H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer
Laser capture microdissection, polymerase chain reactionrestriction fragment length polymorphism analysis, and DNA sequencing was used to detect H-ras codon 12 and 13 mutations during the stages of mammary gland cancer development in rats exposed to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat. Ten oral doses of PhIP (75 mg/kg, p.o., once per day) were administered to adolescent female Sprague-Dawley rats and mammary glands examined histologically
... intraductal proliferations (IDPs), carcinoma in situ and carcinomas 7-14 weeks later. Mammary gland epithelial cells from normal tissue and distinct lesions were collected from glass slides and analyzed for mutations. H-ras codon 12/13 mutations were detected in 73%, 75%, 100%, and 100% of normal mammary glands, IDPs, carcinoma in situ, and carcinoma, respectively, after PhIP treatment. The spectrum of activating mutations included G 35 to A or C base substitution mutations in codon 12, and G 37 to T or A base substitution mutations in codon 13. The spectrum of H-ras mutations was similar among normal mammary gland from PhIP treated rats, preneoplastic lesions, and carcinomas. Furthermore, the spectrum of mutations was consistent with the involvement of PhIPguanine adduct formation. The results support the notion that mutations in H-ras codons 12 and 13 are largely PhIP-DNA adduct-induced and involved in the initiation and development of mammary gland cancer in rats exposed to PhIP. Abbreviations: dG-C8-PhIP, N-deoxyguanosin-8-yl-PhIP; DMBA, 7,12dimethylbenz[a]anthracene; IDPs, intraductal proliferations; LCM, laser capture microdissection; NMU, nitrosomethylurea; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine .