Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet.

Forty Fischer-344 male rats were given a high-risk diet (HRD) that was high in fat, low in fiber and low in calcium. After 4 weeks, the rats were given two weekly s.c. injections of azoxymethane (AOM, 15 mg/kg body wt), and remained on the same diet till death. Eight rats were killed at 12 weeks and again at 20 weeks in order to microdissect aberrant crypt foci (ACF) containing four or more crypts/focus from their colons. The remaining 24 rats were killed at 30 weeks to harvest colonic tumors. The polymerase chain reaction (PCR) was used to amplify specific DNA segments in the K-ras gene from ACF and colonic tumors. The PCR-amplified DNAs were sequenced to identify the point mutations in codons 12 and 13. All the mutations detected in the ACF and colonic tumors were G to A transitions in the second position of codon 12. These mutations were present in the ACF of 2/8 (25%) and 3/8 (37%) rats at 12 and 20 weeks respectively. The mutations were present in colonic tumors of 7/24 (29%) rats. These results provide important evidence for the significance of K-ras mutations in ACF (> 4 crypts/focus) as early markers of malignant potential in the colons of F344 rats exposed to AOM while receiving a high-risk western style diet.