the in vivo situation than isolated cells. Whether or not possible genotoxic effects of nicotine are dose dependent, cumulative, and repaired sufficiently after repetitive exposure should be elucidated in MOC with the aid of the Comet assay, a sensitive short-term genetic toxicity test detecting strand breaks, alkali-labile sites, and incomplete DNA repair in human mucosa cells, thereby indicating a possible step in tumor initiation (Tice et al., 2000).
MATERIALS AND METHODS
Preparation of mini-organ cultures.
During surgery of the nasal air passage, human mucosa specimens from the lower ridge of the inferior nasal turbinate of 14 male patients were harvested (35.3 years on average). All tissue donors consumed less than 60 g alcohol per day and were healthy in other respects (Table 1). The mucosa was resected to improve nasal breathing and to diminish the patient's snoring. The study was approved by the Ethics Commission of the University of Regensburg Medical Faculty, and all participants provided written informed consent.
Fresh specimens were immediately transported to the laboratory and dissected into cubes of about 1 mm3 restricted only to mucosa and excluding deeper layers. Connective tissue and bony structures were resected. This preparation technique ensures that the cube contained respiratory epithelium with cilia, goblet cells, and mucosal gland cells, exclusively. The cubes were washed three times in Airway Epithelial Cell Growth Medium (AECGM; PromoCell, Heidelberg, Germany) and transferred into 24-well plates, one fragment per well. Thus the sample extent decided on the number of possible experimental days. Adhesion to the dish surface was prevented by coating the wells with 0.75% Agar Noble (BD Becton Dickinson, Heidelberg, Germany) dissolved in Dulbecco's modified eagle medium (DMEM; Gibco-BRL, Eggenstein, Germany) containing 10% fetal calf serum (FCS), nonessential amino acids, penicillin, and streptomycin (Biochrom, Berlin, Germany). The mini-organ cultures (MOC) were incubated in 250 μl AECGM at 37°C, 5% CO2, and 95% relative humidity. The medium was exchanged every other day. Until the seventh day of culture the mucosa fragments were completely coated with partially ciliated epithelium.
Exposure.
The experimental protocol is outlined in Figure 1. Epithelization of the surface area of the MOC was allowed to occur without experimental disruption up to the seventh day. On days 7, 9 and 11, the cultured MOC were repeatedly exposed to nicotine (MP Biomedicals, Eschwege, Germany) for 60 min. According to results of preliminary pilot studies on single cells of the inferior nasal turbinate (Sassen et al., 2003), nicotine dissolved in phosphate buffered saline (PBS) (Sigma-Aldrich, Taufkirchen, Germany) was added at 0, 2, and 4 mM. Subsequently, the Comet assay was performed on the exposed MOC. Recovery from genotoxic effects was controlled in cubes placed onto fresh plates after nicotine exposure on days 8, 10, and 12 and incubated for 24 h in AECGM.
The directly alkylating mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.02 mM, 60 min; Sigma-Aldrich) was used as a positive control to point out technical malfunctions, e.g., failures during electrophoresis.
Cell separation.
The samples were digested enzymatically by treatment with collagenase P (1 mg/ml), hyaluronidase from bovine testes (1 mg/ml) (Roche, Mannheim, Germany), and pronase E Type XIV from Streptomyces griseus (5 mg/ml) (Sigma-Aldrich), dissolved in AECGM, for 45 min at 37°C in a shaking water bath (Pool-Zobel et al., 1994). Following digestion, the reaction tubes were vortexed and residual connective tissue was removed. The tubes were centrifuged and the supernatant discarded. The enzymes were neutralized by fetal bovine serum and the cells subsequently washed in PBS.
Alkaline single cell microgel electrophoresis (Comet) assay.
After examining the viability of the cells using trypan blue staining, the cells were subjected to the Comet assay (e.g., Tice et al., 2000; Kleinsasser et al., 2001, 2003, 2005a). 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) with pH > 13. A 20 min "unwinding" period was followed by an 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-Aldrich). A DMLB microscope (Leica, Heerbrugg, Switzerland) equipped with an adapted CCD camera (Cohu Inc., San Diego, CA) was used to examine the slides. The software Komet 4.0 (Kinetic Imaging, Liverpool, UK) was applied for planimetric evaluation of fluorescence intensity.
To quantify the level of induced DNA damage, 80 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 tail (DT), and the tail length (TL) (Lee et al., 2004).
Statistics.
Evaluation was based on mean OTM values of individual MOC from patient 1–14. Missing data was not available because of technical conditions, e.g., insufficient sample material. Statistical analyses using the Wilcoxon Test and ANOVA for repeated measures were performed using the SPSSTM 10.0 program (SPSS Inc., Chicago, IL, USA).
RESULTS
Ciliary beats were not restricted by prolonged culture. Cell viability tested by trypan blue exclusion remained unchanged between 90 and 99%. Neither parameter was affected by single, double, or triple exposure to either 2 and 4 mM nicotine or 0.02 mM MNNG as the positive control.
Individual results for all three parameters of DNA damage, OTM, DT, and TL, together with mean and standard deviation are shown in Tables 2–4. Box plots of OTM values are shown for comparison of DNA damage in controls, nicotine-, and MNNG-treated MOC in Figure 2. In untreated MOC, DNA damage remained low between culture days 7 and 12.
Treatment with the direct mutagen MNNG for 1 h on day 7 (Fig. 2) resulted in a highly significant increase in DNA damage compared to controls (52-fold, p < 0.001). During the following 24 h recovery period, the cells showed significant DNA repair (p < 0.01) resulting in a nearly two-fold reduction in DNA damage. After the second and third treatment with MNNG, OTM increased further (65-fold and 110-fold, p < 0.001) as compared to the controls. ANOVA for repeated analysis for days 7, 9, and 11 confirmed highly significant differences (p < 0.001). On days 10 and 12 MNNG-exposed MOC again showed significant recovery from DNA damage (p < 0.01).
A single 1-h treatment of MOC with 2 and 4 mM nicotine on day 7 resulted in a dose-dependent increase in OTM values (3.9- and 5.6-fold, p < 0.001). A second treatment of the same MOC on days 9 and 11 did not further increase DNA damage at either nicotine concentration. At 2 mM, no significant recovery from DNA damage was observed in MOC 24 h after either single, double, or triple 1-h exposures. After the high dose of 4 mM nicotine, a slight but significant recovery from DNA damage was observed on days 10 and 12, 24 h after the second (1.3-fold reduction, p < 0.05) and third (1.5-fold reduction, p < 0.05) 1-h treatment, respectively. Throughout days 7 to 12, nicotine-treated MOC had significantly higher dose-dependent OTM values compared to the controls (p < 0.01).
Comparing the negative controls of smokers with a history of 4.5–55 package years (n = 7; patient 2, 4, 6, 7, 12–14) and nonsmokers or smokers with a history of less than 2 package years (n = 7; patient 1, 3, 5, 8–11), the Mann-Whitney-U-test showed no differences (p = 0.805).
DISCUSSION
In the present study, genotoxic effects of nicotine were investigated with the aid of a three-dimensional mini-organ culture (MOC) of human upper aerodigestive tract epithelia. The integrity of cells with their intercellular organ-specific formation allows a better simulation of the in vivo situation than assaying single cells (Kleinsasser et al., 2001, 2004; Steinsvg et al., 1991). Repetitive exposures to nicotine, followed by recovery phases, give a more precise depiction of the genotoxic consequences of nicotine. Viability of the cultures did not change within the experimental period and was not affected by nicotine treatment. In the negative control, DNA migration did not increase with prolonged incubation in PBS.
Nicotine showed a dose-dependent increase in DNA fragmentation in MOC, affirming recent results in lymphatic tissue of the palatine tonsils and peripheral lymphocytes (Kleinsasser et al., 2005a). In these cells, nicotine induced a dose-dependent increase in DNA migration by 0.125 to 4 mM, with statistical differences from controls starting at 0.5 mM. Genotoxic effects due to artifactitious conditions, such as increasing pH with increasing nicotine concentrations due to its alkaline properties, and metabolite formation during storage were excluded. There were also no relevant differences in DNA migration when using nicotine from two different commercial sources (Kleinsasser et al., 2005a). Significant direct genotoxic effects have also been shown for human gingival fibroblasts (Argentin and Cicchetti, 2004) and spermatozoa (Arabi, 2004). To what extent this nicotine-derived DNA damage is triggered by enhanced oxidative stress and free radical production (Crowley-Weber et al., 2003; Wetscher et al., 1995) has to be clarified in further experiments.
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 (Hukkanen et al., 2005; Schneider et al.; 2001; Teneggi et al., 2002). The lowest concentration in the present study, 2 mM nicotine, elicited significant DNA damage in mucosa cells of the nose after 1 h incubation (Fig. 2). This concentration is about 80-fold higher than saliva concentrations. However, dose-dependency of DNA damage by nicotine in MOC was not studied in the present experiments. For carcinogenic substances acting through direct genotoxic mechanisms, no threshold exists. Especially after prolonged exposure in habitual tobacco users, lower concentrations of nicotine may lead to irreversible changes in genetic material. The net genotoxic effect of nicotine after exposure to a complex mixture of compounds in cigarette smoke cannot be predicted from single exposure experiments (Lee et al., 1996).
No cumulative effects of 2 and 4 mM nicotine were observed after double or triple exposure (Fig. 2). These findings need further evaluation by additional tests. However, the results proved to be quite constant during the present studies. Within 24 h, recovery from nicotine exposure, as seen by a decrease in DNA migration as an indicator of DNA repair, was very weak, reaching significance only after double and triple exposure to the higher nicotine concentration of 4 mM. This is in sharp contrast to the results achieved with the directly alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine MNNG, which was used as a positive control to detect technical failures (Fig. 2). Repetitive exposure to MNNG led to cumulative DNA damage, which was repaired to a significant extent during the recovery phases. A similar two-fold reduction in DNA damage by MNNG within 24 h was reported to occur in V79 cells (Labaj et al., 2003). The obviously different nature of DNA damage caused by nicotine as compared to MNNG requires further advanced investigations with prolonged exposure mimicking the human in vivo situation. For such studies, the MOC model may offer certain advantages. As with nicotine (Kleinsasser et al., 2005a), genotoxic effects detected with the Comet assay in single cells were confirmed in cells from MOC after 7 days of culture for benzo[a]pyrene-7,8-diol-9,10-epoxide and mono(2-ethylhexyl)phthalate (Kleinsasser et al., 2004). The capacity for repair of DNA damage observed with MNNG in the present study, as well as the metabolism of foreign compounds (Teissier et al., 1998), is an indicator of the retained metabolic competence in MOC. This was also demonstrated with the aid of flow cytometry, detecting no decrease in the cytochrome P450 2A6, the most important enzyme of nicotine metabolism (Kleinsasser et al., manuscript in preparation).
No significant differences were found concerning the possible influence of smoking status on DNA migration in untreated controls. This was not unexpected, in view of the lack of differences in the Comet assay between smokers and nonsmokers in our previous study in isolated lymphocytes and cells from lymphatic tissue of the palatine tonsils (Kleinsasser et al., 2005a). These results are in line with Hoffmann and Speit (2005), showing no differences between heavy and nonsmokers in peripheral blood cells in the Comet assay and in the micronucleus test. However, more studies need to be performed as concern further end points of genotoxicity, e.g., the frequency of micronuclei and sister chromatid exchanges.
Nicotine is known to be activated by the cytochrome P450 2A6 system. This metabolic pathway may be anticipated in this cell system of metabolic competent mini-organ cultures. However, at present we are not able to clearly differentiate possible direct genotoxic effects from effects after metabolic activation. In additional studies (data not shown), an enzyme mix led to a diminished effect which may be discussed as a possible hint for a direct genotoxic effect without the necessity of activation.
The increased DNA fragmentation induced by nicotine in mini-organ cultures of human nasal epithelia reflects direct genotoxic effects and warrants further investigations. In order to exclude possible apoptosis or necrosis reactions induced by nicotine, mini-organ cultures will be examined histologically. Furthermore, dose-dependent effects of long-term exposure of MOC to nicotine, simulating the in vivo situation even more effectively, will be performed as well. Finally, in view of possible antimutagenic effects of nicotine (Lee et al., 1996), co-incubation of nicotine with cigarette smoke condensate and established tobacco carcinogens are warranted. In conclusion, the mechanisms of nicotinic DNA damage and its repair are yet not fully understood and demand future investigations.
However, the present results may add to the controversial discussion on filter cigarettes and so-called light cigarettes with reduced fractions of tar and nicotine concerning their possible health benefit (Harris et al., 2004; Hoffmann et al., 2001; Lee and Sanders, 2004; National Cancer Institute, 2001). In view of nicotine compensation, the cancer risk in smokers of light cigarettes could in fact be far less reduced than expected due to the underestimated genotoxic potential of nicotine.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
The technical expertise of Barbara Goricnik is highly appreciated. We would also like to thank Imperial Tobacco Ltd. for financial support of this study.
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