Abstract B26: The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats
Authors: Peiffer Dan , Siddiqui Jibran H. , Kuo Chieh-Ti , Huang Yi-Wen , Zimmerman Noah P. , Wang Li-Shu , Stoner Gary D. , Carmella Steven G. , Ransom Ben , Hecht Stephen S. 
Published: January 2014

Abstract
Background: Black raspberries (BRB) and their component anthocyanins (AC) inhibit esophageal cancer in rats induced by N-nitrosomethylbenzylamine (NMBA). Previous studies indicate that the bioavailability of the anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to protocatechuic acid (PCA). PCA has greater bioavailability than the AC and is chemopreventive in several organ sites. The current study was undertaken to determine if PCA is also effective against NMBA-induced esophageal cancer and if it influences inflammatory cytokine expression. Cytokines were measured using a simultaneous quantification bead assay. The pro-inflammatory cytokines measured were IL-1 β, IL-5, IL-7, erythropoietin (EPO), TNF- α, & interferon-gamma (IFN- γ), and the anti-inflammatory cytokines were IL-10 and T cell-derived IL-2 and the chemokines CCL5 & CCL20.
Methods and Materials: Male Fischer 344 rats aged 3-5 weeks were randomized into groups by body weight & age. Rats were given s.c. injections of either the vehicle (20% DMSO/H2O) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. All rats were fed AIN-76A control diet during injections. Starting at week 6, groups of rats were fed experimental diets. These include Group 1 (DMSO + AIN-76A diet), Group 2 (NMBA + AIN-76A), Group 3 (NMBA + 3.8 umoles AC/ g AIN-76A), Group 4 (NMBA + 6% BRB in AIN-76A), & Group 5 (NMBA + 500 ppm PCA in AIN-76A). To match previous studies, the Group 3 diet contained 3.8 umoles of AC/g diet. A 6% BRB diet for Group 4 matched the anthocyanin content of Group 3, & the Group 5 diet contained 500 ppm PCA to match the estimated amount of Group 3 AC converted to PCA in the gut. 9 animals from each group were sacrificed at weeks 15 & 25, and 30 will be sacrificed at week 35. The esophagus was taken for tumor counts & used for H&E histological scoring. Rat blood was collected in heparin coated tubes, centrifuged, & stored at −80°C. Cytokines and chemokines were quantified using a bead array.
Results: The study is ongoing. At week 15 H&E slide scoring revealed hyperplasia was significantly reduced in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group. No tumors were present at week 15 in any of the NMBA-treated rats. At week 25 there were significant decreases in tumor count, hyperplasia, & dysplasia in Groups 3-5 when compared to Group 2. Preliminary results showed that pro-inflammatory IL-1 β expression was significantly higher in Group 2 when compared to Groups 1, 4, & 5 at week 25. Anti-inflammatory IL-10 expression was decreased in all NMBA treated groups at weeks 15 & 25. At week 15, T-cell derived IL-2 expression was significantly lower in Groups 1, 4, &5 when compared to Group 2, whereas at week 25, IL-2 expression was significantly lower only in Groups 1 & 5 when compared to Group 2.
Conclusions: PCA is chemopreventive against NMBA-induced esophageal tumorigenesis in rats. Similar to BRB and their AC, PCA significantly reduced tumor incidence and the conversion of dysplastic lesions to papillomas. Cytokine quantification assays suggest that PCA, like BRB and AC, significantly influence the expression levels of certain proinflammatory and anti-inflammatory cytokines in a protective manner. Further observations will be presented at the meeting.
Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B26.