mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes

Proliferation, development and DNA adduct levels in the mammary gland of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a high fat diet

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine derived from cooked meat that is a mammary gland carcinogen in rats, A carcinogenic dose-regimen of PhIP (75 mg/kg, p.o., 10 doses, once per day) was administered to 43-day old female Sprague-Dawley rats, and the rats were then placed on a defined high fat (23.5% corn oil) or low fat (5% corn oil) diet for up to 6 weeks. At various times after carcinogen and diet, and prior to carcinogenesis, we examined the percentage of proliferating cells in terminal end bud (TEB) epithelial structures of the rat mammary gland by proliferating cell nuclear antigen staining, mammary gland architecture by whole mounting, and PhIP-DNA adduct levels in mammary epithelial cells by the P-32-post-labeling assay. Immediately after dosing, the percentage of proliferating epithelial cells in TEBs was significantly higher in PhIP-treated rats than in control rats receiving vehicle only [7.5 +/- 0.9% (n = 99) versus 4.2 +/- 0.6% (n = 127), respectively], The mammary glands of PhIP-treated rats showed a significantly lower density of alveolar buds (ABs) and a higher density of TEBs than control rats, which suggests that PhIP exposure partially inhibited the normal glandular differentiation of TEBs to ABs, After 6 weeks on the diet, proliferation in TEBs was statistically higher in rats given PhIP plus a high fat diet than in rats given vehicle plus a low fat diet. The mammary glands from rats on a high fat diet also showed a statistically higher density of TEBs when compared with rats on a low fat diet [2.08 +/- 0.34% versus 1.04 +/- 0.20%, respectively (n = 6)], PhIP-DNA adduct levels were relatively high in mammary epithelial cells of treated rats. At 3 h after the last dose of PhIP, DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SE] were 10.5 +/- 1.7 (n = 8) and 0.9 +/- 0.2 (n = 7) in epithelial cells isolated from mammary gland and in the liver, respectively, DNA adduct removal rates from the mammary gland were not different between rats on the high fat and low fat diets. Adducts were still detected after 6 weeks on either diet. Thus, events that occurred prior to neoplasia in the mammary glands of PhIP-treated rats include formation of PhIP-DNA adducts at relatively high levels, and enhanced proliferation in TEBs (putative sites of origin of mammary gland carcinomas) and partial inhibition of TEE differentiation. The high fat diet, a promoter of PhIP-induced mammary gland carcinogenesis, appeared to sustain the proliferative effect of PhIP in mammary gland TEBs at a time when PhIP-DNA adducts are still detectable. These early events may contribute to the targeting and carcinogenicity of PhIP to the mammary gland of rats

Mammary gland carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Sprague-Dawley rats on high- and low-fat diets

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic amine derived from cooked meat. Mammary gland tumors were induced in female Sprague-Dawley rats given 10 doses of PhIP (75 mg/kg po) once per day from 43 days of age and then placed on a defined high-fat (23.5% corn oil) or low-fat (5% corn oil) diet for 25 weeks. Mammary tumor incidence was 49% (44 of 90 rats) and 31% (27 of 88 rats) in the high- and low-fat groups, respectively. No tumors were found in vehicle control rats on the high- or the low-fat diet (n = 44 and 43, respectively). The higher tumor incidence in the high-fat group was due to an increase specifically in carcinomas (classified as tubulopapillary carcinomas) rather than benign tumors (tubular adenomas and fibroadenomas). The incidence of carcinomas was 45% and 24% in PhIP-treated rats on the high- and low-fat diets, respectively. In addition, the percentage of carcinomas showing stromal invasion was highest in the high-fat diet group (22% vs. 8%, high- vs. low-fat diet). Proliferating cell nuclear antigen immunostaining (PCNA) index revealed six times more proliferation in carcinomas from rats on the high-fat diet than in rats on the low-fat diet. Adenomas from rats on different diets had similar PCNA indexes. The tumor apoptotic index, quantitated by immunohistochemical detection (terminal deoxynucleotidyl transferase nick end labeling), was twice as high in carcinomas from rats on the high-fat diet as in carcinomas from rats on the low-fat diet but was similar between the two groups of adenomas. The PCNA-to-apoptosis ratio was 43 and 17 in carcinomas from rats on the high- and low-fat diets, respectively, indicating that the growth rate of carcinomas was greater in rats on the high-fat diet. The results from this study show that the high-fat diet increases the incidence, invasiveness, and growth of PhIP-induced mammary gland carcinomas