Possible factors leading to a misjudgement of mutant frequencies in HPRT assay.
Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay in in vitro study were analysed. In 12 separate reconstruction experiments with independent pairs of wild type (WT) and mutant (HPRT-) clones, the CE of WT cells (Group 1) and the recovery of mutant cells in absence (Group 2), as well as in the presence of non-irradiated (Group 3), or irradiated (Group 4) WT cells (10(4) cells/well) was determined. The plating of mutant cells with irradiated WT cells improved their CEs by almost 30%. In contrast, the presence of non-irradiated WT cells led to a slight decline (10%) in CE of mutant cells, resulting in a significant difference between groups (p = 0.0083). The extent of decline in survival of mutant cells in the presence of non-irradiated WT cells negatively correlated (r = 0.3496, p < 0.05) with the initial CE of WT cells. The data suggest that the presence of WT cells in the selection plates may suppress the recovery of mutants in HPRT assay, and this negative effect is stronger in samples with high CE. These findings indicate a possible source for a serious underestimation of mutant frequencies (3-fold in the range of CEs from 10% to 60%) in the HPRT assay and may be useful for the interpretation of results from studies on exposure to mutagens in humans.
Khaidakov
M
M
Department of Biology, University of Victoria, B.C., Canada.
Glickman
B W
BW
D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009154:Mutation
D007041:Hypoxanthine Phosphoribosyltransferase