K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrectly incorporate opposite deoxyguanine in DNA, then pair with deoxyadenosine during subsequent replication. It appears to preferentially target the 3′-G of 5′-NGGN-3′ sequences in mammalian cells in culture to induce G → A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tumors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone, while N-nitrosomethylurea (NMU) alone or NMU + BrdUrd resulted in incidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treated with BrdUrd for mutations in K-ras exons 1 and 2 and compared the prevalence and spectrum of mutations with those found in comparable tumors induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12–15 months earlier with BrdUrd (three specimens) or NMU (11 specimens) or both agents sequentially (eight specimens) were amplified by PCR. The base sequence of codons 12–13 and 59–63 of K-ras was determined by the dideoxynucleotide method. Sequencing results were confirmed by allele-specific oligonucleotide hybridization. Two of three tumors that appeared in rats given BrdUrd alone contained both a codon 12 GGT → GAT transition and a codon 61 CAA → CTA transversion. One tumor induced by NMU alone also showed a codon 12 GGT → GAT mutation, while only wild type sequence could be demonstrated in the codon 12–13 region in the remaining ten such tumors. Three NMU-induced tumors also showed codon 61 CAA → CTA mutations, while the remaining tumors had wild type sequence. While the GGT → GAT transitions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA → CTA transversions, the overall low prevalence of mutations, and the lack of any difference in mutation spectrum between tumors induced by NMU and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effect of either agent.