Mechanistic Basis for the Chemopreventive Effects of Black Raspberries at a Late Stage of Rat Esophageal Carcinogenesis Li-Shu Wang, 1 Alan A. Dombkowski, 2 Claire Seguin, 1 Claudia Rocha, 1 Daniela Cukovic, 2 Anju Mukundan, 2 Cassandra Henry, 1 and Gary D. Stoner 1 * 1 Ohio State University Comprehensive Cancer Center, Columbus, Ohio 2 Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA- and NMBA þ BRB- treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA- treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17 846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17 846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell–cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis. ß 2011 Wiley-Liss, Inc. Key words: black raspberries; rat esophagus; microarray; chemoprevention INTRODUCTION The Fischer 344 (F344) rat has been used exten- sively as a model for squamous cell carcinoma (SCC) of the esophagus, the most prevalent type of esophageal cancer worldwide [1]. In this model, esophageal tumors are induced routinely by treat- ment of rats with the nitrosamine carcinogen, N-nitrosomethylbenzylamine (NMBA) [2]. In a typ- ical bioassay, s.c. injections of NMBA at 0.25 – 0.5 mg/ kg body weight three times a week for 5 wk or once per week for 15 wk results in 100% tumor incidence by 20 – 25 wk [3]. Our laboratory has used this model since the early 1980s to identify and determine the mechanism(s) of action of putative chemopreven- tive agents for esophageal cancer [4]. We reported that the addition of black raspberry (BRB) powder to the diet of NMBA-treated rats at concentrations of 5% and 10% results in a 39–64% reduction in the number of esophageal tumors [5]. Recent studies suggest that amongst the most active chemopreven- tive agents in BRBs are the anthocyanins which comprise about 5% of the dry weight of the berries [6]. An investigation of the effects of BRBs on early molecular events in rat esophageal tumorigenesis indicated that the berries elicit a ‘‘genome wide’’ effect on gene expression in NMBA-treated esoph- agus [7]. In this study, 4- to 5-wk-male F344 rats were fed a 5% BRB diet for a period of 3 wk and, in the 3rd wk of berry treatment, the rats received three s.c. injections of NMBA (0.5 mg/kg). Twenty-four hours after the last NMBA treatment, rats were harvested, esophagi removed, and total RNA isolated from the epithelium for cDNA microarray analysis of the expression of 41 000 genes. The three injections of NMBA during 1 wk led to dysregulation of 2261 genes in the esophagus, and the 5% BRB diet MOLECULAR CARCINOGENESIS 50:291–300 (2011) ß 2011 WILEY-LISS, INC. Additional Supporting Information may be found in the online version of this article. Abbreviations: SCC, squamous cell carcinoma; NMBA, N-nitro- somethylbenzylamine; BRBs, freeze-dried black raspberries; PE, preneo- plastic esophagus; DMSO, dimethyl sulfoxide; PGE 2 , prostaglandin E 2 ; LTB 4 , leukotriene B 4 ; MMP10, matrix metalloproteinase 10. *Correspondence to: Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, 2001 Polaris Pkwy, Columbus, OH 43240. Received 10 September 2009; Revised 9 February 2010; Accepted 17 February 2010 DOI 10.1002/mc.20634 Published online in Wiley Online Library (wileyonlinelibrary.com).