Molecular analysis of lacI mutations in Rat2 ™ cells exposed to 7,12-dimethylbenz[ a]anthracene: evidence for DNA sequence and DNA strand biases for mutation

Abstract

The Rat2 ™ cell line carries 50–70 stably integrated copies per cell of a λ/lacI shuttle vector as a target for mutagenicity testing. Rat2 ™ cells were exposed to 1 and 10 μg/ml of 7,12-dimethylbenz[ a]anthracene (DMBA) for 24 h at 37 °C in the presence of primary rat hepatocytes, and grown to confluence. The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined. The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 ±4.9 × 10 −5) and 33-fold (127 ±19.9 × 10 −5) higher, respectively, than the spontaneous mutant frequency (3.8 + 0.7 × 10 −5). DNA sequence analysis of the DMBA-induced lacI −1 mutants indicated that they contained mainly basepair substitution mutations at A : T and G : C, and that A : T → T : A and G : C → T : A transversions were the predominant types. In addition, 23 of 28 (82%) A : T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand. Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5′ by a dC, and 17 of these were A : T → T:A transversions, suggesting a sequence preference for this mutation. Except for a higher proportion of G : C → A : T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar. These results indicate that DMBA mutagenesis in the lacI gene of Rat2 ™ cells displays distinct DNA sequence and DNA strand preferences.