Sensitivity of UVB-Induced Mutant Detection in the [Phi]X174 Transgenic Forward Mutation Assay

A perceived shortcoming of transgenic rodent mutation assays is the relatively high spontaneous mutant frequencies (MFs) of the bacterial transgenes compared to endogenous genes. This background is dominated by G:C->A:T mutation, frequently in a CpG context, mammalian sites of cytosine methylation. The single A:T target site of the ΦX174 transgenic mouse reversion assay might avoid this background, yet in vivo mutagenic sensitivity at this site was poor because of background mutation in the recovery bacteria. In order to determine the actual spontaneous MF of the ΦX174 transgene in the mouse cell, several research tools have been developed: 1) single burst analysis, for distinguishing mouse from bacterial mutation; 2) a transgenic mouse embryonic cell line, PX-2; and 3) a forward mutational assay, which has few CpG sites among its target sites. In this study, single burst analysis was applied to the transgenic cell line for the first time, evaluating the response to UVB irradiation for potential phototoxicity studies. Under appropriate plating conditions, single burst analysis lowered the spontaneous MF10-fold from the original report to 0.17×10-5. The MF 72 h after 70 J/m2 UVB irradiation was 8.3×10-5. The characteristic UVB-induced mutant spectrum included >80% G:C->A:T at dipyrimidine sites (primarily TpC dinucleotides) with 13% of these at a single CpG site, and 20% as multiple mutants, tandem and non-tandem. The spontaneous MF per nucleotide, 3×10-8, was comparable to that of human disease genes in the germline. When normalized for target number and dose, single burst analysis of the ΦX174 forward mutational assay produced a UVB-induced MF that was equivalent to that of the cII transgene in mouse cell culture, but a spontaneous MF an order of magnitude lower. The results suggest this cell line is highly sensitive to UVB irradiation.