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Laser capture microdissection, polymerase chain reaction-restriction 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 for 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 PhIP-guanine 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.

H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer