Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines

Abstract

Lung and liver tumors were induced in female A/J mice after treatment for 7 weeks (3 times/week, i.p.) with either 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (50 mg/kg) or nitrosodimethylamine (NDMA) (3 mg/kg). Both compounds can be activated via alpha-hydroxylation to methylating agents, while NNK may also undergo hydroxylation at the N-methyl carbon to form a pyridyloxobutylated adduct. The purpose of these studies was to identify and characterize the activated oncogenes present in tumors induced by NDMA and NNK. Following transfection of high molecular weight DNA onto NIH/3T3 mouse fibroblasts, transforming genes were detected in 90% of both NNK- (10 of 11) and NDMA- (9 of 10) induced lung tumors. In contrast, transformation of NIH/3T3 fibroblasts was observed only in 40% (2 of 5) and 13% (1 of 8) of the liver tumors from NNK- and NDMA-treated mice, respectively. Southern blot analysis indicated that the transforming gene present in all lung tumors was an activated K-ras oncogene. Both rearranged bands and amplified signals were detected in the transfectants. The one transformant from the NDMA-induced liver tumor contained an activated K-ras gene. In contrast, the two liver transformants from NNK-induced tumors did not contain an activated ras or raf gene. Hybridization with oligonucleotide probes that were centered around either codon 12 or 61 of the K-ras gene were utilized to localize the mutations. Activation of this gene appeared to occur largely via a mutation in codon 12 (15 of 20 transformants) and was observed with a similar frequency in pulmonary tumors induced by either compound. The remaining mutations were found in codon 61. The specific mutation within these two codons was determined by amplifying the exon containing the base change, followed by direct sequencing. With one exception the mutation observed in codon 12 was a GC to AT transition (GGT to GAT). One transformant contained a GC to TA transversion. The activating mutation detected in codon 61 was always an AT to GC transition of the middle A (CAA to CGA). The GC to AT mutation observed in codon 12 is consistent with the formation of the O6-methylguanine adduct. Similar concentrations (23 to 32 pmol/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA.(ABSTRACT TRUNCATED AT 400 WORDS)