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-2Kk and HPRT-deficient T cells was measured in the H-2Kb, kDd,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-2Kk-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 x 10(-6) at 0 Gy to a maximum of 1.13 x 10(-4) at 4 Gy, and decreases to 2.9 x 10(-5) at 6 Gy. The H-2K- MF in the non-irradiated mouse was 8.4 x 10(-7). It increases with dose to a maximum of 8.1 x 10(-6) at 4 Gy and declines to 3.3 x 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-2Kk-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-2Dd 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-2Kb 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.

Studies of thioguanine‐resistant lymphocytes induced by in vivo irradiation of mice

“…The occurrence of dosedependent radiation mutagenesis in mice has been demonstrated in a number of studies [Dempsey and Morley 1986;Lorenz et al, 1990Lorenz et al, , 1993Lorenz et al, , 1994Grdina et al, 1992;Kataoka et al, 1992Kataoka et al, , 1993Gaziev et al, 1995;Klarmann et al, 1995;Dobrovolsky et al, 2002;Liang et al, 2007]. However, the more extensive evaluation of time elapsed after acute g irradiation revealed that mutant frequency dependence on dose shifted between 3 and 5 weeks and 10-53 weeks, from being clearly quadratic at the peak of mutant recovery in weeks 3-5 to a statistically strong but less-defined dose relationship in the later time frame.…”

“…In contrast, in studies of neutron-exposed mice Grdina et al [1992] found that mutant frequency was elevated at 7 weeks, but not at 5 weeks, and was maximal and stable from 8 to 21 weeks after exposure of mice to 1 Gy. In a study of a larger dose range at 8-10 weeks after irradiation, Klarmann et al [1995] detected a maximum frequency after 400 cGy of X-rays and a decreased mutant frequency after 600 cGy that they attributed to dose-dependent selection against mutants. Additional research of the key variables will be needed to determine whether the observed differences in dose dependence as a function of time and dose rate and radiation source are attributable to differences in lymphocyte population dynamics associated with varying cytotoxicity, differences in the molecular spectra of the induced mutations, or combinations of these and additional factors.…”

“…Additional research of the key variables will be needed to determine whether the observed differences in dose dependence as a function of time and dose rate and radiation source are attributable to differences in lymphocyte population dynamics associated with varying cytotoxicity, differences in the molecular spectra of the induced mutations, or combinations of these and additional factors. Cell proliferation-based selection has been postulated to explain the age dependence of Hprt mutant frequencies in unirradiated, heterozygous Hprt 1/2 female mice [Deubel et al, 1996] and also the shape of the dose dependence of hprt Hprt frequency after exposure of mice to acute doses of X-rays (see mentioned earlier, [Klarmann et al, 1995]). Given the differences in design and dose dependence of mutagenesis among the in vivo radiation studies available to date, and our limited understanding of the key variables, caution is advised in making comparisons among studies employing different dose rates, radiation qualities, and sampling times.…”

Methods for Genotoxicity Testing of Environmental Pollutants

“…In the mouse, rat, and human hprt systems, promutagens, direct acting mutagens and X-irradiation readily induce positive responses (Cochrane and Skopek 1994;Klarmann et al 1995;Aidoo et al 1997) indicating that rodent lymphocytes effectively detect the induction of genotoxic damage in vivo. The results of these studies indicate the ability of rat lymphocytes to detect the carcinogenic potential of many tissue-or organ-specific mutagens.…”