title: 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 lambda/lacI shuttle vector as a target for mutagenicity testing. Rat2 cells were exposed to 1 and 10 micrograms/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 lacI- 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.
authors.affiliation: Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, USA. mmanjanatha@nctr.fda.gov
authors.forename: M G
authors.initials: MG
authors.lastname: Manjanatha
authors.forename: J B
authors.initials: JB
authors.lastname: Chen
authors.forename: J G
authors.initials: JG
authors.lastname: Shaddock
authors.forename: A J
authors.initials: AJ
authors.lastname: Harris
authors.forename: S D
authors.initials: SD
authors.lastname: Shelton
authors.forename: D A
authors.initials: DA
authors.lastname: Casciano
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity*; D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000187:drug effects* / Q000235:genetics*; D010582:Bacteriophage lambda / Q000187:drug effects / Q000235:genetics; D002273:Carcinogens / Q000633:toxicity; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D051381:Rats; D012097:Repressor Proteins / Q000187:drug effects* / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019076:Transgenes / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D002273:Carcinogens; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D015127:9,10-Dimethyl-1,2-benzanthracene