Abstract LB-63: (S)-N′-nitrosonornicotine, a constituent of smokeless tobacco, is a potent oral tumorigen in rats
Authors: Balbo Silvia , James-Yi Sandra , O'Sullivan M. Gerard , Stepanov Irina , Wang Mingyao , Zhang Siyi , Kassie Fekadu , Carmella Steven , Wettlaufer Courtney , Hohol Kelsey , Knezevich Aleks , Upadhyaya Pramod , Hecht Stephen S. 
Published: August 2012

Abstract
Smokeless tobacco products, consisting most commonly of moist snuff placed in the mouth, either directly or in sachets, are gaining popularity in the U.S. In 2009, 7.0% of men and 11.0% of male high school students were current users. While smokeless tobacco use is unquestionably less harmful than cigarette smoking, it is nevertheless a recognized cause of oral cancer. A quantitatively important carcinogen in all smokeless tobacco products is N′-nitrosonornicotine (NNN), occurring at levels ranging from 0.4 - 17 µg/g dry weight in current products consumed in the U.S. These amounts are far higher than those of nitrosamines in other consumer products. NNN has a chiral center at its 2′-position and consequently exists as enantiomers. The major enantiomer in tobacco products is (S)-NNN. Racemic NNN, administered to F-344 rats in the drinking water, is known to induce tumors of the esophagus in rats, but there are no reports in the literature on the carcinogenicity of (S)-NNN. Based on DNA binding studies of (S)-NNN that demonstrated relatively high adduct levels in both the rat esophagus and oral cavity, we initiated a carcinogenicity study. Groups of 24 male F-344 rats, 7 weeks of age, were treated with (S)-NNN or (R)-NNN (15 ppm in the drinking water) or racemic NNN (15 rats, 30 ppm), or were given tap water. All rats in the groups treated with (S)-NNN or racemic NNN began losing weight after one year of treatment and had died or were euthanized for humane reasons by 17 months of treatment, while the rats given (R)-NNN and the controls were terminated at 20 months. Tumors were counted blinded to treatment. Necropsy of 20 (S)-NNN treated rats demonstrated a 100% incidence of oral tumors and a total of 91 oral tumors, including tumors of the tongue (1.5 tumors per rat), buccal mucosa (1.0), soft palate (0.5), and pharynx (0.75) in addition to esophageal tumors in all rats (6.1 tumors per rat). Some of the oral tumors were &gt;4 mm in size: tongue (9 tumors); buccal mucosa (2); soft palate (4); pharynx (5). (R)-NNN induced oral tumors in only 5 of 24 rats and esophageal tumors in 3, while racemic NNN was also highly active causing 153 esophageal tumors and 96 oral tumors in 12 necropsied rats. Preliminary histopathological analysis of the tumors indicated that they encompass a spectrum from benign squamous papillomas to malignant squamous cell carcinomas. These results clearly demonstrate, for the first time, the strong carcinogenicity of (S)-NNN in the rat oral cavity. Thus, (S)-NNN is the only potent oral carcinogen identified in smokeless tobacco. There is an urgent need to eliminate this powerful carcinogen from tobacco products.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-63. doi:1538-7445.AM2012-LB-63