Brand specific responses to smokeless tobacco in a rat lip canal model Joel L. Schwartz 1,2 , Klaus D. Brunnemann 3 , Alexander J. Adami 1 , Suchismita Panda 1 , Sara C. Gordon 1 , Dietrich Hoffmann 2 , Guy R. Adami 1,4 1 Department of Oral Medicine and Diagnostics, University of Illinois at Chicago, Chicago, IL, USA; 2 American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, NY, USA [no longer operating]; 3 Department of Pathology, New York Medical College, Valhalla, NY, USA; 4 Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, IL, USA BACKGROUND: Different compositions of smokeless tobacco (ST) are widely thought to cause oral carcinoma at different rates but there is little direct evidence for this hypothesis. METHODS: We used a rat lip canal model to examine the mucosal changes induced by chronic daily exposure to four different brands of ST: Skoal, Copenhagen, Ettan Swedish Snus, and Stonewall, differing in measured levels of: tobacco specific nitrosamines (TSNAs), unprotonated nicotine, moisture, and pH. RESULTS: Exposure to the lip canal for 12 months pro- duced changes in the mucosa marked by increases in S phase and M phase cells for the Skoal and Copenhagen exposed rats. This correlated with the high level of TSNAs and nicotine in these products. All the tobacco products, to different degrees, induced sites of moderate to severe dysplasia some with extensive rete peg out- growth from the oral mucosa not seen in the controls. Many of these sites showed a loss of p16 expression. CONCLUSIONS: While all ST products caused dyspla- sia, the products with lower levels of TSNAs and un- protonated nicotine caused less, consistent with the model that tobacco with low levels of nitrosamines might potentially induce fewer carcinomas in human users. J Oral Pathol Med (2010) 39: 453–459 Keywords: dysplasia; lip canal; nitrosamines; smokeless tobacco; squamous cell carcinoma Introduction Smokeless tobacco (ST) can cause experimental and clinical oral squamous cell carcinoma (OSCC) (Surgeon General Report 2003) and is associated with snuff dippers keratosis, gingivitis, periodontitis, gingival recession, epithelial attachment, and caries (1). A recent Federal Trade Commission report stated the number of pounds of ST sold in the United States as of 2005 had increased by 3% since 2002. In part as a result of increases in the number of municipal bans on tobacco smoke, and increased taxing of tobacco smoke products, a shift to ST products is occurring. Usage of ST in the form of loose tobacco or snuff correlates with increased rates of oral carcinogenesis in humans, although at lower rates than that caused by smoking tobacco (2–5). Animal models have been used to explore a causative role. Rats surgerized to create a lip canal lined by oral mucosa, where ST is placed twice daily showed increased rates of malignant oral tumors (3, 6). The surgery and subsequent inflammation, along with mechanical injury resulting from tobacco place- ment, may have contributed to the tumor induction, although controls with cotton instead of ST exposure showed no dysplasia or tumors (6, 7). Additional studies, with the application of purified nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and NO nitrosonornicotine (NNN), the predominant carcinogenic tobacco specific nitrosamines (TSNAs) in ST, have identified these chemicals as genotoxins and as oral carcinogens (8). These TSNAs, in combination with nicotine, hydrogen peroxide or additional agents in- duced oral tumors in non-surgerized rat mucosa and in the hamster cheek pouch, and with snuff in the rat lip canal (9, 10). This is suggestive of a causative role of TSNAs and ST in the oral carcinogenesis in humans. Relative short term usage of snuff ST by humans induces characteristic non-neoplastic mucosal change known as snuff dippers’ lesion. This is frequently a white corrugated lesion of the oral mucosa typically at the site of ST placement which can remain for years with continued usage. Microscopically, increased keratin (hyperkeratosis) and epithelial thickening (which can be hyperplasia) are seen which is rarely accompanied by Correspondence: Joel L. Schwartz, Department of Oral Medicine and Diagnostics, University of Illinois at Chicago, 801 S. Paulina St. M ⁄ C 838. Chicago, IL 60612-7213, USA. Tel: +1 312 355 4311, Fax: +1 312 355 2688, E-mail: joschwar@uic.edu Accepted for publication February 7, 2010 doi: 10.1111/j.1600-0714.2010.00892.x J Oral Pathol Med (2010) 39: 453–459 ª 2010 John Wiley & Sons A/S Æ All rights reserved interscience.wiley.com/journal/jop