ANALYSIS OF MUTATIONS IN THE K-RAS AND P53 GENES OF LUNG-TUMORS AND IN THE HPRT GENE OF 6-THIOGUANINE-RESISTANT T-LYMPHOCYTES FROM RATS TREATED WITH 1,6-DINITROPYRENE

Direct pulmonary instillation of 1,6-dinitropyrene (DNP) into male Fis cher 344 rats results in a dose-dependent induction of lung tumors and 6-thioguanine-resistant (TG(r)) T-lymphocytes. The treatment also res ults in DNP binding to dG in the lung and in T-lymphocytes. In the pre sent study, we have examined the types of mutations associated with th ese responses to DNP. Sequencing of DNA amplification products from 20 DNP-induced lung tumors identified 5 mutations in K-ras codon 12, 4 G GT --> TGT transversions and one GGT --> GAT transition. No mutations were found in K-ras codons 13 or 61. Single-strand conformation polymo rphism analysis of p53 exons 5-8 revealed mobility shifts indicative o f mutation in 9 of the 20 tumor samples. Eight of the mutations were s ubstitutions at G:C base pairs, and one was a deletion of a single G:C base pair. DNA from 161 TG(r) lymphocyte colonies cultured from DNP-t reated rats was examined for point mutations by amplification of hprt exons 2, 3, and 8, and screening the products for mutant:wild-type het eroduplex formation by denaturing gradient-gel electrophoresis. Only t hree mutations were found, a G --> T transversion in exon 3, a G --> A transition in exon 8, and a complex mutation consisting of a tandem G --> T transversion and a one base deletion in exon 3. The mutations i dentified in the DNP-induced lung tumors and TG(r) T-lymphocytes are c onsistent with the formation of dG-DNA adducts by DNP. The extremely l ow recovery of point mutations from TG(r) lymphocytes suggests that DN P induces a substantial number of mutations by other mechanisms. (C) 1 997 Elsevier Science B.V.