DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue® rats treated with 7,12-dimethylbenz[a]anthracene

Recently we compared the lad and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue(R) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lad MF and types of lad mutations. seven-week-old female BE rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lad MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment, The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18.3 +/- 4 x 10(-6) p.f.u.). P-32-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts, Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lad mutations were base pair sutbstitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations:) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lad gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lad in mammary tissue and that adducts with bath G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.