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 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.
Smith
B A
BA
National Center for Toxicological Research, Jefferson, AR 72079, USA. BASmith@NCTR.FDA.GOV
Manjanatha
M G
MG
Pogribny
I P
IP
Mittelstaedt
R A
RA
Chen
T
T
Fullerton
N F
NF
Beland
F A
FA
Heflich
R H
RH
D000818:Animals; D002999:Clone Cells; D004252:DNA Mutational Analysis; D004351:Drug Resistance; D005091:Exons / Q000187:drug effects; D016158:Genes, p53 / Q000187:drug effects*; D011905:Genes, ras / Q000187:drug effects*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects / Q000235:genetics*; D008175:Lung Neoplasms / Q000139:chemically induced / Q000235:genetics* / Q000276:immunology; D008297:Male; D009153:Mutagens; D017354:Point Mutation / Q000187:drug effects; D011721:Pyrenes / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D013866:Thioguanine / Q000494:pharmacology*
D009153:Mutagens; D011721:Pyrenes; C033309:1,6-dinitropyrene; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine