SPLICING MUTATIONS IN THE CHO DHFR GENE PREFERENTIALLY INDUCED BY (+/-)-3-ALPHA,4-BETA-DIHYDROXY-1-ALPHA,2-ALPHA-EPOXY-1,2,3,4-TETRAHYDROBENZO[C]PHENANTHRENE

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3α,4β-dihydroxy-1α,2α-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 μM yielded a 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10-4, 103-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A·T → T·A and G·C → T·A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG. Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.