Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line

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 1-24h. Under neutral conditions high myosmine concentrations of 25-50mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Co-incubation of 25 mM myosmine with 1mM H(2)O(2) for 1h significantly enhanced the genotoxicity of H(2)O(2) 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 1-10mM myosmine after 4h 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-methyl-N'-nitro-N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10mM 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.