title: Three origins of phiX174 am3 revertants in transgenic cell culture.
abstract: Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors. We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS). In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions. PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU). An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56. In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of <14. The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively. These results show that only in vivo revertants reflect mutagen treatment. They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.
authors.affiliation: Mammalian Mutagenesis Group, Laboratory of Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA. malling@niehs.nih.gov
authors.forename: Heinrich V
authors.initials: HV
authors.lastname: Malling
authors.forename: Robert R
authors.initials: RR
authors.lastname: Delongchamp
authors.forename: Carrie R
authors.initials: CR
authors.lastname: Valentine
mesh_terms: D000465:Algorithms; D000818:Animals; D030801:Animals, Genetically Modified; D010584:Bacteriophage phi X 174 / Q000235:genetics*; D018929:Cell Culture Techniques; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D004926:Escherichia coli / Q000378:metabolism; D005038:Ethylnitrosourea; D006720:Homozygote; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D009153:Mutagens; D005038:Ethylnitrosourea