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

The Rat2(TM) cell line carries 50-70 stably integrated copies per cell of a lambda/lacl shuttle vector asa target for mutagenicity testing. Rat2(TM) cells were exposed to 1 and 10 mu g/ml of 7,12-dimethylbenz[a]anthracene (DMBA) for 24 h at 37 degrees 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 x 10(-5)) and 33-fold (127 +/- 19.9 x 10(-5)) higher, respectively, than the spontaneous mutant frequency (3.8 +/- 0.7 x 10(-5)). DNA sequence analysis of the DMBA-induced lacl(-) 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 lacl gene of Rat2(TM) cells displays distinct DNA sequence and DNA strand preferences.