This study examined the effects of lyophilized black raspberries (BRB) on azoxymethane (AOM)-induced aberrant crypt foci (ACF), colon tumors, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in male Fischer 344 rats. AOM was injected (15 mg/kg body wt i.p.) once per week for 2 wk. At 24 h after the final injection, AOM-treated rats began consuming diets containing 0%, 2.5%, 5%, or 10% (wt/wt) BRB. Vehicle controls received 5% BRB or diet only. Rats were sacrificed after 9 and 33 wk of BRB feeding for ACF enumeration and tumor analysis. ACF multiplicity decreased 36%, 24%, and 21% (P < 0.01 for all groups) in the 2.5%, 5%, and 10% BRB groups, respectively, relative to the AOM-only group. Total tumor multiplicity declined 42%, 45%, and 71% (P < 0.05 for all groups). Although not significant, a decrease in tumor burden (28%, 42%, and 75%) was observed in all BRB groups. Adenocarcinoma multiplicity decreased 28%, 35%, and 80% (P < 0.01) in the same treatment groups. Urinary 8-OHdG levels were reduced by 73%, 81%, and 83% (P < 0.01 for all groups). These results indicate that BRB inhibit several measures of AOM-induced colon carcinogenesis and modulate an important marker of oxidative stress in the Fischer 344 rat.

Angle-resolved low-coherence interferometry ͑a/LCI͒ is used to obtain quantitative, depth-resolved nuclear morphology measurements. We compare the average diameter and texture of cell nuclei in rat esophagus epithelial tissue to grading criteria established in a previous a/LCI study to prospectively grade neoplastic progression. We exploit the depth resolution of a/LCI to exclusively examine the basal layer of the epithelium, approximately 50 to 100 m beneath the tissue surface, without the need for exogenous contrast agents, tissue sectioning, or fixation. The results of two studies are presented that compare the performance of two a/LCI modalities. Overall, the combined studies show 91% sensitivity and 97% specificity for detecting dysplasia, using histopathology as the standard. In addition, the studies enable the effects of dietary chemopreventive agents, difluoromethylornithine ͑DFMO͒ and curcumin, to be assessed by observing modulation in the incidence of neoplastic change. We demonstrate that a/LCI is highly effective for monitoring neoplastic change and can be applied to assessing the efficacy of chemopreventive agents in the rat esophagus.

Our laboratory has used a rodent model of human esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In the present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks. Beginning 1 week later, they were fed a synthetic diet containing 5% black raspberries (BRB) for the duration of the bioassay (25 weeks). Rats were sacrificed at weeks 9, 15, and 25. Esophageal tissues were collected, and tumor data were recorded. The expression and enzymatic activities of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of c-Jun in the esophagi, were evaluated to investigate the mechanism(s) by which black raspberries modulate tumorigenesis. At week 25, BRB inhibited tumor multiplicity, the standard end point in this tumor model, from 3.78 F 0.41 tumors per rat in NMBAtreated animals to 2.23 F 0.21 tumors per rat in animals treated with NMBA plus BRB (P < 0.005). BRB reduced mRNA and protein expression levels of COX-2, iNOS, and c-Jun as well as the level of prostaglandin E 2 in preneoplastic lesions of the esophagus at week 25. The berries inhibited mRNA expression of iNOS and c-Jun, but not COX-2, in papillomatous lesions of the esophagus. Prostaglandin E 2 and total nitrite levels were also decreased by BRB in papillomas. These results suggest a novel tumor suppressive role of BRB through inhibition of COX-2, iNOS, and c-Jun.

For several years, our laboratory has been evaluating the ability of lyophilized (freeze-dried) black raspberries (Rubus occidentalis, BRBs), blackberries (R. fructicosus, BBs), and strawberries (Fragaria ananasia, STRWs) to inhibit carcinogen-induced cancer in the rodent esophagus. To assure "standardized" berry preparations for study, each berry type is of the same cultivar, picked at about the same degree of ripeness, washed and frozen within 2-4 h of the time of picking, and freeze-dried under conditions that preserve the components in the berries. Some of the known chemopreventive agents in berries include vitamins A, C, and E and folic acid; calcium and selenium; β-carotene, α-carotene, and lutein; polyphenols such as ellagic acid, ferulic acid, pcoumaric acid, quercetin, and several anthocyanins; and phytosterols such as β-sitosterol, stigmasterol, and kaempferol. In initial bioassays, freeze-dried STRW, BRB, and BB powders were mixed into AIN-76A synthetic diet at concentrations of 5% and 10% and fed to Fischer 344 rats before, during, and after treatment with the esophageal carcinogen Nnitrosomethylbenzylamine (NMBA). At 25 wk of the bioassay, all three berry types were found to inhibit the number of esophageal tumors (papillomas) in NMBA-treated animals by 24-56% relative to NMBA controls. This inhibition correlated with reductions in the formation of the NMBA-induced O 6 -methylguanine adduct in esophageal DNA, suggesting that the berries influenced the metabolism of NMBA leading to reduced DNA damage. Studies are ongoing to determine the mechanisms by which berries influence NMBA metabolism and DNA adduct formation. BRBs and STRWs were also tested in a postinitiation scheme and were found to inhibit NMBA-induced esophageal tumorigenesis by 31-64% when administered in the diet following treatment of the animals with NMBA. Berries, therefore, inhibit tumor promotion and progression events as well as tumor initiation. In vivo mechanistic studies with BRBs indicate that they reduce the growth rate of premalignant esophageal cells, in part, through down-regulation of cyclooxygenase-2 leading to reduced prostaglandin production and of inducible nitric oxide synthase leading to reduced nitrate/nitrite levels in the esophagus. Based upon the preclinical data on rodents, we have initiated prevention trials in humans to determine if berries might exhibit chemopreventive effects in the esophagus.

Angiogenesis, the formation of new blood vessels, is critical to tumor growth and metastasis. Vascular endothelial growth factor (VEGF), an important angiogenic activator, is essential for angiogenesis. Our laboratory has used a rodent model of human esophageal squamous cell carcinoma (ESCC) to identify putative chemopreventive agents for this disease and determine their mechanisms of action. We reported that dietary black raspberry powder (BRB) inhibits N-nitrosomethylbenzylamine (NMBA)-induced tumor development in the rat esophagus by inhibiting the formation of DNA adducts and reducing the proliferation rate of preneoplastic cells. On a molecular level, BRB downregulates the expression of c-Jun, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In this study we analyzed the effect of BRB on angiogenesis. VEGF expression was determined by real-time RT-PCR and immunohistochemical analysis of microvessel density (MVD). BRB significantly suppressed VEGF-C expression from a 2.38 (+/- 0.34)-fold increase in animals treated with NMBA alone to a 1.08 (+/- 0.22)-fold increase in animals treated with NMBA plus BRB (P < 0.005). The MVD of esophagus was decreased from 53.7 +/- 5.6 vessels/cm in animals treated with NMBA alone to 22.6 +/- 2.6 vessels/cm in animals treated with NMBA plus BRB (P < 0.0001). Our data also suggest that downregulation of VEGF is correlated with suppression of COX-2 (r2 = 0.86, P < 0.001) and iNOS (r2 = 0.81, P < 0.005). As high vascularity is a risk factor for metastasis and tumor recurrence, BRB may have cancer therapeutic effects in human esophageal cancer.