thioguanine-anhydrous and 1-6-dinitropyrene

Direct pulmonary instillation of 1,6-dinitropyrene (DNP) into male Fischer 344 rats results in a dose-dependent induction of lung tumors and 6-thioguanine-resistant (TGr) T-lymphocytes. The treatment also results in DNP binding to dG in the lung and in T-lymphocytes. In the present study, we have examined the types of mutations associated with these responses to DNP. Sequencing of DNA amplification products from 20 DNP-induced lung tumors identified 5 mutations in K-ras codon 12, 4 GGT-->TGT transversions and one GGT-->GAT transition. No mutations were found in K-ras codons 13 or 61. Single-strand conformation polymorphism analysis of p53 exons 5-8 revealed mobility shifts indicative of mutation in 9 of the 20 tumor samples. Eight of the mutations were substitutions at G:C base pairs, and one was a deletion of a single G:C base pair. DNA from 161 TGr lymphocyte colonies cultured from DNP-treated rats was examined for point mutations by amplification of hprt exons 2, 3, and 8, and screening the products for mutant: wild-type heteroduplex formation by denaturing gradient-gel electrophoresis. Only three 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 identified in the DNP-induced lung tumors and TGr T-lymphocytes are consistent with the formation of dG-DNA adducts by DNP. The extremely low recovery of point mutations from TGr lymphocytes suggests that DNP induces a substantial number of mutations by other mechanisms.

Topics: Animals; Clone Cells; DNA Mutational Analysis; Drug Resistance; Exons; Genes, p53; Genes, ras; Hypoxanthine Phosphoribosyltransferase; Lung Neoplasms; Male; Mutagens; Point Mutation; Pyrenes; Rats; Rats, Inbred F344; T-Lymphocytes; Thioguanine

The ability of Chinese hamster ovary (CHO) cells to convert dinitropyrenes (DNPs) to mutagenic species has been investigated by examining the effects of 1,6-DNP and 1,8-DNP on three distinct end-points in this cell line. At concentrations ranging from 0.05 to 5 micrograms/ml both analogues induced increases in the frequency of 6-thioguanine-resistant mutants in CHO cells when exposure was limited to 3 h. This treatment time was also sufficient to permit the induction of sister-chromatid exchange and chromosome aberrations in CHO cells. The results obtained suggest that CHO cells, unlike mouse lymphoma L5178Y cells, possess an endogenous metabolic activity which is capable of bringing about the conversion of DNPs to their mutagenic form without a requirement for prolonged exposure periods and as such may be a more suitable cell line for the study of this class of compounds.

Topics: Animals; Cell Line; Cell Survival; Chromosome Aberrations; Cricetinae; Cricetulus; Drug Resistance; Female; Fibroblasts; Hypoxanthine Phosphoribosyltransferase; Mutagenicity Tests; Ovary; Pyrenes; Sister Chromatid Exchange; Thioguanine