MOLECULAR ANALYSIS OF LACI MUTATIONS IN RAT2(TM) CELLS EXPOSED TO 7,12-DIMETHYLBENZ[A]ANTHRACENE - EVIDENCE FOR DNA-SEQUENCE AND DNA STRANDBIASES 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 resc ued from untreated and mutagen-treated cells and mutant frequencies we re determined. The low and high doses of DMBA induced mutant frequenci es 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 m utations at A:T and G:C, and that A:T --> T:A and G:C --> T:A transver sions were the predominant types. In addition, 23 of 28 (82%) A:T base pair substitution mutations occurred with the mutated dA, the putative ly 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 transitio ns in the low dose data, the mutational profiles from low and high dos es 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.