Mutations induced by (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells.

Abstract

The polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) is an exceptionally potent carcinogen. Its direct DNA-reactive metabolite, the fjord region (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide [(-)-anti-DB[a,l]PDE], was used to investigate induction of mutations in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells. Cells exposed to 1-10 nM (-)-anti-DB[a,l]PDE exhibited a close dose-responsive increase in the frequency of mutant clones resistant to 6-thioguanine. RNA was isolated from mutant clones and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the Hprt gene was amplified by polymerase chain reaction and sequenced. Analysis of the DNA base sequence changes induced by (-)-anti-DB[a,l]PDE indicated that base substitutions were the most prevalent mutations, followed by exon deletions. Among the groups of V79 cells treated with low concentrations of (-)-anti-DB[a,l]PDE, most displayed high selectivity for both A:T-->T:A transversions and A:T-->G:C transitions, while cells exposed to a higher dose (10 nM) formed predominantly G:C-->T:A transversions. Also, the number of base substitutions per mutant clone increased with dose. In general, the mutation profiles induced by (-)-anti-DB[a,l]PDE exhibited a wide spectrum; in addition to base substitutions, deletions, insertions, frameshift mutations, as well as tandem mutations were detected. Analysis of the DNA adduct levels induced by (-)-anti-DB[a,l]PDE revealed that a concentration-dependent increase in the level of adduct formation preceded the concentration-dependent increase in mutational events in these cells and that an increasing proportion of DNA adducts at deoxyadenosine were formed with dose. The results of this study demonstrate a correspondence between the concentration and types of DNA adducts and the frequency and types of mutations induced by (-)-anti-DB[a,l]PDE in V79 cells.