Characterization of mutant spectra generated by a forward mutational assay for gene A of ΦX174 from ENU‐treated transgenic mouse embryonic cell line PX‐2†

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Abstract
The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for ΦX174 that requires an altered, functional ΦX174 protein and therefore should have fewer targets (sense, base‐pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N‐ethyl‐N‐nitrosourea (ENU). We identified 25 target sites and 33 different mutations in ΦX174 gene A after sequencing over 350 spontaneous and ENU‐induced mutants, mostly from mouse embryonic cell line PX‐2 isolated from mice transgenic for ΦX174 am3,cs70 (line 54). All six types of base‐pair substitution were represented among both the spontaneous and ENU‐treated mutant spectra. The mutant spectra from cells treated with 200 and 400 μg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R2 = 0.79), with a ninefold increase in mutant frequency at the 400 μg/ml dose. The spontaneous mutant frequency was 1.9 × 10−5 and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 targets were identified only from ENU‐treated cells. Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases. The results suggest that the ΦX174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay. Environ. Mol. Mutagen. 39:55–68, 2002 Published 2002 Wiley‐Liss, Inc.

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