Identification of strong oral carcinogen in smokeless tobacco

The chemical (S)-N'-nitrosonornicotine, or (S)-NNN, which is present in smokeless tobacco products, is a strong oral carcinogen, according to results presented at the AACR Annual Meeting 2012, being held March 31 - April 4. Although smokeless tobacco products have long been linked with certain cancers, including oral cavity cancers and esophageal cancers, this is the first study to identify a specific chemical present in smokeless tobacco products that induces oral cancer in animals, according to Silvia Balbo, Ph.D., research associate at the Masonic Cancer Center of the University of Minnesota in Minneapolis, Minn. "(S)-NNN is the only chemical in smokeless tobacco known to cause oral cancer," Balbo said. "This finding provides mechanistic underpinning for the epidemiologic observations that smokeless tobacco products cause oral cancer." Balbo and colleagues administered two forms of NNN called (S)-NNN and (R)-NNN to four groups of 24 rats. The rats were given either (S)-NNN alone, (R)-NNN alone, a combination of both or tap water. The total dose was approximately equivalent to the amount of (S)-NNN to which a smokeless tobacco user would be exposed from chronic use of these products. All rats assigned to (S)-NNN alone or the combination began losing weight after one year of exposure and died by 17 months. Rats assigned to (R)-NNN or tap water were terminated at 20 months. All rats assigned to (S)-NNN had esophageal tumors and demonstrated 100 percent incidence of oral tumors including tumors of the tongue, buccal mucosa, soft palate and pharynx. In contrast, researchers found oral tumors in only five of 24 rats given (R)-NNN and esophageal tumors in three of 24 rats assigned to (R)-NNN. Twelve rats given the combination of (S)-NNN and (R)-NNN had 153 esophageal tumors and 96 oral tumors. "Measures should be taken to reduce this chemical in smokeless tobacco," Balbo said. "If it is not possible to stop the use of smokeless tobacco products, we should advocate for a reduction of this chemical in these products." Because the Food and Drug Administration regulates tobacco products, Balbo said she hoped these results will inform regulatory decisions. Moving forward, she and her colleagues hope to identify other chemicals that may be carcinogens in smokeless tobacco and to understand what level of these chemicals is present in smokeless tobacco products. "In addition, we have to understand how this research translates to human beings," Balbo added. "We have to understand the uptake of NNN from smokeless tobacco products in humans and develop better biomarkers, such as urinary biomarkers, to have a tool to monitor the levels to which smokeless tobacco users are exposed." Balbo believes these findings are yet another affirmation that tobacco products should be avoided. 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 >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.