Effects of Black Raspberry diet on N-Nitrosomethylbenzylamine-altered gene expression in Rat Esophagus

Our recent study (Reen et al., Cancer Res 2007;67:6484-92) identified 2261 dysregulated genes in the esophagi of rats that received a one week exposure to the carcinogen, N-Nitrosomethylbenzylamine (NMBA). We further reported that 1323 of these genes were positively modulated in the esophagus of NMBA-treated animals that also consumed dietary phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables. Herein, we report our companion study wherein litter mate rats either received regular chow or chow with 5% freeze-dried black raspberries (BRB) for three weeks. During the third week one-half of these animals received three subcutaneous injections of NMBA (0.5 mg/kg body weight). All animals were sacrificed 24h after the last dose of NMBA, and their esophagi excised and processed for histological grading and RNA microarray profiling. Two hundred and fifty nine of the 2261 genes that were dysregulated by NMBA-alone were expressed at control levels in the esophagus of BRB-fed rats. Overall, the data indicate that a BRB diet has a genome-wide modulating effect on NMBA-caused dysregulation of gene expression in rat esophagus, including genes involved in phase I and phase II metabolism, oxidative damage, inflammation, differentiation, chromosome partitioning, cell adhesion, motility and cytoskeleton formation, and genes known to act as oncogenes and tumor suppressor genes regulating apoptosis, cell-cycling and proliferation, and angiogenesis. Keywords: Toxicogenomics, reference design. For each treatment we used a pooling strategy to enable the inclusion of samples from numerous animals. Three replicate microarrays were completed for each of the four treatments (control, BRB, NMBA and NMBA+BRB) for a total of twelve microarrays. Each microarray was hybridized using a pooled RNA sample, and each pool was created from equal amounts of total RNA from two or three independent RNA samples, representing individual animals. Each of these samples was obtained from a different animal, and no sample was included in more than one pool. In total, samples from nine animals per group were pooled for control, NMBA, BRB and NMBA+BRB microarrays. To facilitate comparisons of gene expression across all treatments, we utilized a reference design in which each microarray was co-hybridized with a common reference sample labeled with Cy3, and RNA from the treatment pool was labeled with Cy5. Stratagene’s Universal Rat reference RNA (Stratagene, La Jolla, CA) was used as the common reference in all microarrays.