title: Characterization of mutant spectra generated by a forward mutational assay for gene A of Phi X174 from ENU-treated transgenic mouse embryonic cell line PX-2.
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 Phi X174 that requires an altered, functional Phi X174 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 Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 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 microg/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 (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose. The spontaneous mutant frequency was 1.9 x 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 Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.
authors.affiliation: Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079-9501, USA. cvalentine@nctr.fda.gov
authors.forename: Carrie R
authors.initials: CR
authors.lastname: Valentine
authors.forename: Beverly A
authors.initials: BA
authors.lastname: Montgomery
authors.forename: Scott G
authors.initials: SG
authors.lastname: Miller
authors.forename: Robert R
authors.initials: RR
authors.lastname: Delongchamp
authors.forename: Bentley A
authors.initials: BA
authors.lastname: Fane
authors.forename: Heinrich V
authors.initials: HV
authors.lastname: Malling
mesh_terms: D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics; D020137:Base Pair Mismatch; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005038:Ethylnitrosourea / Q000633:toxicity*; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation
chemical_list: D009153:Mutagens; D005038:Ethylnitrosourea