The in vivo but not the in vitro am3 revertant frequencies increase linearly with increased ethylnitrosourea doses in spleen of mice transgenic for phiX174 am3, cs70 using the single burst assay.
The am3 revertant frequencies (RF) in spleens from male mice transgenic for phiX174 am3, cs70 were analyzed 14 weeks after ethylnitrosourea (ENU) treatment, both by the single burst assay (SBA) and the mixed burst assay (MBA). The mean in vivo (burst size >30/assay plate) revertant frequency (MRF) for the vehicle control was 2.6x10(-7). The ENU induced in vivo RF were linear over the dose range 0-150mg/kg, (r(2)=0.999). The concomitant in (burst size <or=30/assay plate) was independent of dose (r(2)=0.216). The only viable revertants are base pair substitutions of the center base pair in the am3 nonsense (TAG) codon in the phiX174 lysis E gene. Sequenced revertants chosen randomly from in vitro plates and in vivo untreated control plates were A-->G transitions. Sequence analysis of in vivo revertants from ENU treated animals revealed revertants that were 17% A-->G transitions and 83% A-->T transversions, the latter being consistent with the reported A:T base pair alterations induced by ENU. No A-->C transitions were seen. This suggests the occurrence of an ENU-induced O(2) ET-dT lesion leading to a dT base mismatch. The observations in this report both confirm and validate the use of the SBA for distinguishing between in vivo mutations that are fixed in the animal and in vitro mutations that arise from other sources. The ability of the SBA to distinguish the in vivo from the in vitro origin of mutations has increased the specificity, sensitivity and utility of the phiX transgenic system.
Weaver
Robert P
RP
Mammalian Mutagenesis Group, Laboratory of Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Malling
Heinrich V
HV
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics*; D001835:Body Weight / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005038:Ethylnitrosourea / Q000633:toxicity*; D005787:Gene Frequency; D016014:Linear Models; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009929:Organ Size / Q000187:drug effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000033:anatomy & histology / Q000187:drug effects*
D009153:Mutagens; D005038:Ethylnitrosourea