Accelerated⁴⁸Ti Ions Induce Autosomal Mutations in Mouse Kidney Epithelium at Low Dose and Fluence

Abstract

Exposure to high-energy charged particles (HZE ions) at low fluence could significantly affect astronaut health after prolonged missions in deep space by inducing mutations and related cancers. We tested the hypothesis that the mutagenic effects of HZE ions could be detected at low fluence in a mouse model that detects autosomal mutations in vivo. Aprt heterozygous mice were exposed to 0.2, 0.4 and 1.4 Gy of densely ionizing (48)Ti ions (1 GeV/amu, LET = 107 keV/μm). We observed a dose-dependent increase in the Aprt mutant fraction in kidney epithelium at the two lowest doses (an average of 1 or 2 particles/cell nucleus) that plateaued at the highest dose (7 particles/cell nucleus). Mutant cells were expanded to determine mutation spectra and translocations affecting chromosome 8, which encodes Aprt. A PCR-based analysis for loss of heterozygosity (LOH) events on chromosome 8 demonstrated a significant shift in the mutational spectrum from Ti ion exposure, even at low fluence, by revealing "radiation signature" mutations in mutant cells from exposed mice. Likewise, a cytogenetic assay for nonreciprocal chromosome 8 translocations showed an effect of exposure. A genome-wide LOH assay for events affecting nonselected chromosomes also showed an effect of exposure even for the lowest dose tested. Considered in their entirety, these results show that accelerated (48)Ti ions induce large mutations affecting one or more chromosomes at low dose and fluence.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change

“…Approximately half of all spontaneous mutations are due to chromosome loss (53.7%), with LOH patterns consistent with intragenic events (23.5%) and true mitotic recombination (16.8%) as the other two major categories. The classification of spontaneous mitotic recombination events was confirmed by a cytogenetic analysis in our prior study [14] and in a study by others with mouse ear fibroblasts [32]. …”

“…The discontinuous LOH pattern is defined as a LOH tract that is not physically linked to the mutation leading to loss of Aprt expression. We use the qualifier “apparent” for the mitotic recombination LOH patterns because prior studies have shown that a substantial fraction of these patterns after charged particle exposure are due to non-reciprocal chromosome translocations [14, 27] (also, see below).…”

“…We showed in recent studies that a significant fraction of Aprt mutants induced by charged particles (5 Gy of protons or 0.2, 0.4, or 1.4 Gy Ti ions) exhibiting the “apparent” mitotic recombination LOH patterns were due to non-reciprocal chromosome translocations involving chromosome 8 [14, 27]. Here we examined a subset of Aprt mutants from Fe ion-exposed kidneys (0.25–1.0 Gy) exhibiting the mitotic recombination pattern with fluorescent in situ hybridization (FISH).…”

“…In prior work with protons (4 and 5 Gy) and Ti ions (0.2, 0.4, and 1.4 Gy), we showed that Aprt mutant cells isolated from irradiated mouse kidneys display LOH events on other chromosomes at higher frequencies than observed in spontaneous mutants [14, 29]. We use the term “genomic LOH” to describe these mutants.…”

“…Each clone was plated in one well of a 24 well plate and when confluent the cells were split into three wells. Finally, when the three wells were confluent, the cells were seeded into one T25 cm 2 flask and split 1:2 thereafter for DNA preparations and cytogenetic analyses, which was performed as described elsewhere [14]. …”