title: K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors.
abstract: 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.
authors.affiliation: Office of Special Nutritionals, US Food and Drug Administration, Laurel, MD 20708, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Calvert
authors.forename: G S
authors.initials: GS
authors.lastname: Buzard
authors.forename: V N
authors.initials: VN
authors.lastname: Anisimov
authors.forename: J M
authors.initials: JM
authors.lastname: Rice
mesh_terms: D000225:Adenine; D000818:Animals; D001973:Bromodeoxyuridine; D002273:Carcinogens; D003062:Codon / Q000235:genetics*; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D006147:Guanine; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000235:genetics*; D008297:Male; D008637:Mesenchymoma / Q000139:chemically induced / Q000235:genetics*; D008770:Methylnitrosourea; D009374:Neoplasms, Experimental / Q000139:chemically induced / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051381:Rats
chemical_list: D002273:Carcinogens; D003062:Codon; D006147:Guanine; D008770:Methylnitrosourea; D001973:Bromodeoxyuridine; D000225:Adenine