Mutant Frequency at the H-2K Class 1 and HPRT Genes in T Lymphocytes from the X-ray-exposed Mouse

Abstract

The frequency of H-2K^k and HPRT-deficient T cells was measured in the H-2K^bkD^d,k genotype mouse 8–10 weeks after X-ray exposure at doses up to 6 Gy to compare the mutant frequency (MF) of an autosomal gene with that of an X-chromosomal gene. H-2K mutants were enriched by magnetic cell separation (MACS) using the H-2K^k-specific monoclonal antibody H100.5/28 and were isolated by limiting dilution cloning. Finally, the mutant phenotype was verified by flow cytometric analysis in a representative number of clones. The frequency of HPRT-deficient T cells rises from 2·5 × 10⁻⁶ at 0 Gy to a maximum of 1·3 × 10⁻⁴ at 4 Gy, and decreases to 2·9 × 10⁻⁵ at 6 Gy. The H-2K⁻ MF in the non-irradiated mouse was 8·4 × 10⁻⁷. It increases with dose to a maximum of 8·1 × 10⁻⁶ at 4 Gy and declines to 3·3 × 10⁻⁶ at 6 Gy. The H-2K⁻ MF measured depends on the monoclonal antibody used for the isolation of mutants. In a pilot study with another H-2K^k-specific monoclonal antibody (11.4.1), the spontaneous MF was four times higher than in experiments with the H100.5/28 monoclonal antibody. The expression of other class 1 antigens was investigated in H-2K⁻ clones. The H-2D^d antigen had also disappeared in six of 41 clones from irradiated animals. This gene is situated at a distance of 1500 kb from the K-locus. The H-2K^b antigen was present in every investigated clone. In the discussion a model is presented that explains the shape of the dose–response curve of MF by selection against mutants in vivo systems under homeostasis. The results of the present investigation indicate that observed X-ray mutagenicity depends on many factors and that several genes have to be explored before reliable risk estimates are possible.