cyclin-d1 and 2-amino-1-methyl-6-phenylimidazo(4-5-b)pyridine

Species differences in the metabolism of xenobiotics by cytochrome P450 are critical in evaluating the use of experimental animals in studying toxic compounds relevant to human diseases. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced by high-temperature cooking of fish and meat, is activated to become a carcinogen by cytochrome P4501A2 (CYP1A2) through N

Topics: Animals; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Carcinogens; Colonic Neoplasms; Cooking; Cyclin D1; Cyclin G1; Cyclin-Dependent Kinase Inhibitor p21; Cytochrome P-450 CYP1A2; DNA Damage; Female; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Imidazoles; Immediate-Early Proteins; Inactivation, Metabolic; Insulin-Like Growth Factor I; Male; Mice; Mice, Transgenic; Signal Transduction; Transgenes; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines formed by cooking meat and fish at high temperature. PhIP induces colorectal adenoma risk in male rats when administered orally. This study used male Fisher 344 rats to investigate the impact of dietary Chlorella on PhIP metabolism and aberrant colonic gene expression following short-term PhIP treatment. High-performance liquid chromatography analysis revealed that fecal excretion of unmetabolized PhIP was significantly increased in rats whose diets were supplemented with Chlorella compared to rats in a PhIP-only group (P<0.001). Quantitative realtime PCR confirmed that the increase in beta-catenin and cyclin D1 mRNA in the colon induced by PhIP was ameliorated in rats pre-fed with Chlorella (P=0.052 for beta-catenin; P=0.005 for cyclin D1). The increase in DNA shearing that is a hallmark of caspase-8-mediated apoptosis by PhIP was also significantly diminished in the colons of rats pre-fed Chlorella (P=0.012). These results suggested that administering dietary Chlorella with a Western-style diet concomitantly or immediately before mutagen exposure might be beneficial in blocking the absorption of food mutagens such as PhIP.

Topics: Animals; Apoptosis; beta Catenin; Caspase 8; Chlorella; Colon; Cyclin D1; Dietary Supplements; DNA Fragmentation; Feces; Gene Expression; Imidazoles; Male; Meat; Mutagens; Rats; Rats, Inbred F344; Real-Time Polymerase Chain Reaction; RNA, Messenger

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet. Herein, the expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ER beta) and progesterone receptor (PR) was examined in mammary gland carcinomas induced by PhIP in female Sprague-Dawley rats. Quantitative real-time polymerase chain reaction demonstrated that ER alpha, ER beta and PR were statistically elevated by 3-, 4- and 8-fold in carcinomas compared with normal mammary glands. By immunohistochemistry, carcinomas showed statistically higher nuclear expression of all three steroid receptors with the majority of carcinomas showing at least 10% of epithelial cells stained for ER alpha (49/55, 89%), ER beta (41/55, 75%) and PR (48/55, 87%). Furthermore, the level of expression of the three steroid hormone receptors was positively correlated with each other across the bank of carcinomas (Spearman analysis, P < 0.05). The expression of ER alpha in carcinomas was associated with tumor grade, extent of nuclear pleomorphism and cellular proliferation as measured by proliferating cell nuclear antigen (PCNA) and phospho-Rb immunostaining (Spearman analysis, P < 0.05). Confocal microscopy was used to measure the percentage of epithelial cells showing nuclear colocalization of receptors, PCNA, and cyclin D1. Colocalization of the receptors, and the colocalization of the receptors with PCNA and cyclin D1 was strikingly higher in carcinomas than in the normal mammary gland. In carcinoma cells, 37% of ER alpha positive epithelial cells were colocalized with PCNA in contrast to just 0.25% of cells in the normal mammary gland. The findings from this study indicate that ER alpha, ER beta and PR were co-upregulated and nuclear localized in epithelial cells from rat mammary carcinomas compared with normal mammary glands, and that the co-upregulation was positively correlated with proliferation and cell cycle progression in carcinomas.

Topics: Animals; Carcinogens; Cell Cycle; Cell Nucleus; Cell Proliferation; Cyclin D1; Epithelial Cells; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Imidazoles; Immunoenzyme Techniques; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Receptors, Progesterone; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Chemopreventive activity by retinoic acid (RA) has been demonstrated previously in rat colon. The spontaneous tumourigenesis in the Min/+ mouse, which harbours a germline mutation in the tumour suppressor gene adenomatous polyposis coli (Apc), is characterized by inactivation of Apc, nuclear accumulation of beta-catenin and the enhanced expression of specific genes activated by T cell factor (TCF)/beta-catenin signalling. Recently it was reported that beta-catenin interacts with retinoic acid receptor in a retinoid-dependent manner, reducing beta-catenin/TCF regulated transcription. Our hypothesis was therefore that dietary supplementation with all-trans RA may inhibit the Apc-driven tumourigenesis in Min/+ mice. Surprisingly, in two different experiments the results showed that dietary RA significantly stimulated both the formation and growth of small intestinal tumours. In the first experiment Min/+ mice were exposed to 50 mg 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine/kg bodyweight at day 3-6 after birth and then treated with 50 mg/kg dietary RA in 1-3 weeks from the age of 2 weeks. In the second experiment the mice were not treated with carcinogen, and the diet was supplemented with 5 or 10 mg/kg RA from the age of 4 weeks until termination of the experiment at 11 weeks. Immunohistochemical studies revealed no differences in beta-catenin, cyclin D1 or proliferating cell nuclear antigen staining following RA treatment. There was no intestinal toxicity in mice fed 10 mg/kg RA, indicating that the increased tumourigenesis in Min/+ mice is a specific effect of all-trans RA.

Topics: Animals; beta Catenin; Body Weight; Cyclin D1; Cytoskeletal Proteins; Dietary Supplements; Female; Genes, APC; Germ-Line Mutation; Imidazoles; Intestinal Neoplasms; Male; Mice; Mice, Inbred C57BL; Proliferating Cell Nuclear Antigen; Trans-Activators; Tretinoin

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a suspected human breast carcinogen found in cooked meat that induces mammary gland cancer in rats. By real time PCR analysis, PhIP-induced rat mammary gland carcinomas showed statistically higher expression of the G(1)-S cyclin D1 (5-fold) and its kinase partner cyclin-dependent kinase (Cdk)-4 (37-fold) in comparison with normal mammary gland, whereas cyclin D2, cyclin D3, and Cdk6 were not statistically changed. Amplification of cyclin D1 was observed by real time PCR in 24% of carcinomas (15 of 63). Only 1 of 47 carcinomas showed Cdk4 amplification. By Western blotting, the level of phospho-Rb was >2-fold higher in carcinomas than in normal mammary gland. By immunohistochemical analysis, cyclin D1, Cdk4, and phospho-Rb nuclear protein expression was 5.7-, 3.9-, and 2.3-fold higher, respectively, in carcinomas than in normal mammary gland, whereas the expression of cyclin D2, cyclin D3, and Cdk6 was similar. Among carcinomas, Cdk4 and phospho-Rb levels were positively correlated with cell proliferation. Previous studies by this laboratory indicated that these carcinomas harbor a high frequency of H-ras mutations. The H-ras pathway is linked to the cell cycle via cyclin D1. The results from the current study implicate cyclin D1/Cdk4, phospho-Rb as a central pathway in PhIP-induced rat mammary gland carcinogenesis.

Topics: Animals; Blotting, Western; Carcinogens; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Female; Gene Amplification; Imidazoles; Immunohistochemistry; Mammary Neoplasms, Experimental; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; RNA, Messenger; Signal Transduction