- Email: [email protected]
Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line
Toxicology 222 (2006) 71–79
Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line Sarah Vogt, Katharina Fuchs, Elmar Richter ∗ Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University, Goethestrasse 33, D-80336 Munich, Germany Received 3 January 2006; received in revised form 23 January 2006; accepted 24 January 2006 Available online 28 February 2006
Abstract The incidence of esophageal adenocarcinoma is rapidly rising in Western populations. Gastroesophageal reflux disease (GERD) is thought to be one of the most important risk factors. However, the mechanisms by which GERD enhances tumor formation at the gastroesophageal junction are not well understood. Myosmine is a tobacco alkaloid which has also a wide spread occurrence in human diet. It is readily activated by nitrosation and peroxidation giving rise to the same hydroxypyridylbutanone-releasing DNA adducts as the esophageal carcinogen N -nitrosonornicotine. Therefore, the genotoxicity of myosmine was tested in a human esophageal adenocarcinoma cell line (OE33). DNA damage was assessed by single-cell gel electrophoresis (Comet assay). DNA strand breaks, alkali labile sites and incomplete excision repair were expressed using the Olive tail moment (OTM). The Fapy glycosylase (Fpg) enzyme was incorporated into the assay to reveal additional oxidative DNA damage. DNA migration was determined after incubation of the cells for 1–24 h. Under neutral conditions high myosmine concentrations of 25–50 mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5 mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Coincubation of 25 mM myosmine with 1 mM H2 O2 for 1 h significantly enhanced the genotoxicity of H2 O2 but not the oxidative lesions additionally detected with the Fpg enzyme. In the presence of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) a dose-dependent significant genotoxic effect was obtained with 1–10 mM myosmine after 4 h incubation. NS-398, a selective inhibitor of cyclooxygenase 2, did not affect the SIN-1 stimulated genotoxicity of myosmine. Finally, the 23 h repair of N-methylN -nitro-N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10 mM myosmine. In conclusion, myosmine exerts significant genotoxic effects in esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Esophageal cells; Myosmine; Genotoxicity; Comet assay
1. Introduction The esophageal adenocarcinoma (EAC) has received considerable attention in the last years because of its
∗
Corresponding author. Tel.: +49 89 2180 75742; fax: +49 89 2180 75743. E-mail address: [email protected] (E. Richter).
rapid increase in incidence (Chen and Yang, 2001). In Western populations a yearly increase of ∼10% can be observed and at present EAC is more prevalent than esophageal squamous cell carcinoma (ESCC; Powell et al., 2002). Barrett’s esophagus (BE), a columnar metaplasia of the lower esophageal epithelium, is accepted to be the premalignant condition from which EAC results almost exclusively (Wild and Hardie, 2003). A number of risk factors are known for the development of
0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2006.01.025
72
S. Vogt et al. / Toxicology 222 (2006) 71–79
BE and EAC including gastroesophageal reflux disease (GERD), high body mass index, a diet low in fruits and vegetables and to some extent smoking (Chow et al., 1998; Lagergren et al., 1999; Wu et al., 2001; Terry et al., 2001; DeMeester, 2005). Nevertheless the molecular mechanisms leading to tumor formation are not well understood so far. Genetic instability has been shown to occur early in this process. Loss of heterozygosity, inactivation of tumor suppressor genes and microsatellite instability have all been identified in BE and associated EAC (Olliver et al., 2005; Wilson et al., 1998; Banki et al., 2005; Tselepis et al., 2003). The tobacco-specific nitrosamines (TSNA) 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N -nitrosonornicotine (NNN) arising from nitrosation of nicotine and nornicotine are implicated in smoking-related human cancer (Hecht, 2003). NNN induces esophageal and nasal tumors in rats and is likely to play an important role in the development of esophageal cancer in smokers (Hecht, 1998). Upon metabolic activation by 2 -hydroxylation NNN leads to a pyridyloxobutylating agent which binds to DNA. Thus among others O6 -[4-oxo-4-(3pyridyl)butyl]guanine (O6 -popGua) can be formed, that is highly mutagenic in human cells causing predominantly G → A transitions (Pauly et al., 2002). Additionally O6 -popGua is a substrate for the human O6 -alkylguanine-DNA alkyltransferase (AGT), resulting in increased persistence of promutagenic O6 methylguanine (O6 -mGua; Wang et al., 1997). After DNA hydrolysis 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) is released by the majority of the pyridyloxobutyladducts. These HPB-releasing adducts have been
detected in target tissues of animals treated with NNK and NNN as well as in human lung tissue from smokers (Hecht, 1998; Foiles et al., 1991; Sticha et al., 2002; Boysen et al., 2003). Recently, high levels of HPB-releasing DNA adducts were found not only in the lung but also in the mucosa of esophagus and cardia from sudden death victims. Interestingly, these adducts were independent of the smoking status (H¨olzle et al., 2003). Myosmine, 3-(1-pyrroline-2-yl)pyridine, is a minor tobacco alkaloid but has been detected in a wide variety of food products such as cereals, potatoes, nuts, cocoa and milk (Zwickenpflug et al., 1998; Tyroller et al., 2002). Upon nitrosation myosmine leads to NNN but also directly to a reactive intermediate which pyridyloxobutylates DNA and proteins (Fig. 1; Zwickenpflug, 2000). In vitro experiments showed that myosmine is rapidly nitrosated at pH 2–4 by about 60–80% within 4–8 h (Wilp et al., 2002). These conditions occur in the esophagus by reflux of gastric acid (Moriya et al., 2002). Alternatively, myosmine can also be activated by peroxidation, just as well leading to the formation of HPB adducts (Zwickenpflug and Tyroller, 2005). Genotoxic effects of myosmine have been shown in human lymphocytes and upper aerodigestive tract epithelial cells (Kleinsasser et al., 2003). Alkaline single cell gel electrophoresis (Comet assay) has proven to be a sensitive tool for detecting DNA single and double strand breaks, alkali labile sites and incomplete excision repair (Collins et al., 1997; Faust et al., 2004; Shao et al., 2005). Incorporation of the Fapy glycosylase (Fpg) enzyme into the assay reveals additional damage including 8-hydroxydeoxyguanosine, a common marker of oxidative DNA damage. The enzyme
Fig. 1. Myosmine contributes to HPB releasing DNA and hemoglobin adducts without requiring metabolic activation such as the tobacco-specific nitrosamines NNK and NNN.
S. Vogt et al. / Toxicology 222 (2006) 71–79
recognizes the damaged DNA base and generates strand brakes that can be measured in the comet assay (Collins and Horvathova, 2001).
73
tion (400 mM Tris, pH 7,5), the cells were stained with 50 l ethidium bromide (20 g/ml). 2.3. Analysis and statistics
2. Materials and methods 2.1. Cell culture and drug treatment The cell line OE33 (a gift of Dr. von Rahden, Technical University Munich; ECACC No. 96070808) was established from the adenocarcinoma of the lower esophagus (Barrett metaplasia) of a 73-year-old female patient. Cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen-Gibco, Karlsruhe, Germany) at 37 ◦ C with 5% CO2 . Twenty-four hours prior to drug treatment 2 × 105 cells/well in 1 ml medium were plated in 12well plates. The cells were treated in medium without serum with myosmine (synthesized by Dr. Wolfgang Zwickenpflug in our institute), H2 O2 , 3-morpholinosydnonimine (SIN-1), N[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS398), N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (all from Sigma–Aldrich, Taufkirchen, Germany) for 1–24 h with concentrations indicated in the results. Myosmine and H2 O2 were dissolved in sterile filtered water (membraPure GmbH, Bodenheim, Germany), SIN-1 in phosphate buffered saline (PBS) at pH 5.5 and NS-398 in dimethyl sulfoxide (DMSO, Sigma–Aldrich). Cells for the negative controls were treated with the corresponding solvents. Following treatment, cells were washed with PBS, trypsinized and resuspended in 1 ml complete medium. Cells were separated by centrifugation (10 min, 800 U/min, 4 ◦ C) and resuspended in 25 l PBS. The viability of the treated cells was determined by trypan blue exclusion. Only cells with a viability >70% were used for the comet assay.
For each slide 50 cells were analyzed using a fluorescence microscope (Olympus, Hamburg, Germany) with a CCD camera. The comets were measured using an image analysis system (Comet Assay II Version 2.11, Perceptive Instruments Ltd, Haverhill, UK). To quantify the DNA damage the Olive tail moment (OTM) was used, which reflects the percentage of DNA in the tail of the comet multiplied with the median migration distance. For statistical analysis the Wilcoxon Signed Rank test of the Prism 4 for Windows program (GraphPad Software Inc., San Diego, CA, USA) was applied to compare DNA migration. All experiments were run at least six times. Values are given as mean and the standard error of the mean, respectively.
3. Results Under neutral conditions high myosmine concentrations of 25–50 mM were necessary to elicit a weak genotoxic effect (data not shown). Myosmine is known to be activated under peroxidative and nitrosative conditions (Fig. 1). These conditions are prevalent at the gastroesophageal junction. Chemical nitrosation of myosmine proceeds well at pH values at or below 5 (Wilp et al., 2002). Incubations with myosmine at pH 6 (Fig. 2), the lowest pH value OE33 cells tolerate for longer incubation times, showed a significant dose-dependent increase of DNA migration. OTM values in the comet assay were identical in the negative controls after 4 h (0.51 ± 0.06) and 24 h (0.50 ± 0.11) incubation. A significant increase (P < 0.05) was noted after 4 h incubation with myosmine
2.2. Comet assay Slides with a frosting of 5 mm along the long edges (76 × 26 mm, Langenbrinck, Emmendingen, Germany) were prepared with 85 l of 0.5% agarose (Biozym, Hessisch Oldendorf, Germany) at least one week before the experiment. The cell suspension was mixed with 75 l Low Melting Agarose (Biozym) and applied to the prepared slides. Alkaline lysis (10% DMSO, 1% Triton × 100 in alkaline lysis buffer: 2.5 M NaCl, 10 mM Tris, 100 mM EDTA, pH 10) followed overnight. Fpg (Sigma–Aldrich) treatment was performed according to Collins et al. (1997) with modifications. After 1 h lysis the slides were incubated with 50 l Fpg (1 g/ml) for 30 min. The slides were placed in a horizontal gel electrophoresis chamber (Labtech International, Burkhardtsdorf, Germany), positioned close to the anode and covered with electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13). After a 30 min DNA “unwinding” period, the electrophoresis was performed at 25 V and 300 mA for 30 min. Following 3 min × 5 min neutraliza-
Fig. 2. Concentration dependent DNA damage in OE33 cells after 4 and 24 h incubation with myosmine at pH 6 determined as OTM in the comet assay (mean ± S.E. of six experiments). a Significantly different from the corresponding control values, P < 0.05.
74
S. Vogt et al. / Toxicology 222 (2006) 71–79
Fig. 3. Effect of 1 mM H2 O2 on DNA damage by 25 mM myosmine in OE33 cells after 1 h incubation with or without post-treatment with the Fpg enzyme (mean ± S.E. of 5–13 experiments). a significantly different from controls, P < 0.001; b significantly different from H2 O2 only, P < 0.01; c significantly different from experiments without posttreatment with the Fpg enzyme, P < 0.05.
at 10 mM (1.7-fold to 0.87 ± 0.13) and at 25 mM (4-fold to 2.04 ± 0.56) and after 24 h incubation with myosmine at 5 mM (1.3-fold to 0.67 ± 0.09) and at 25 mM (7-fold to 3.46 ± 1.24), respectively. The differences between 4 and 24 h of incubation with myosmine did not reach significance. The possible influence of peroxidative conditions on myosmine activation was studied by 1 h incubation of 25 mM myosmine in the presence or absence of 1 mM H2 O2 (Fig. 3). Whereas myosmine alone did not increase DNA damage significantly (0.63 ± 0.22 versus 0.45 ± 0.13 in controls; 1.4-fold, n.s.) a highly significant 11-fold increase was observed with H2 O2 alone (4.82 ± 0.82, P < 0.001). However, co-incubation with myosmine increased OTM values 1.7-fold (8.27 ± 1.10) compared to H2 O2 alone (P < 0.01). To evaluate the contribution of oxidative damage, the DNA was incubated after cell lysis with the Fpg enzyme. In the negative control (1.15-fold increase to 0.52 ± 0.08, P < 0.05) and with 25 mM myosmine (1.16-fold decrease to 0.54 ± 0.09, P < 0.05) only minor changes of OTM values were observed. However, as expected, DNA damage by H2 O2 alone was increased 2.5-fold by post-incubation with Fpg (11.8 ± 3.35, P < 0.05). Interestingly, after coincubation with myosmine the Fpg-stimulated DNA damage by H2 O2 was not further increased (12.0 ± 3.1, n.s.). Nitrosative stress, which activates myosmine in vitro, occurs in tissues under conditions of chronic inflammation, where the NO• synthase is induced and NO• and peroxynitrite are released (Bryan et al., 2004). After 4 h incubation with 1 mM of the NO• and/or peroxynitrite donor SIN-1 added to the medium at 0 and 2 h (Doulias
Fig. 4. Effect of 1 mM SIN-1 on DNA damage by myosmine in OE33 cells after 4 h incubation (mean ± S.E. of 6–19 experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a,b Significantly different from control values, P < 0.05 (a) and P < 0.001 (b); c,d significantly different from myosmine only, P < 0.01 (c) and P < 0.001 (d); e,f significantly different from SIN-1 only, P < 0.01 (e) and P < 0.001 (f).
et al., 2001; Martin-Romero et al., 2004), a significantly increased DNA migration was observed compared to the controls (1.59 ± 0.27 versus 0.50 ± 0.07 in controls, 3.2fold, P < 0.001; Fig. 4). Addition of myosmine at concentrations as low as 1, 5 and 10 mM dose-dependently increased the DNA fragmentation versus SIN-1 only by 1.4-fold (2.31 ± 0.89, n.s.), 3.7-fold (5.88 ± 1.62, P < 0.01) and 5.1-fold (8.15 ± 0.99, P < 0.001), respectively. Evaluation of the contribution of oxidative damage by post-treatment of DNA with the Fpg enzyme confirmed the absence of any effect of myosmine (Fig. 5). In
Fig. 5. Effect of 1 mM SIN-1 on DNA damage by myosmine in OE33 cells after 4 h incubation with or without post-treatment with the Fpg enzyme (mean ± S.E. of six experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a Significantly different from control values without post-treatment with the Fpg enzyme, P < 0.05; b significantly different from myosmine only, P < 0.05; c significantly different from corresponding values obtained with myosmine only and SIN-1 only, P < 0.05.
S. Vogt et al. / Toxicology 222 (2006) 71–79
Fig. 6. Effect of 24 h pre-treatment with 50 M of the COX-2 inhibitor NS-398 on DNA damage in OE33 cells after 4 h incubation with 10 mM myosmine alone or in combination with 1 mM SIN-1 (mean ± S.E. of six experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a Significantly different from corresponding control values and from values obtained with myosmine only. P < 0.05.
contrast, DNA damage after treatment with SIN-1 alone increased 2.4-fold after Fpg treatment (2.97 ± 1.46 versus 1.25 ± 0.45, n.s.). Epidemiological, experimental and early clinical evidence indicate that cyclooxygenase-2 (COX-2) represents a potential molecular target for the treatment and/or prevention of esophageal cancer (Altorki, 2004). The protective effect of non-steroidal anti-inflammatory drugs in Barrett’s esophageal carcinogenesis most probably reflects COX-2 inhibition. The selective COX-2 inhibitor NS-398 was shown to reduce COX-2 mRNA expression in OE33 cells in a time- and dose-dependent manner (Cheong et al., 2004). To determine the role of COX-2 for the genotoxicity of myosmine, OE33 cells were treated with 50 M NS-398 24 h before 4 h incubation with myosmine or myosmine and SIN-1 (Fig. 6). Pre-incubation with NS-398 did not affect the DNA damage in controls and in cells treated with 10 mM myosmine. The DNA damaging effect of myosmine and SIN-1 in combination was confirmed (8.34 ± 2.56 versus 0.42 ± 0.05 in controls, 20-fold, P < 0.05). Pretreatment with NS-398 further increased the DNA damage by this combination (12.48 ± 5.87, 1.5-fold, n.s.) but this effect did not reach significance. Finally, the effect of myosmine on the repair of MNNG-induced DNA damage was studied. OE33 cells were incubated for 1 h with 1 M MNNG to induce methyl adducts (Fig. 7). Subsequently cells were allowed to recover in fresh medium for 23 h. During this repair time cells were additionally incubated with 10 mM myosmine, 1 mM SIN-1 or both in combination. After 1 h incubation with 1 M MNNG OTM values increased 53-fold compared to controls (10.6 ± 0.6
75
Fig. 7. Effect 10 mM myosmine and 1 mM SIN-1 alone or in combination on 23 h repair of DNA damage in OE33 cells pretreated for 1 h with 1 M MNNG (mean ± S.E. of six experiments). a Significantly different from control values, P < 0.05; b significantly different from values obtained after 1 h treatment with MNNG, P < 0.05; c significantly different from corresponding values obtained after 1 h treatment with MNNG and 23 h recovery, P < 0.05; d significantly different from values obtained with myosmine only, P < 0.05.
versus 0.20 ± 0.05 in controls, P < 0.05). Incubation of MNNG-treated cells in fresh medium for 23 h significantly decreased DNA damage (0.90 ± 0.16, 12-fold lower than 1 h MNNG and 4.5-fold higher than controls, P < 0.05). In the presence of 10 mM myosmine the MNNG-induced DNA damage decreased only 5.7fold (1.83 ± 0.31, P < 0.05 versus both MNNG only and MNNG + 23 h recovery). Incubation with 1 mM SIN-1 inhibited the repair within 23 h even more efficiently than myosmine (3.85 ± 0.46, 2.7-fold lower than after 1 h MNNG and 4.3-fold higher than after 23 h recovery, both P < 0.05). After co-incubation of myosmine and SIN-1 repair of MNNG-induced DNA damage was lowest (4.44 ± 0.85, 2.4-fold lower than after 1 h MNNG and 5-fold higher than after 23 h recovery, both P < 0.05) but the effect was not significantly different from treatment with SIN-1 only. 4. Discussion Myosmine is present to a considerable extent in sources other than tobacco (Zwickenpflug et al., 1998; Tyroller et al., 2002). In human saliva up to 5 g/L are found independent from the smoking status (Maier et al., 2005). Myosmine can be readily activated by nitrosation or peroxidation under physiological conditions occurring in the mucosa of the lower esophagus especially under reflux conditions. The main nitrosation products are NNN and HPB (Zwickenpflug, 2000; Wilp et al.,
76
S. Vogt et al. / Toxicology 222 (2006) 71–79
2002). NNN is a known esophageal carcinogen and HPB-releasing DNA-adducts are associated with NNNderived esophageal tumors (Hecht, 1998). The ability of myosmine to form DNA adducts was shown in vitro with calf thymus DNA (Wilp et al., 2002). In human lymphocytes and upper aerodigestive tract epithelial cells, myosmine showed dose- and time-dependent genotoxic effects (Kleinsasser et al., 2003). In the present study, myosmine was less genotoxic in OE33 cells compared to freshly isolated human cells. At neutral pH levels, where myosmine is barely nitrosated, incubation of OE33 cells with myosmine lead only to a low increase of DNA damage in the comet assay. Chemical nitrosation of myosmine proceeds well at pH values of 2–5 (Wilp et al., 2002). However, OE33 cells do not tolerate such low pH values. Therefore, incubations with myosmine could only be performed at pH 6. Under these conditions about 20–30% of myosmine are nitrosated within 24 h in the presence of 41 mM nitrite (Wilp et al., 2002). Lowering the pH to 6 considerably enhanced the genotoxic effects of myosmine in OE33 cells leading to a time- and dose-dependent increase of DNA damage with significant effects already at 5 mM myosmine (Fig. 2). This indicates that even under very slight acidic conditions myosmine could have genotoxic effects in the lower esophagus. The concentration of myosmine eliciting a genotoxic effect in vitro is several orders of magnitude higher than the highest concentration detected so far in saliva and plasma from humans approaching 30 nM (Maier et al., 2005). However, there exists no threshold for initiators of carcinogenicity such as nitrosamines (Lijinsky, 1986; Peto et al., 1991). Therefore, in the absence of a no-effect level, myosmine could have a relevant genotoxic effect in vivo after long-term exposure to much lower doses. At lower pH values as frequently occurring in chronic reflux disease, activation of myosmine should be much more effective. The gastric cardia and, during acid reflux, Barrett’s segments are the anatomical sites with maximal potential for acid catalyzed nitrosation (Suzuki et al., 2003, 2005). This could explain the presence of high levels of DNA adducts releasing HPB upon acid hydrolysis in the mucosae of human esophagus and cardia (H¨olzle et al., 2003). Interestingly, these adduct levels were independent from the smoking status as were the concentrations of myosmine in human saliva (Maier et al., 2005). Oxidative stress has an important role for formation and development of adenocarcinoma (Chen and Yang, 2001). Enzymatic changes and decreased levels of glutathione in BE are consistent with increased formation of hydrogen peroxide and oxygen free radicals in patients with reflux disease (Sihvo et al., 2002; M¨ork et al.,
2003). This is in line with epidemiological data showing a protective effect of a high intake of antioxidants with fruits and vegetables (Terry et al., 2000). To determine if enhanced endogenous levels of H2 O2 under oxidative stress could affect myosmine-induced genotoxicity via peroxidation, OE33 cells were co-incubated with myosmine and H2 O2 (Fig. 3). A non-genotoxic concentration of myosmine significantly increased DNA damage by H2 O2 . Since H2 O2 mainly gives rise to oxidative DNA damage, the effect of myosmine could be due to an increase of oxidative stress. However, post-incubation with the Fpg enzyme revealing oxidative DNA damage after treatment with H2 O2 alone did not result in a further increase after co-incubation of H2 O2 with myosmine. Therefore, the increased DNA damage by myosmine is most probably not due to additional oxidative DNA damage. It is rather likely that H2 O2 activates myosmine to genotoxic intermediates (Zwickenpflug and Tyroller, 2005). Chronic gastroesophageal reflux and BE are accompanied by inflammation which leads to the release of reactive species. Expression of inducible NO• synthase is increased in mucosal tissue from patients with BE and EAC (Wilson et al., 1998; Bove et al., 2005). In a rat model of EAC increased nitrotyrosine immunostaining as a marker of peroxynitrite formation was demonstrated (Goldstein et al., 1998). 3morpholinosydnonimine (SIN-1), the reactive metabolite of molsidomine, is often used for in vitro release of NO• , O2 − , peroxynitrite and hydroxyl radicals. After a single dose of 1 mM SIN-1 no genotoxic effect was observed (data not shown). However, in experiments lasting 4 h a second dose of 1 mM SIN-1 added to the medium after 2 h significantly increased DNA damage (Fig. 4). Under these conditions, co-incubation with myosmine caused a strong concentration-dependent increase of DNA damage elicited by SIN-1 alone. Similar to the experiments with H2 O2 , post-treatment with the Fpg enzyme increased the OTM values about twofold (Fig. 5) confirming the oxidative nature of DNA damage by the peroxynitrite donor SIN-1 (Yu et al., 2005). Again, co-treatment with myosmine did not show this Fpg-dependent increase of OTM values. Therefore, SIN-1 may also increase the formation of reactive intermediates from myosmine. From the present experiment it is not clear whether this increase is mediated by nitrosative or peroxidative mechanisms. In an aqueous oxygen-saturated solution of neutral pH, SIN-1 decomposes rapidly to nitric oxide and superoxide anions, generating peroxynitrite. However, at relatively low in vivo oxygen concentrations, SIN-1 reacts with alternative electron acceptors and behaves more like a NO•
S. Vogt et al. / Toxicology 222 (2006) 71–79
donor (Singh et al., 1999). In experiments with cultured cells, the oxygen above the cell media will be consumed after approximately half an hour (Doulias et al., 2001). Observational studies evaluating the association of aspirin/nonsteroidal anti-inflammatory drug use and esophageal cancer indicate a reduced risk. This is most probably mediated by inhibition of cyclooxygenase (COX)-2 (Corley et al., 2003; Zimmermann et al., 1999). COX-2 is predominantly expressed at sites of inflammation and catalyzes the production of prostaglandins that stimulate cancer cell proliferation, inhibit apoptosis and enhance cancer induced angiogenesis and invasiveness (Altorki, 2004). COX-2 is up-regulated in Barrett’s esophageal carcinogenesis (Wilson et al., 1998; Zimmermann et al., 1999; Buskens et al., 2002; Cheong et al., 2003; von Rahden et al., 2005). Recently, nicotine has been shown to activate COX-2 in a gastric adenocarcinoma cell line (Shin et al., 2004). On the other hand, cyclooxygenases are able to activate carcinogens such as NNK (Rioux and Castonguay, 1998). Therefore, myosmine could possibly promote its own activation by induction of COX-2. The selective COX-2 inhibitor NS-398 suppresses COX-2 mRNA expression in OE33 cells in a time- and dose-dependent manner (Cheong et al., 2004). Therefore, pre-incubation with NS-398 should have a protective effect in OE33 cells if COX-2 is involved in myosmine genotoxicity. This was clearly not the case (Fig. 6). DNA damage in cells incubated with myosmine and SIN-1 was even higher after 24 h pre-incubation with the inhibitor. O6 -Alkylguanine is formed in DNA by alkylating compounds and it may mispair with thymine during replication thereby leading to mutagenesis and malignant transformation. In human cells O6 -methylguanine (O6 -mG) and other alkylated bases are repaired by O6 alkylguanine-DNA alkyltransferase (AGT) which is a suicide enzyme that is inactivated by the process of repair (Gerson, 2004; Laval and Wink, 1994). One of the TSNA-induced HPB-releasing adducts has been identified as O6 -[1-oxo-1-(3-pyridyl)but-4-yl]-d-guanosine (O6 -pobG). This highly mutagenic adduct (Pauly et al., 2002) may not only stem from metabolic activation of NNN and NNK (Wang et al., 2003; Upadhyaya et al., 2003) but probably also from nitrosative and/or peroxidative activation of myosmine (Fig. 1). In addition to its inherent mutagenic activity, O6 -pobG enhances the carcinogenesis of NNK in A/J mice through inhibition of O6 -mGua repair (Liu et al., 1999; Pegg, 2000). The inhibition by O6 -pobG is similar to that by other bulky O6 alkylguanine adducts which are adjuvants in chemotherapy (Mijal et al., 2004; Mijal et al., 2005; Nelson et al., 2004) Therefore it was tested if myosmine also inhibits
77
the repair of O6 -mG in OE33 cells (Fig. 7). After 1 h incubation with the methylating agent MNNG, cells were grown for 23 h with fresh medium with or without addition of 10 mM myosmine and/or 1 mM SIN-1. The addition of myosmine doubled the remaining damage, indicating a inhibition of AGT. The even stronger effect of SIN-1 was expected because Laval and Wink (1994) showed that exogenously released NO• inhibits the AGT in cells. In conclusion, myosmine is significantly genotoxic to esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation. By inhibition of O6 -alkylguanine-DNA alkyltransferase, myosmine could also enhance the carcinogenicity of other alkylating agents. Acknowledgements Supported in part by Philip Morris USA Inc. and Philip Morris International. References Altorki, N., 2004. COX-2: a target for prevention and treatment of esophageal cancer. J. Surg. Res. 117, 114–120. Banki, F., DeMeester, S.R., Mason, R.J., Campos, G., Hagen, J.A., Peters, J.H., Bremner, C.G., DeMeester, T.R., 2005. Barrett’s esophagus in females: a comparative analysis of risk factors in females and males. Am. J. Gastroenterol. 100, 560–567. Bove, M., Vieth, M., Casselbrant, A., Ny, L., Lundell, L., Ruth, M., 2005. Acid challenge to the esophageal mucosa: effects on local nitric oxide formation and its relation to epithelial functions. Digest. Dis. Sci. 50, 640–648. Boysen, G., Kenney, P.M.J., Upadhyaya, P., Wang, M., Hecht, S.S., 2003. Effects of benzyl isothiocyanate and 2-phenethyl isothiocyanate on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone metabolism in F-344 rats. Carcinogenesis 24, 517–525. Bryan, N.S., Rassaf, T., Maloney, R.E., Rodriguez, C.M., Saijo, F., Rodriguez, J.R., Feelisch, M., 2004. Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc. Natl. Acad. Sci. U.S.A. 101, 4308–4313. Buskens, C.J., van Rees, B.P., Sivula, A., Reitsma, J.B., Haglund, C., Bosma, P.J., Offerhaus, G.J., van Lanschot, J.J., Ristim¨aki, A., 2002. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus. Gastroenterology 122, 1800–1807. Chen, X.X., Yang, C.S., 2001. Esophageal adenocarcinoma: a review and perspectives on the mechanism of carcinogenesis and chemoprevention. Carcinogenesis 22, 1119–1129. Cheong, E., Igali, L., Harvey, I., Mole, M., Lund, E., Johnson, I.T., Rhodes, M., 2003. Cyclo-oxygenase-2 expression in Barrett’s oesophageal carcinogenesis: an immunohistochemical study. Aliment. Pharmacol. Therap. 17, 379–386. Cheong, E., Ivory, K., Doleman, J., Parker, M.L., Rhodes, M., Johnson, I.T., 2004. Synthetic and naturally occurring COX-2 inhibitors sup-
78
S. Vogt et al. / Toxicology 222 (2006) 71–79
press proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest. Carcinogenesis 25, 1945–1952. Chow, W.-H., Blot, W.J., Vaughn, T.L., Risch, H.A., Gammon, M.D., Stanford, J.L., Dubrow, R., Schoenberg, J.B., Mayne, S.T., Farrow, D.C., Ahsan, H., West, A.B., Rotterdam, H., Niwa, S., Fraumeni, J.F., 1998. Body mass index and risk of adenocarcinomas of the esophagus and gastric cardia. J. Natl. Cancer Inst. 90, 150–155. Collins, A.R., Dobson, V.L., Dusinsk´a, M., Kennedy, G., Stetina, R., 1997. The comet assay: what can it really tell us? Mutat. Res. 375, 183–193. Collins, A.R., Horvathova, E., 2001. Oxidative DNA damage, antioxidants and DNA repair: applications of the comet assay. Biochem. Soc. Trans. 29, 337–341. Corley, D.A., Kerlikowske, K., Verma, R., Buffler, P., 2003. Protective association of aspirin/NSAIDs and esophageal cancer: a systematic review and meta-analysis. Gastroenterology 124, 47–56. DeMeester, S., 2005. Adenocarcinoma of the esophagus and cardia: a review of the disease and its treatment. Ann. Surg. Oncol. 13, 1–19. Doulias, P.T., Barbouti, A., Galaris, D., Ischiropoulos, H., 2001. SIN-1induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay). Free Radic. Biol. Med. 30, 679–685. Faust, F., Kassie, F., Knasm¨uller, S., Boedecker, R.H., Mann, M., Mersch-Sundermann, V., 2004. The use of the alkaline comet assay with lymphocytes in human biomonitoring studies. Mutat. Res. 566, 209–229. Foiles, P.G., Akerkar, S.A., Carmella, S.G., Kagan, M., Stoner, G.D., Resau, J.H., Hecht, S.S., 1991. Mass spectrometric analysis of tobacco-specific nitrosamine DNA-adducts in smokers and nonsmokers. Chem. Res. Toxicol. 4, 364–368. Gerson, S.L., 2004. MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev. Cancer 4, 296–307. Goldstein, S.R., Yang, G.-Y., Chen, X., Curtis, S.K., Yang, C.S., 1998. Studies of iron deposits, inducible nitric oxide synthase and nitrotyrosine in a rat model for esophageal adenocarcinoma. Carcinogenesis 19, 1445–1449. Hecht, S.S., 1998. Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 559–603. Hecht, S.S., 2003. Tobacco carcinogens, their biomarkers and tobaccoinduced cancer. Nat. Rev. Cancer 3, 733–744. H¨olzle, D., Schl¨obe, D., Richter, E., Ostermeier-Hatz, D., von Meyer, L., Tricker, A.R., 2003. 4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung, esophagus and cardia of sudden death victims. Proc. Am. Ass. Cancer Res. 44 (2nd ed.), 1281–1282. Kleinsasser, N.H., Wallner, B.C., Harr´eus, U.A., Zwickenpflug, W., Richter, E., 2003. Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells. Toxicology 192, 171–177. Lagergren, J., Bergstr¨om, R., Nyr´en, O., 1999. Association between body mass and adenocarcinoma of the esophagus and gastric cardia. Ann. Intern. Med. 130, 883–890. Laval, F., Wink, D.A., 1994. Inhibition by nitric oxide of the repair protein, O6 -methylguanine-DNA-methyltransferase. Carcinogenesis 15, 443–447. Lijinsky, W., 1986. The significance of N-nitroso compounds as environmental carcinogens. J. Environ. Sci. Health C4, 1–45. Liu, L.L., Qin, X., Gerson, S.L., 1999. Reduced lung tumorigenesis in human methylguanine DNA-methyltransferase transgenic mice
achieved by expression of transgene within the target cell. Carcinogenesis 20, 279–284. Maier, M., Schulze, A., Richter, E., 2005. Biomonitoring of myosmine in human milk and saliva. Toxicol. Lett. 158, S201–S202. Martin-Romero, F.J., Gutierrez-Martin, Y., Henao, F., GutierrezMerino, C., 2004. Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123. J. Fluoresc. 14, 17–23. Mijal, R.S., Loktionova, N.A., Vu, C.C., Pegg, A.E., Peterson, L.A., 2005. O6 -Pyridyloxobutylguanine adducts contribute to the mutagenic properties of pyridyloxobutylating agents. Chem. Res. Toxicol. 18, 1619–1625. Mijal, R.S., Thomson, N.M., Fleischer, N.L., Pauly, G.T., Moschel, R.C., Kanugula, S., Fang, Q., Pegg, A.E., Peterson, L.A., 2004. The repair of the tobacco specific nitrosamine derived adduct O6 -[4-oxo-4-(3-pyridyl)butyl]guanine by O6 -alkylguanine-DNA alkyltransferase variants. Chem. Res. Toxicol. 17, 424–434. Moriya, A., Grant, J., Mowat, C., Williams, C., Carswell, A., Preston, T., Anderson, S., Iijima, K., McColl, K.E.L., 2002. In vitro studies indicate that acid catalysed generation of N-nitrosocompounds from dietary nitrate will be maximal at the gastro-oesophageal junction and cardia. Scand. J. Gastroenterol. 37, 253–261. M¨ork, H., Scheurlen, M., Al-Taie, O., Zierer, A., Kraus, M., Sch¨ottker, K., Jakob, F., K¨ohrle, J., 2003. Glutathione peroxidase isoforms as part of the local antioxidative defense system in normal and Barrett’s esophagus. Int. J. Cancer 105, 300–304. Nelson, M.E., Loktionova, N.A., Pegg, A.E., Moschel, R.C., 2004. 2-amino-O4 -benzylpteridine derivatives: potent inactivators of O6 -alkylguanine-DNA alkyltransferase. J. Med. Chem. 47, 3887–3891. Olliver, J.R., Hardie, L.J., Gong, Y., Dexter, S., Chalmers, D., Harris, K.M., Wild, C.P., 2005. Risk factors, DNA damage, and disease progression in Barrett’s esophagus. Cancer Epidemiol. Biomar. Prev. 14, 620–625. Pauly, G.T., Peterson, L.A., Moschel, R.C., 2002. Mutagenesis by O6 [4-oxo-4-(3-pyridyl)butyl]guanine in Escherichia coli and human cells. Chem. Res. Toxicol. 15, 165–169. Pegg, A.E., 2000. Repair of O(6)-alkylguanine by alkyltransferases. Mutat. Res. 462, 83–100. Peto, R., Gray, R., Brantom, P., Grasso, P., 1991. Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of N-nitrosodiethylamine or Nnitrosodimethylamine. Cancer Res. 51, 6452–6469. Powell, J., McConkey, C.C., Gillison, E.W., Spychal, R.T., 2002. Continuing rising trend in oesophageal adenocarcinoma. Int. J. Cancer 102, 422–427. Rioux, N., Castonguay, A., 1998. Prevention of NNK-induced lung tumorigenesis in A/J mice by acetylsalicylic acid and NS-398. Cancer Res. 58, 5354–5360. Shao, L., Lin, J., Huang, M., Ajani, J.A., Wu, X., 2005. Predictors of esophageal cancer risk: assessment of susceptibility to DNA damage using comet assay. Genes Chromosomes Cancer 44, 415–422. Shin, V.Y., Wu, W.K.K., Ye, Y.-N., So, W.H.L., Koo, M.W.L., Liu, E.S.L., Luo, J.-C., Cho, C.-H., 2004. Nicotine promotes gastric tumor growth and neovascularization by activating extracellular signal-regulated kinase and cyclooxygenase-2. Carcinogenesis 25, 2487–2495. Sihvo, E.I.T., Salminen, J.T., Rantanen, T.K., R¨am¨o, O.J., Ahotupa, M., F¨arkkil¨a, M., Auvinen, M.I., Salo, J.A., 2002. Oxidative stress has a role in malignant transformation in Barrett’s oesophagus. Int. J. Cancer 102, 551–555.
S. Vogt et al. / Toxicology 222 (2006) 71–79 Singh, R.J., Hogg, N., Joseph, J., Konorev, E., Kalyanaraman, B., 1999. The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch. Biochem. Biophys. 361, 331–339. Sticha, K.R.K., Kenney, P.M.J., Boysen, G., Liang, H., Su, X., Wang, M., Upadhyaya, P., Hecht, S.S., 2002. Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone in A/J mouse lung. Carcinogenesis 23, 1433–1439. Suzuki, H., Iijima, K., Moriya, A., McElroy, K., Scobie, G., Fyfe, V., McColl, K.E.L., 2003. Conditions for acid catalysed luminal nitrosation are maximal at the gastric cardia. Gut 52, 1095–1101. Suzuki, H., Iijima, K., Scobie, G., Fyfe, V., McColl, K.E.L., 2005. Nitrate and nitrosative chemistry within Barrett’s oesophagus during acid reflux. Gut 54, 1527–1535. Terry, P., Lagergren, J., Hansen, H., Wolk, A., Nyr´en, O., 2001. Fruit and vegetable consumption in the prevention of oesophageal and cardia cancers. Eur. J. Cancer Prev. 10, 365–369. Terry, P., Lagergren, J., Ye, W., Nyr´en, O., Wolk, A., 2000. Antioxidants and cancers of the esophagus and gastric cardia. Int. J. Cancer 87, 750–754. Tselepis, C., Morris, C.D., Wakelin, D., Hardy, R., Perry, I., Luong, Q.T., Harper, E., Harrison, R., Attwood, S.E., Jankowski, J.A., 2003. Upregulation of the oncogene c-myc in Barrett’s adenocarcinoma: induction of c-myc by acidified bile acid in vitro. Gut 52, 174–180. Tyroller, S., Zwickenpflug, W., Richter, E., 2002. New sources of dietary myosmine uptake from cereals, fruits, vegetables and milk. J. Agric. Food Chem. 50, 4909–4915. Upadhyaya, P., Sturla, S.J., Tretyakova, N., Ziegel, R., Villalta, P.W., Wang, M.Y., Hecht, S.S., 2003. Identification of adducts produced by the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1butanol with deoxyguanosine and DNA. Chem. Res. Toxicol. 16, 180–190. von Rahden, B.H.A., Stein, H.J., P¨uhringer, F., Koch, I., Langer, R., Piontek, G., Siewert, J.R., H¨ofler, H., Sarbia, M., 2005. Coexpression of cyclooxygenases (COX-1, COX-2) and vascular endothelial growth factors (VEGF-A, VEGF-C) in esophageal adenocarcinoma. Cancer Res. 65, 5038–5044. Wang, L., Spratt, T.E., Liu, X.-K., Hecht, S.S., Pegg, A.E., Peterson, L.A., 1997. Pyridyloxobutyl adduct O6 [4-oxo-4-(3-
79
pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone treated DNA and is a substrate of O6 -alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 10, 562–567. Wang, M., Cheng, G., Sturla, S.J., Shi, Y., McIntee, E.J., Villalta, P.W., Upadhyaya, P., Hecht, S.S., 2003. Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens. Chem. Res. Toxicol. 16, 616–626. Wild, C.P., Hardie, L.J., 2003. Reflux, Barrett’s oesophagus and adenocarcinoma: burning questions. Nature Rev. Cancer 3, 676–685. Wilp, J., Zwickenpflug, W., Richter, E., 2002. Nitrosation of dietary myosmine as risk factor of human cancer. Food Chem. Toxicol. 40, 1223–1228. Wilson, K.T., Fu, S., Ramanujam, K.S., Meltzer, S.J., 1998. Increased expression of inducible nitric oxide synthase and cyclooxygenase2 in Barrett’s esophagus and associated adenocarcinomas. Cancer Res. 58, 2929–2934. Wu, A.H., Wan, P., Bernstein, L., 2001. A multiethnic population-based study of smoking, alcohol and body size and risk of adenocarcinomas of the stomach and esophagus (United States). Cancer Cause Control 12, 721–732. Yu, H., Venkatarangan, L., Wishnok, J.S., Tannenbaum, S.R., 2005. Quantitation of four Guanine oxidation products from reaction of DNA with varying doses of peroxynitrite. Chem. Res. Toxicol. 18, 1849–1857. Zimmermann, K.C., Sarbia, M., Weber, A.-A., Borchard, F., Gabbert, H.E., Schr¨or, K., 1999. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res. 59, 198– 204. Zwickenpflug, W., 2000. N-nitrosation of myosmine yields HPB (4-hydroxy-1-(3-pyridyl)-1-butanone) and NNN (Nnitrosonornicotine). J. Agric. Food Chem. 48, 392–394. Zwickenpflug, W., Meger, M., Richter, E., 1998. Occurrence of the tobacco alkaloid myosmine in nuts and nut products of Arachus hypogaea and Corylus avellana. J. Agric. Food Chem. 46, 2703–2706. Zwickenpflug, W., Tyroller, S., 2005. Reaction of the tobacco alkaloid myosmine with hydrogen peroxide. Chem. Res. Toxicol. [epub ahead].
Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line Sarah Vogt, Katharina Fuchs, Elmar Richter ∗ Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University, Goethestrasse 33, D-80336 Munich, Germany Received 3 January 2006; received in revised form 23 January 2006; accepted 24 January 2006 Available online 28 February 2006
Abstract The incidence of esophageal adenocarcinoma is rapidly rising in Western populations. Gastroesophageal reflux disease (GERD) is thought to be one of the most important risk factors. However, the mechanisms by which GERD enhances tumor formation at the gastroesophageal junction are not well understood. Myosmine is a tobacco alkaloid which has also a wide spread occurrence in human diet. It is readily activated by nitrosation and peroxidation giving rise to the same hydroxypyridylbutanone-releasing DNA adducts as the esophageal carcinogen N -nitrosonornicotine. Therefore, the genotoxicity of myosmine was tested in a human esophageal adenocarcinoma cell line (OE33). DNA damage was assessed by single-cell gel electrophoresis (Comet assay). DNA strand breaks, alkali labile sites and incomplete excision repair were expressed using the Olive tail moment (OTM). The Fapy glycosylase (Fpg) enzyme was incorporated into the assay to reveal additional oxidative DNA damage. DNA migration was determined after incubation of the cells for 1–24 h. Under neutral conditions high myosmine concentrations of 25–50 mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5 mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Coincubation of 25 mM myosmine with 1 mM H2 O2 for 1 h significantly enhanced the genotoxicity of H2 O2 but not the oxidative lesions additionally detected with the Fpg enzyme. In the presence of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) a dose-dependent significant genotoxic effect was obtained with 1–10 mM myosmine after 4 h incubation. NS-398, a selective inhibitor of cyclooxygenase 2, did not affect the SIN-1 stimulated genotoxicity of myosmine. Finally, the 23 h repair of N-methylN -nitro-N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10 mM myosmine. In conclusion, myosmine exerts significant genotoxic effects in esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Esophageal cells; Myosmine; Genotoxicity; Comet assay
1. Introduction The esophageal adenocarcinoma (EAC) has received considerable attention in the last years because of its
∗
Corresponding author. Tel.: +49 89 2180 75742; fax: +49 89 2180 75743. E-mail address: [email protected] (E. Richter).
rapid increase in incidence (Chen and Yang, 2001). In Western populations a yearly increase of ∼10% can be observed and at present EAC is more prevalent than esophageal squamous cell carcinoma (ESCC; Powell et al., 2002). Barrett’s esophagus (BE), a columnar metaplasia of the lower esophageal epithelium, is accepted to be the premalignant condition from which EAC results almost exclusively (Wild and Hardie, 2003). A number of risk factors are known for the development of
0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2006.01.025
72
S. Vogt et al. / Toxicology 222 (2006) 71–79
BE and EAC including gastroesophageal reflux disease (GERD), high body mass index, a diet low in fruits and vegetables and to some extent smoking (Chow et al., 1998; Lagergren et al., 1999; Wu et al., 2001; Terry et al., 2001; DeMeester, 2005). Nevertheless the molecular mechanisms leading to tumor formation are not well understood so far. Genetic instability has been shown to occur early in this process. Loss of heterozygosity, inactivation of tumor suppressor genes and microsatellite instability have all been identified in BE and associated EAC (Olliver et al., 2005; Wilson et al., 1998; Banki et al., 2005; Tselepis et al., 2003). The tobacco-specific nitrosamines (TSNA) 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N -nitrosonornicotine (NNN) arising from nitrosation of nicotine and nornicotine are implicated in smoking-related human cancer (Hecht, 2003). NNN induces esophageal and nasal tumors in rats and is likely to play an important role in the development of esophageal cancer in smokers (Hecht, 1998). Upon metabolic activation by 2 -hydroxylation NNN leads to a pyridyloxobutylating agent which binds to DNA. Thus among others O6 -[4-oxo-4-(3pyridyl)butyl]guanine (O6 -popGua) can be formed, that is highly mutagenic in human cells causing predominantly G → A transitions (Pauly et al., 2002). Additionally O6 -popGua is a substrate for the human O6 -alkylguanine-DNA alkyltransferase (AGT), resulting in increased persistence of promutagenic O6 methylguanine (O6 -mGua; Wang et al., 1997). After DNA hydrolysis 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) is released by the majority of the pyridyloxobutyladducts. These HPB-releasing adducts have been
detected in target tissues of animals treated with NNK and NNN as well as in human lung tissue from smokers (Hecht, 1998; Foiles et al., 1991; Sticha et al., 2002; Boysen et al., 2003). Recently, high levels of HPB-releasing DNA adducts were found not only in the lung but also in the mucosa of esophagus and cardia from sudden death victims. Interestingly, these adducts were independent of the smoking status (H¨olzle et al., 2003). Myosmine, 3-(1-pyrroline-2-yl)pyridine, is a minor tobacco alkaloid but has been detected in a wide variety of food products such as cereals, potatoes, nuts, cocoa and milk (Zwickenpflug et al., 1998; Tyroller et al., 2002). Upon nitrosation myosmine leads to NNN but also directly to a reactive intermediate which pyridyloxobutylates DNA and proteins (Fig. 1; Zwickenpflug, 2000). In vitro experiments showed that myosmine is rapidly nitrosated at pH 2–4 by about 60–80% within 4–8 h (Wilp et al., 2002). These conditions occur in the esophagus by reflux of gastric acid (Moriya et al., 2002). Alternatively, myosmine can also be activated by peroxidation, just as well leading to the formation of HPB adducts (Zwickenpflug and Tyroller, 2005). Genotoxic effects of myosmine have been shown in human lymphocytes and upper aerodigestive tract epithelial cells (Kleinsasser et al., 2003). Alkaline single cell gel electrophoresis (Comet assay) has proven to be a sensitive tool for detecting DNA single and double strand breaks, alkali labile sites and incomplete excision repair (Collins et al., 1997; Faust et al., 2004; Shao et al., 2005). Incorporation of the Fapy glycosylase (Fpg) enzyme into the assay reveals additional damage including 8-hydroxydeoxyguanosine, a common marker of oxidative DNA damage. The enzyme
Fig. 1. Myosmine contributes to HPB releasing DNA and hemoglobin adducts without requiring metabolic activation such as the tobacco-specific nitrosamines NNK and NNN.
S. Vogt et al. / Toxicology 222 (2006) 71–79
recognizes the damaged DNA base and generates strand brakes that can be measured in the comet assay (Collins and Horvathova, 2001).
73
tion (400 mM Tris, pH 7,5), the cells were stained with 50 l ethidium bromide (20 g/ml). 2.3. Analysis and statistics
2. Materials and methods 2.1. Cell culture and drug treatment The cell line OE33 (a gift of Dr. von Rahden, Technical University Munich; ECACC No. 96070808) was established from the adenocarcinoma of the lower esophagus (Barrett metaplasia) of a 73-year-old female patient. Cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen-Gibco, Karlsruhe, Germany) at 37 ◦ C with 5% CO2 . Twenty-four hours prior to drug treatment 2 × 105 cells/well in 1 ml medium were plated in 12well plates. The cells were treated in medium without serum with myosmine (synthesized by Dr. Wolfgang Zwickenpflug in our institute), H2 O2 , 3-morpholinosydnonimine (SIN-1), N[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS398), N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (all from Sigma–Aldrich, Taufkirchen, Germany) for 1–24 h with concentrations indicated in the results. Myosmine and H2 O2 were dissolved in sterile filtered water (membraPure GmbH, Bodenheim, Germany), SIN-1 in phosphate buffered saline (PBS) at pH 5.5 and NS-398 in dimethyl sulfoxide (DMSO, Sigma–Aldrich). Cells for the negative controls were treated with the corresponding solvents. Following treatment, cells were washed with PBS, trypsinized and resuspended in 1 ml complete medium. Cells were separated by centrifugation (10 min, 800 U/min, 4 ◦ C) and resuspended in 25 l PBS. The viability of the treated cells was determined by trypan blue exclusion. Only cells with a viability >70% were used for the comet assay.
For each slide 50 cells were analyzed using a fluorescence microscope (Olympus, Hamburg, Germany) with a CCD camera. The comets were measured using an image analysis system (Comet Assay II Version 2.11, Perceptive Instruments Ltd, Haverhill, UK). To quantify the DNA damage the Olive tail moment (OTM) was used, which reflects the percentage of DNA in the tail of the comet multiplied with the median migration distance. For statistical analysis the Wilcoxon Signed Rank test of the Prism 4 for Windows program (GraphPad Software Inc., San Diego, CA, USA) was applied to compare DNA migration. All experiments were run at least six times. Values are given as mean and the standard error of the mean, respectively.
3. Results Under neutral conditions high myosmine concentrations of 25–50 mM were necessary to elicit a weak genotoxic effect (data not shown). Myosmine is known to be activated under peroxidative and nitrosative conditions (Fig. 1). These conditions are prevalent at the gastroesophageal junction. Chemical nitrosation of myosmine proceeds well at pH values at or below 5 (Wilp et al., 2002). Incubations with myosmine at pH 6 (Fig. 2), the lowest pH value OE33 cells tolerate for longer incubation times, showed a significant dose-dependent increase of DNA migration. OTM values in the comet assay were identical in the negative controls after 4 h (0.51 ± 0.06) and 24 h (0.50 ± 0.11) incubation. A significant increase (P < 0.05) was noted after 4 h incubation with myosmine
2.2. Comet assay Slides with a frosting of 5 mm along the long edges (76 × 26 mm, Langenbrinck, Emmendingen, Germany) were prepared with 85 l of 0.5% agarose (Biozym, Hessisch Oldendorf, Germany) at least one week before the experiment. The cell suspension was mixed with 75 l Low Melting Agarose (Biozym) and applied to the prepared slides. Alkaline lysis (10% DMSO, 1% Triton × 100 in alkaline lysis buffer: 2.5 M NaCl, 10 mM Tris, 100 mM EDTA, pH 10) followed overnight. Fpg (Sigma–Aldrich) treatment was performed according to Collins et al. (1997) with modifications. After 1 h lysis the slides were incubated with 50 l Fpg (1 g/ml) for 30 min. The slides were placed in a horizontal gel electrophoresis chamber (Labtech International, Burkhardtsdorf, Germany), positioned close to the anode and covered with electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13). After a 30 min DNA “unwinding” period, the electrophoresis was performed at 25 V and 300 mA for 30 min. Following 3 min × 5 min neutraliza-
Fig. 2. Concentration dependent DNA damage in OE33 cells after 4 and 24 h incubation with myosmine at pH 6 determined as OTM in the comet assay (mean ± S.E. of six experiments). a Significantly different from the corresponding control values, P < 0.05.
74
S. Vogt et al. / Toxicology 222 (2006) 71–79
Fig. 3. Effect of 1 mM H2 O2 on DNA damage by 25 mM myosmine in OE33 cells after 1 h incubation with or without post-treatment with the Fpg enzyme (mean ± S.E. of 5–13 experiments). a significantly different from controls, P < 0.001; b significantly different from H2 O2 only, P < 0.01; c significantly different from experiments without posttreatment with the Fpg enzyme, P < 0.05.
at 10 mM (1.7-fold to 0.87 ± 0.13) and at 25 mM (4-fold to 2.04 ± 0.56) and after 24 h incubation with myosmine at 5 mM (1.3-fold to 0.67 ± 0.09) and at 25 mM (7-fold to 3.46 ± 1.24), respectively. The differences between 4 and 24 h of incubation with myosmine did not reach significance. The possible influence of peroxidative conditions on myosmine activation was studied by 1 h incubation of 25 mM myosmine in the presence or absence of 1 mM H2 O2 (Fig. 3). Whereas myosmine alone did not increase DNA damage significantly (0.63 ± 0.22 versus 0.45 ± 0.13 in controls; 1.4-fold, n.s.) a highly significant 11-fold increase was observed with H2 O2 alone (4.82 ± 0.82, P < 0.001). However, co-incubation with myosmine increased OTM values 1.7-fold (8.27 ± 1.10) compared to H2 O2 alone (P < 0.01). To evaluate the contribution of oxidative damage, the DNA was incubated after cell lysis with the Fpg enzyme. In the negative control (1.15-fold increase to 0.52 ± 0.08, P < 0.05) and with 25 mM myosmine (1.16-fold decrease to 0.54 ± 0.09, P < 0.05) only minor changes of OTM values were observed. However, as expected, DNA damage by H2 O2 alone was increased 2.5-fold by post-incubation with Fpg (11.8 ± 3.35, P < 0.05). Interestingly, after coincubation with myosmine the Fpg-stimulated DNA damage by H2 O2 was not further increased (12.0 ± 3.1, n.s.). Nitrosative stress, which activates myosmine in vitro, occurs in tissues under conditions of chronic inflammation, where the NO• synthase is induced and NO• and peroxynitrite are released (Bryan et al., 2004). After 4 h incubation with 1 mM of the NO• and/or peroxynitrite donor SIN-1 added to the medium at 0 and 2 h (Doulias
Fig. 4. Effect of 1 mM SIN-1 on DNA damage by myosmine in OE33 cells after 4 h incubation (mean ± S.E. of 6–19 experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a,b Significantly different from control values, P < 0.05 (a) and P < 0.001 (b); c,d significantly different from myosmine only, P < 0.01 (c) and P < 0.001 (d); e,f significantly different from SIN-1 only, P < 0.01 (e) and P < 0.001 (f).
et al., 2001; Martin-Romero et al., 2004), a significantly increased DNA migration was observed compared to the controls (1.59 ± 0.27 versus 0.50 ± 0.07 in controls, 3.2fold, P < 0.001; Fig. 4). Addition of myosmine at concentrations as low as 1, 5 and 10 mM dose-dependently increased the DNA fragmentation versus SIN-1 only by 1.4-fold (2.31 ± 0.89, n.s.), 3.7-fold (5.88 ± 1.62, P < 0.01) and 5.1-fold (8.15 ± 0.99, P < 0.001), respectively. Evaluation of the contribution of oxidative damage by post-treatment of DNA with the Fpg enzyme confirmed the absence of any effect of myosmine (Fig. 5). In
Fig. 5. Effect of 1 mM SIN-1 on DNA damage by myosmine in OE33 cells after 4 h incubation with or without post-treatment with the Fpg enzyme (mean ± S.E. of six experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a Significantly different from control values without post-treatment with the Fpg enzyme, P < 0.05; b significantly different from myosmine only, P < 0.05; c significantly different from corresponding values obtained with myosmine only and SIN-1 only, P < 0.05.
S. Vogt et al. / Toxicology 222 (2006) 71–79
Fig. 6. Effect of 24 h pre-treatment with 50 M of the COX-2 inhibitor NS-398 on DNA damage in OE33 cells after 4 h incubation with 10 mM myosmine alone or in combination with 1 mM SIN-1 (mean ± S.E. of six experiments). SIN-1 was added to the incubation medium at 0 and 2 h. a Significantly different from corresponding control values and from values obtained with myosmine only. P < 0.05.
contrast, DNA damage after treatment with SIN-1 alone increased 2.4-fold after Fpg treatment (2.97 ± 1.46 versus 1.25 ± 0.45, n.s.). Epidemiological, experimental and early clinical evidence indicate that cyclooxygenase-2 (COX-2) represents a potential molecular target for the treatment and/or prevention of esophageal cancer (Altorki, 2004). The protective effect of non-steroidal anti-inflammatory drugs in Barrett’s esophageal carcinogenesis most probably reflects COX-2 inhibition. The selective COX-2 inhibitor NS-398 was shown to reduce COX-2 mRNA expression in OE33 cells in a time- and dose-dependent manner (Cheong et al., 2004). To determine the role of COX-2 for the genotoxicity of myosmine, OE33 cells were treated with 50 M NS-398 24 h before 4 h incubation with myosmine or myosmine and SIN-1 (Fig. 6). Pre-incubation with NS-398 did not affect the DNA damage in controls and in cells treated with 10 mM myosmine. The DNA damaging effect of myosmine and SIN-1 in combination was confirmed (8.34 ± 2.56 versus 0.42 ± 0.05 in controls, 20-fold, P < 0.05). Pretreatment with NS-398 further increased the DNA damage by this combination (12.48 ± 5.87, 1.5-fold, n.s.) but this effect did not reach significance. Finally, the effect of myosmine on the repair of MNNG-induced DNA damage was studied. OE33 cells were incubated for 1 h with 1 M MNNG to induce methyl adducts (Fig. 7). Subsequently cells were allowed to recover in fresh medium for 23 h. During this repair time cells were additionally incubated with 10 mM myosmine, 1 mM SIN-1 or both in combination. After 1 h incubation with 1 M MNNG OTM values increased 53-fold compared to controls (10.6 ± 0.6
75
Fig. 7. Effect 10 mM myosmine and 1 mM SIN-1 alone or in combination on 23 h repair of DNA damage in OE33 cells pretreated for 1 h with 1 M MNNG (mean ± S.E. of six experiments). a Significantly different from control values, P < 0.05; b significantly different from values obtained after 1 h treatment with MNNG, P < 0.05; c significantly different from corresponding values obtained after 1 h treatment with MNNG and 23 h recovery, P < 0.05; d significantly different from values obtained with myosmine only, P < 0.05.
versus 0.20 ± 0.05 in controls, P < 0.05). Incubation of MNNG-treated cells in fresh medium for 23 h significantly decreased DNA damage (0.90 ± 0.16, 12-fold lower than 1 h MNNG and 4.5-fold higher than controls, P < 0.05). In the presence of 10 mM myosmine the MNNG-induced DNA damage decreased only 5.7fold (1.83 ± 0.31, P < 0.05 versus both MNNG only and MNNG + 23 h recovery). Incubation with 1 mM SIN-1 inhibited the repair within 23 h even more efficiently than myosmine (3.85 ± 0.46, 2.7-fold lower than after 1 h MNNG and 4.3-fold higher than after 23 h recovery, both P < 0.05). After co-incubation of myosmine and SIN-1 repair of MNNG-induced DNA damage was lowest (4.44 ± 0.85, 2.4-fold lower than after 1 h MNNG and 5-fold higher than after 23 h recovery, both P < 0.05) but the effect was not significantly different from treatment with SIN-1 only. 4. Discussion Myosmine is present to a considerable extent in sources other than tobacco (Zwickenpflug et al., 1998; Tyroller et al., 2002). In human saliva up to 5 g/L are found independent from the smoking status (Maier et al., 2005). Myosmine can be readily activated by nitrosation or peroxidation under physiological conditions occurring in the mucosa of the lower esophagus especially under reflux conditions. The main nitrosation products are NNN and HPB (Zwickenpflug, 2000; Wilp et al.,
76
S. Vogt et al. / Toxicology 222 (2006) 71–79
2002). NNN is a known esophageal carcinogen and HPB-releasing DNA-adducts are associated with NNNderived esophageal tumors (Hecht, 1998). The ability of myosmine to form DNA adducts was shown in vitro with calf thymus DNA (Wilp et al., 2002). In human lymphocytes and upper aerodigestive tract epithelial cells, myosmine showed dose- and time-dependent genotoxic effects (Kleinsasser et al., 2003). In the present study, myosmine was less genotoxic in OE33 cells compared to freshly isolated human cells. At neutral pH levels, where myosmine is barely nitrosated, incubation of OE33 cells with myosmine lead only to a low increase of DNA damage in the comet assay. Chemical nitrosation of myosmine proceeds well at pH values of 2–5 (Wilp et al., 2002). However, OE33 cells do not tolerate such low pH values. Therefore, incubations with myosmine could only be performed at pH 6. Under these conditions about 20–30% of myosmine are nitrosated within 24 h in the presence of 41 mM nitrite (Wilp et al., 2002). Lowering the pH to 6 considerably enhanced the genotoxic effects of myosmine in OE33 cells leading to a time- and dose-dependent increase of DNA damage with significant effects already at 5 mM myosmine (Fig. 2). This indicates that even under very slight acidic conditions myosmine could have genotoxic effects in the lower esophagus. The concentration of myosmine eliciting a genotoxic effect in vitro is several orders of magnitude higher than the highest concentration detected so far in saliva and plasma from humans approaching 30 nM (Maier et al., 2005). However, there exists no threshold for initiators of carcinogenicity such as nitrosamines (Lijinsky, 1986; Peto et al., 1991). Therefore, in the absence of a no-effect level, myosmine could have a relevant genotoxic effect in vivo after long-term exposure to much lower doses. At lower pH values as frequently occurring in chronic reflux disease, activation of myosmine should be much more effective. The gastric cardia and, during acid reflux, Barrett’s segments are the anatomical sites with maximal potential for acid catalyzed nitrosation (Suzuki et al., 2003, 2005). This could explain the presence of high levels of DNA adducts releasing HPB upon acid hydrolysis in the mucosae of human esophagus and cardia (H¨olzle et al., 2003). Interestingly, these adduct levels were independent from the smoking status as were the concentrations of myosmine in human saliva (Maier et al., 2005). Oxidative stress has an important role for formation and development of adenocarcinoma (Chen and Yang, 2001). Enzymatic changes and decreased levels of glutathione in BE are consistent with increased formation of hydrogen peroxide and oxygen free radicals in patients with reflux disease (Sihvo et al., 2002; M¨ork et al.,
2003). This is in line with epidemiological data showing a protective effect of a high intake of antioxidants with fruits and vegetables (Terry et al., 2000). To determine if enhanced endogenous levels of H2 O2 under oxidative stress could affect myosmine-induced genotoxicity via peroxidation, OE33 cells were co-incubated with myosmine and H2 O2 (Fig. 3). A non-genotoxic concentration of myosmine significantly increased DNA damage by H2 O2 . Since H2 O2 mainly gives rise to oxidative DNA damage, the effect of myosmine could be due to an increase of oxidative stress. However, post-incubation with the Fpg enzyme revealing oxidative DNA damage after treatment with H2 O2 alone did not result in a further increase after co-incubation of H2 O2 with myosmine. Therefore, the increased DNA damage by myosmine is most probably not due to additional oxidative DNA damage. It is rather likely that H2 O2 activates myosmine to genotoxic intermediates (Zwickenpflug and Tyroller, 2005). Chronic gastroesophageal reflux and BE are accompanied by inflammation which leads to the release of reactive species. Expression of inducible NO• synthase is increased in mucosal tissue from patients with BE and EAC (Wilson et al., 1998; Bove et al., 2005). In a rat model of EAC increased nitrotyrosine immunostaining as a marker of peroxynitrite formation was demonstrated (Goldstein et al., 1998). 3morpholinosydnonimine (SIN-1), the reactive metabolite of molsidomine, is often used for in vitro release of NO• , O2 − , peroxynitrite and hydroxyl radicals. After a single dose of 1 mM SIN-1 no genotoxic effect was observed (data not shown). However, in experiments lasting 4 h a second dose of 1 mM SIN-1 added to the medium after 2 h significantly increased DNA damage (Fig. 4). Under these conditions, co-incubation with myosmine caused a strong concentration-dependent increase of DNA damage elicited by SIN-1 alone. Similar to the experiments with H2 O2 , post-treatment with the Fpg enzyme increased the OTM values about twofold (Fig. 5) confirming the oxidative nature of DNA damage by the peroxynitrite donor SIN-1 (Yu et al., 2005). Again, co-treatment with myosmine did not show this Fpg-dependent increase of OTM values. Therefore, SIN-1 may also increase the formation of reactive intermediates from myosmine. From the present experiment it is not clear whether this increase is mediated by nitrosative or peroxidative mechanisms. In an aqueous oxygen-saturated solution of neutral pH, SIN-1 decomposes rapidly to nitric oxide and superoxide anions, generating peroxynitrite. However, at relatively low in vivo oxygen concentrations, SIN-1 reacts with alternative electron acceptors and behaves more like a NO•
S. Vogt et al. / Toxicology 222 (2006) 71–79
donor (Singh et al., 1999). In experiments with cultured cells, the oxygen above the cell media will be consumed after approximately half an hour (Doulias et al., 2001). Observational studies evaluating the association of aspirin/nonsteroidal anti-inflammatory drug use and esophageal cancer indicate a reduced risk. This is most probably mediated by inhibition of cyclooxygenase (COX)-2 (Corley et al., 2003; Zimmermann et al., 1999). COX-2 is predominantly expressed at sites of inflammation and catalyzes the production of prostaglandins that stimulate cancer cell proliferation, inhibit apoptosis and enhance cancer induced angiogenesis and invasiveness (Altorki, 2004). COX-2 is up-regulated in Barrett’s esophageal carcinogenesis (Wilson et al., 1998; Zimmermann et al., 1999; Buskens et al., 2002; Cheong et al., 2003; von Rahden et al., 2005). Recently, nicotine has been shown to activate COX-2 in a gastric adenocarcinoma cell line (Shin et al., 2004). On the other hand, cyclooxygenases are able to activate carcinogens such as NNK (Rioux and Castonguay, 1998). Therefore, myosmine could possibly promote its own activation by induction of COX-2. The selective COX-2 inhibitor NS-398 suppresses COX-2 mRNA expression in OE33 cells in a time- and dose-dependent manner (Cheong et al., 2004). Therefore, pre-incubation with NS-398 should have a protective effect in OE33 cells if COX-2 is involved in myosmine genotoxicity. This was clearly not the case (Fig. 6). DNA damage in cells incubated with myosmine and SIN-1 was even higher after 24 h pre-incubation with the inhibitor. O6 -Alkylguanine is formed in DNA by alkylating compounds and it may mispair with thymine during replication thereby leading to mutagenesis and malignant transformation. In human cells O6 -methylguanine (O6 -mG) and other alkylated bases are repaired by O6 alkylguanine-DNA alkyltransferase (AGT) which is a suicide enzyme that is inactivated by the process of repair (Gerson, 2004; Laval and Wink, 1994). One of the TSNA-induced HPB-releasing adducts has been identified as O6 -[1-oxo-1-(3-pyridyl)but-4-yl]-d-guanosine (O6 -pobG). This highly mutagenic adduct (Pauly et al., 2002) may not only stem from metabolic activation of NNN and NNK (Wang et al., 2003; Upadhyaya et al., 2003) but probably also from nitrosative and/or peroxidative activation of myosmine (Fig. 1). In addition to its inherent mutagenic activity, O6 -pobG enhances the carcinogenesis of NNK in A/J mice through inhibition of O6 -mGua repair (Liu et al., 1999; Pegg, 2000). The inhibition by O6 -pobG is similar to that by other bulky O6 alkylguanine adducts which are adjuvants in chemotherapy (Mijal et al., 2004; Mijal et al., 2005; Nelson et al., 2004) Therefore it was tested if myosmine also inhibits
77
the repair of O6 -mG in OE33 cells (Fig. 7). After 1 h incubation with the methylating agent MNNG, cells were grown for 23 h with fresh medium with or without addition of 10 mM myosmine and/or 1 mM SIN-1. The addition of myosmine doubled the remaining damage, indicating a inhibition of AGT. The even stronger effect of SIN-1 was expected because Laval and Wink (1994) showed that exogenously released NO• inhibits the AGT in cells. In conclusion, myosmine is significantly genotoxic to esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation. By inhibition of O6 -alkylguanine-DNA alkyltransferase, myosmine could also enhance the carcinogenicity of other alkylating agents. Acknowledgements Supported in part by Philip Morris USA Inc. and Philip Morris International. References Altorki, N., 2004. COX-2: a target for prevention and treatment of esophageal cancer. J. Surg. Res. 117, 114–120. Banki, F., DeMeester, S.R., Mason, R.J., Campos, G., Hagen, J.A., Peters, J.H., Bremner, C.G., DeMeester, T.R., 2005. Barrett’s esophagus in females: a comparative analysis of risk factors in females and males. Am. J. Gastroenterol. 100, 560–567. Bove, M., Vieth, M., Casselbrant, A., Ny, L., Lundell, L., Ruth, M., 2005. Acid challenge to the esophageal mucosa: effects on local nitric oxide formation and its relation to epithelial functions. Digest. Dis. Sci. 50, 640–648. Boysen, G., Kenney, P.M.J., Upadhyaya, P., Wang, M., Hecht, S.S., 2003. Effects of benzyl isothiocyanate and 2-phenethyl isothiocyanate on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone metabolism in F-344 rats. Carcinogenesis 24, 517–525. Bryan, N.S., Rassaf, T., Maloney, R.E., Rodriguez, C.M., Saijo, F., Rodriguez, J.R., Feelisch, M., 2004. Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc. Natl. Acad. Sci. U.S.A. 101, 4308–4313. Buskens, C.J., van Rees, B.P., Sivula, A., Reitsma, J.B., Haglund, C., Bosma, P.J., Offerhaus, G.J., van Lanschot, J.J., Ristim¨aki, A., 2002. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus. Gastroenterology 122, 1800–1807. Chen, X.X., Yang, C.S., 2001. Esophageal adenocarcinoma: a review and perspectives on the mechanism of carcinogenesis and chemoprevention. Carcinogenesis 22, 1119–1129. Cheong, E., Igali, L., Harvey, I., Mole, M., Lund, E., Johnson, I.T., Rhodes, M., 2003. Cyclo-oxygenase-2 expression in Barrett’s oesophageal carcinogenesis: an immunohistochemical study. Aliment. Pharmacol. Therap. 17, 379–386. Cheong, E., Ivory, K., Doleman, J., Parker, M.L., Rhodes, M., Johnson, I.T., 2004. Synthetic and naturally occurring COX-2 inhibitors sup-
78
S. Vogt et al. / Toxicology 222 (2006) 71–79
press proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest. Carcinogenesis 25, 1945–1952. Chow, W.-H., Blot, W.J., Vaughn, T.L., Risch, H.A., Gammon, M.D., Stanford, J.L., Dubrow, R., Schoenberg, J.B., Mayne, S.T., Farrow, D.C., Ahsan, H., West, A.B., Rotterdam, H., Niwa, S., Fraumeni, J.F., 1998. Body mass index and risk of adenocarcinomas of the esophagus and gastric cardia. J. Natl. Cancer Inst. 90, 150–155. Collins, A.R., Dobson, V.L., Dusinsk´a, M., Kennedy, G., Stetina, R., 1997. The comet assay: what can it really tell us? Mutat. Res. 375, 183–193. Collins, A.R., Horvathova, E., 2001. Oxidative DNA damage, antioxidants and DNA repair: applications of the comet assay. Biochem. Soc. Trans. 29, 337–341. Corley, D.A., Kerlikowske, K., Verma, R., Buffler, P., 2003. Protective association of aspirin/NSAIDs and esophageal cancer: a systematic review and meta-analysis. Gastroenterology 124, 47–56. DeMeester, S., 2005. Adenocarcinoma of the esophagus and cardia: a review of the disease and its treatment. Ann. Surg. Oncol. 13, 1–19. Doulias, P.T., Barbouti, A., Galaris, D., Ischiropoulos, H., 2001. SIN-1induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay). Free Radic. Biol. Med. 30, 679–685. Faust, F., Kassie, F., Knasm¨uller, S., Boedecker, R.H., Mann, M., Mersch-Sundermann, V., 2004. The use of the alkaline comet assay with lymphocytes in human biomonitoring studies. Mutat. Res. 566, 209–229. Foiles, P.G., Akerkar, S.A., Carmella, S.G., Kagan, M., Stoner, G.D., Resau, J.H., Hecht, S.S., 1991. Mass spectrometric analysis of tobacco-specific nitrosamine DNA-adducts in smokers and nonsmokers. Chem. Res. Toxicol. 4, 364–368. Gerson, S.L., 2004. MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev. Cancer 4, 296–307. Goldstein, S.R., Yang, G.-Y., Chen, X., Curtis, S.K., Yang, C.S., 1998. Studies of iron deposits, inducible nitric oxide synthase and nitrotyrosine in a rat model for esophageal adenocarcinoma. Carcinogenesis 19, 1445–1449. Hecht, S.S., 1998. Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 559–603. Hecht, S.S., 2003. Tobacco carcinogens, their biomarkers and tobaccoinduced cancer. Nat. Rev. Cancer 3, 733–744. H¨olzle, D., Schl¨obe, D., Richter, E., Ostermeier-Hatz, D., von Meyer, L., Tricker, A.R., 2003. 4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung, esophagus and cardia of sudden death victims. Proc. Am. Ass. Cancer Res. 44 (2nd ed.), 1281–1282. Kleinsasser, N.H., Wallner, B.C., Harr´eus, U.A., Zwickenpflug, W., Richter, E., 2003. Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells. Toxicology 192, 171–177. Lagergren, J., Bergstr¨om, R., Nyr´en, O., 1999. Association between body mass and adenocarcinoma of the esophagus and gastric cardia. Ann. Intern. Med. 130, 883–890. Laval, F., Wink, D.A., 1994. Inhibition by nitric oxide of the repair protein, O6 -methylguanine-DNA-methyltransferase. Carcinogenesis 15, 443–447. Lijinsky, W., 1986. The significance of N-nitroso compounds as environmental carcinogens. J. Environ. Sci. Health C4, 1–45. Liu, L.L., Qin, X., Gerson, S.L., 1999. Reduced lung tumorigenesis in human methylguanine DNA-methyltransferase transgenic mice
achieved by expression of transgene within the target cell. Carcinogenesis 20, 279–284. Maier, M., Schulze, A., Richter, E., 2005. Biomonitoring of myosmine in human milk and saliva. Toxicol. Lett. 158, S201–S202. Martin-Romero, F.J., Gutierrez-Martin, Y., Henao, F., GutierrezMerino, C., 2004. Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123. J. Fluoresc. 14, 17–23. Mijal, R.S., Loktionova, N.A., Vu, C.C., Pegg, A.E., Peterson, L.A., 2005. O6 -Pyridyloxobutylguanine adducts contribute to the mutagenic properties of pyridyloxobutylating agents. Chem. Res. Toxicol. 18, 1619–1625. Mijal, R.S., Thomson, N.M., Fleischer, N.L., Pauly, G.T., Moschel, R.C., Kanugula, S., Fang, Q., Pegg, A.E., Peterson, L.A., 2004. The repair of the tobacco specific nitrosamine derived adduct O6 -[4-oxo-4-(3-pyridyl)butyl]guanine by O6 -alkylguanine-DNA alkyltransferase variants. Chem. Res. Toxicol. 17, 424–434. Moriya, A., Grant, J., Mowat, C., Williams, C., Carswell, A., Preston, T., Anderson, S., Iijima, K., McColl, K.E.L., 2002. In vitro studies indicate that acid catalysed generation of N-nitrosocompounds from dietary nitrate will be maximal at the gastro-oesophageal junction and cardia. Scand. J. Gastroenterol. 37, 253–261. M¨ork, H., Scheurlen, M., Al-Taie, O., Zierer, A., Kraus, M., Sch¨ottker, K., Jakob, F., K¨ohrle, J., 2003. Glutathione peroxidase isoforms as part of the local antioxidative defense system in normal and Barrett’s esophagus. Int. J. Cancer 105, 300–304. Nelson, M.E., Loktionova, N.A., Pegg, A.E., Moschel, R.C., 2004. 2-amino-O4 -benzylpteridine derivatives: potent inactivators of O6 -alkylguanine-DNA alkyltransferase. J. Med. Chem. 47, 3887–3891. Olliver, J.R., Hardie, L.J., Gong, Y., Dexter, S., Chalmers, D., Harris, K.M., Wild, C.P., 2005. Risk factors, DNA damage, and disease progression in Barrett’s esophagus. Cancer Epidemiol. Biomar. Prev. 14, 620–625. Pauly, G.T., Peterson, L.A., Moschel, R.C., 2002. Mutagenesis by O6 [4-oxo-4-(3-pyridyl)butyl]guanine in Escherichia coli and human cells. Chem. Res. Toxicol. 15, 165–169. Pegg, A.E., 2000. Repair of O(6)-alkylguanine by alkyltransferases. Mutat. Res. 462, 83–100. Peto, R., Gray, R., Brantom, P., Grasso, P., 1991. Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of N-nitrosodiethylamine or Nnitrosodimethylamine. Cancer Res. 51, 6452–6469. Powell, J., McConkey, C.C., Gillison, E.W., Spychal, R.T., 2002. Continuing rising trend in oesophageal adenocarcinoma. Int. J. Cancer 102, 422–427. Rioux, N., Castonguay, A., 1998. Prevention of NNK-induced lung tumorigenesis in A/J mice by acetylsalicylic acid and NS-398. Cancer Res. 58, 5354–5360. Shao, L., Lin, J., Huang, M., Ajani, J.A., Wu, X., 2005. Predictors of esophageal cancer risk: assessment of susceptibility to DNA damage using comet assay. Genes Chromosomes Cancer 44, 415–422. Shin, V.Y., Wu, W.K.K., Ye, Y.-N., So, W.H.L., Koo, M.W.L., Liu, E.S.L., Luo, J.-C., Cho, C.-H., 2004. Nicotine promotes gastric tumor growth and neovascularization by activating extracellular signal-regulated kinase and cyclooxygenase-2. Carcinogenesis 25, 2487–2495. Sihvo, E.I.T., Salminen, J.T., Rantanen, T.K., R¨am¨o, O.J., Ahotupa, M., F¨arkkil¨a, M., Auvinen, M.I., Salo, J.A., 2002. Oxidative stress has a role in malignant transformation in Barrett’s oesophagus. Int. J. Cancer 102, 551–555.
S. Vogt et al. / Toxicology 222 (2006) 71–79 Singh, R.J., Hogg, N., Joseph, J., Konorev, E., Kalyanaraman, B., 1999. The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch. Biochem. Biophys. 361, 331–339. Sticha, K.R.K., Kenney, P.M.J., Boysen, G., Liang, H., Su, X., Wang, M., Upadhyaya, P., Hecht, S.S., 2002. Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone in A/J mouse lung. Carcinogenesis 23, 1433–1439. Suzuki, H., Iijima, K., Moriya, A., McElroy, K., Scobie, G., Fyfe, V., McColl, K.E.L., 2003. Conditions for acid catalysed luminal nitrosation are maximal at the gastric cardia. Gut 52, 1095–1101. Suzuki, H., Iijima, K., Scobie, G., Fyfe, V., McColl, K.E.L., 2005. Nitrate and nitrosative chemistry within Barrett’s oesophagus during acid reflux. Gut 54, 1527–1535. Terry, P., Lagergren, J., Hansen, H., Wolk, A., Nyr´en, O., 2001. Fruit and vegetable consumption in the prevention of oesophageal and cardia cancers. Eur. J. Cancer Prev. 10, 365–369. Terry, P., Lagergren, J., Ye, W., Nyr´en, O., Wolk, A., 2000. Antioxidants and cancers of the esophagus and gastric cardia. Int. J. Cancer 87, 750–754. Tselepis, C., Morris, C.D., Wakelin, D., Hardy, R., Perry, I., Luong, Q.T., Harper, E., Harrison, R., Attwood, S.E., Jankowski, J.A., 2003. Upregulation of the oncogene c-myc in Barrett’s adenocarcinoma: induction of c-myc by acidified bile acid in vitro. Gut 52, 174–180. Tyroller, S., Zwickenpflug, W., Richter, E., 2002. New sources of dietary myosmine uptake from cereals, fruits, vegetables and milk. J. Agric. Food Chem. 50, 4909–4915. Upadhyaya, P., Sturla, S.J., Tretyakova, N., Ziegel, R., Villalta, P.W., Wang, M.Y., Hecht, S.S., 2003. Identification of adducts produced by the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1butanol with deoxyguanosine and DNA. Chem. Res. Toxicol. 16, 180–190. von Rahden, B.H.A., Stein, H.J., P¨uhringer, F., Koch, I., Langer, R., Piontek, G., Siewert, J.R., H¨ofler, H., Sarbia, M., 2005. Coexpression of cyclooxygenases (COX-1, COX-2) and vascular endothelial growth factors (VEGF-A, VEGF-C) in esophageal adenocarcinoma. Cancer Res. 65, 5038–5044. Wang, L., Spratt, T.E., Liu, X.-K., Hecht, S.S., Pegg, A.E., Peterson, L.A., 1997. Pyridyloxobutyl adduct O6 [4-oxo-4-(3-
79
pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone treated DNA and is a substrate of O6 -alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 10, 562–567. Wang, M., Cheng, G., Sturla, S.J., Shi, Y., McIntee, E.J., Villalta, P.W., Upadhyaya, P., Hecht, S.S., 2003. Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens. Chem. Res. Toxicol. 16, 616–626. Wild, C.P., Hardie, L.J., 2003. Reflux, Barrett’s oesophagus and adenocarcinoma: burning questions. Nature Rev. Cancer 3, 676–685. Wilp, J., Zwickenpflug, W., Richter, E., 2002. Nitrosation of dietary myosmine as risk factor of human cancer. Food Chem. Toxicol. 40, 1223–1228. Wilson, K.T., Fu, S., Ramanujam, K.S., Meltzer, S.J., 1998. Increased expression of inducible nitric oxide synthase and cyclooxygenase2 in Barrett’s esophagus and associated adenocarcinomas. Cancer Res. 58, 2929–2934. Wu, A.H., Wan, P., Bernstein, L., 2001. A multiethnic population-based study of smoking, alcohol and body size and risk of adenocarcinomas of the stomach and esophagus (United States). Cancer Cause Control 12, 721–732. Yu, H., Venkatarangan, L., Wishnok, J.S., Tannenbaum, S.R., 2005. Quantitation of four Guanine oxidation products from reaction of DNA with varying doses of peroxynitrite. Chem. Res. Toxicol. 18, 1849–1857. Zimmermann, K.C., Sarbia, M., Weber, A.-A., Borchard, F., Gabbert, H.E., Schr¨or, K., 1999. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res. 59, 198– 204. Zwickenpflug, W., 2000. N-nitrosation of myosmine yields HPB (4-hydroxy-1-(3-pyridyl)-1-butanone) and NNN (Nnitrosonornicotine). J. Agric. Food Chem. 48, 392–394. Zwickenpflug, W., Meger, M., Richter, E., 1998. Occurrence of the tobacco alkaloid myosmine in nuts and nut products of Arachus hypogaea and Corylus avellana. J. Agric. Food Chem. 46, 2703–2706. Zwickenpflug, W., Tyroller, S., 2005. Reaction of the tobacco alkaloid myosmine with hydrogen peroxide. Chem. Res. Toxicol. [epub ahead].