beta-catenin is strongly elevated in rat colonic epithelium following short-term intermittent treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and a high-fat diet

Abstract

Colon tumors expressing high levels of beta-catenin and c-myc have been reported in male F344 rats given three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) alternating with a high-fat (HF) diet. Using the same experimental protocol, rats were euthanized 24 h after the last dose of PhIP so as to examine early changes in colonic crypt homeostasis and beta-catenin expression, before the onset of frank tumors. PhIP/HF dosing caused a significant increase in the bromodeoxyuridine labeling index throughout the entire colon, and within the colonic crypt column cleaved caspase-3 was elevated in the basal and central zones, but reduced in the luminal region. In vehicle/HF controls, beta-catenin was immunolocalized primarily at the border between cells at the top of the crypt, whereas in rats given PhIP/HF diet there was strong cytoplasmic staining, which appeared as a gradient of increased beta-catenin extending from the base of the crypt column to the luminal region. Quantitative real-time PCR and immunoblot analyses confirmed that beta-catenin and c-myc were increased significantly in the colonic mucosa of rats given PhIP/HF diet. Collectively, these findings suggest that PhIP/HF cycling alters beta-catenin and c-myc expression in the colonic mucosa, resulting in expansion of the proliferative zone and redistribution of apoptotic cells from the lumen to the central and basal regions of the colonic crypt. Thus, during the early stages of colon carcinogenesis, alternating exposure to heterocyclic amines and a high-fat diet might facilitate molecular changes resulting in dysregulated beta-catenin and c-myc expression.

Figure 1

Experimental protocol for short‐term intermittent 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) treatment alternating with high‐fat diet. This figure was modified from a recent report by Wang et al., which provided full details of the treatment protocol.^{( 12 )} In brief, male F344 rats were given PhIP by daily oral gavage at a dose of 50 mg/kg body weight, whereas controls received vehicle alone (0.8% dimethyl sulfoxide [DMSO] in ultra‐pure water, adjusted to pH 3.5 with 0.1 N HCl). Following 2 weeks of PhIP or vehicle treatment, rats were placed on AIN93G diet supplemented with Primex hydrogenated vegetable oil, comprising 23% fat‐derived calories (high‐fat [HF] diet). After 4 weeks on HF diet, animals were returned to standard AIN93G diet and treated again with PhIP or vehicle for 2 weeks, followed by an additional 4 weeks on HF diet. A third cycle of PhIP or vehicle then was completed, and 24 h later the rats were euthanized by CO₂ inhalation. Note: in our prior study, which lasted 1 year,^{( 12 )} rats were switched as recommended from AIN93G (growth) diet to AIN94M (maintenance) diet at week 18.

Figure 2

Cycling of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)/high‐fat diet induces cell proliferation in the colon. Rats were treated with PhIP and a high‐fat diet (see Fig. 1) and the bromodeoxyuridine (BrdU) labeling index was determined as described in Materials and Methods. Data bars, mean ± SE; ***P < 0.001, by Students t‐test, PhIP (n = 4) versus corresponding vehicle controls (n = 3).

Figure 3

Cycling of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)/high‐fat diet increases cleaved caspase‐3 labeling in the central and basal regions of the colonic crypt column and reduces it in the luminal region. The cleaved caspase‐3 labeling index was determined as described in Materials and Methods. Data bars, mean ± SE; ***P < 0.001, by Students t‐test, PhIP (n = 4) versus corresponding vehicle controls (n = 3).

Figure 4

Cycling of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)/high‐fat diet increases β‐catenin expression in the colon. Immunohistochemistry revealed low overall expression levels of β‐catenin in vehicle controls, but high levels in PhIP‐treated rats. In vehicle controls, β‐catenin was restricted to the borders between cells (arrows), with little or no cytoplasmic and nuclear staining, whereas in PhIP‐treated rats there was strong cytoplasmic β‐catenin expression (arrow heads).

Figure 5

Cycling of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)/high‐fat diet increases cytoplasmic β‐catenin expression in the colon. The β‐catenin labeling index was scored as the number of cells stained positively for cytoplasmic (and/or nuclear) versus membrane‐associated expression, divided by the total number of cells in that region of the colon. Data bars, mean ± SE; PhIP n = 4, vehicle controls n = 3. **P < 0.01; ***P < 0.001, PhIP group significantly different from the corresponding vehicle controls.

Figure 6

Cycling of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)/HF diet increases β‐catenin and c‐myc mRNA expression in the rat colon. Quantitative real‐time PCR was performed as described in Materials and Methods, for β‐catenin (Ctnnb1) and three β‐catenin/Tcf target genes, namely c‐myc, c‐jun and cyclin D1, normalized to Gapdh. Data bars, mean ± SE, PhIP n = 5, vehicle controls n = 6. *P < 0.05, PhIP group significantly different from corresponding vehicle controls.

Figure 7

Immunodetection of increased β‐catenin and c‐myc in the rat colon before the onset of frank tumors. Representative immunoblots of β‐catenin and c‐myc in colonic mucosa from rats treated with three cycles of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) (or vehicle) alternating with high‐fat diet. A PhIP‐induced colon tumor from the corresponding 1‐year carcinogenicity study,^{( 12 )} is shown in the furthest right lane as a positive control. β‐Actin, loading control.