X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells

Abstract

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here, we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase beta, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks, to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally regulated phenotypes, such as mitotic index.

Figure 1. Prenatal X-irradiation increases the Aprt but not the Hprt mutant frequency in T cells

A. Time course of mutagenic effects of X-rays on T-cells of B6C3F1 mice. Pregnant C57BL/6 mice carrying B6C3F1 fetuses were irradiated with a single dose of 1 Gy X-rays on days 16 − 18 of gestation. T cells were isolated from irradiated offspring and assayed for mutations at Hprt and Aprt at 20, 40 and 60 days after exposure. Each point represents the mean mutant frequency (N = 5 − 22 mice/group). The error bar represents the standard error of the mean. B. Effect of X-ray dose on the Aprt and Hprt MFs in T-cells from B6C3F1 mice irradiated in utero. B6C3F1 fetuses were irradiated with a single dose of 0.5, 1, or 2 Gy of X-rays on days 16 − 18 of gestation. T cells were isolated 60 days after prenatal exposure. Each point represents the mean mutant frequency (N = 5 − 22/group). The error bar shows standard error of the mean.

Figure 2. Mitotic recombination, but not point mutation, is the major cause of the increased Aprt MF in T cells from mice irradiated in utero

A minimum of 120 DAP^r colonies per treatment group were characterized as described in the Methods and Materials. The value of each bar represents the Aprt MF caused by a specific mutation type and is calculated by multiplying the Aprt MF by the fraction of each given mutation type.

Figure 3. Fetal hematopoietic cells have a lower level of DNA polymerase β-dependent BER activity than adult hematopoietic cells

A. In vitro BER activity in extracts of cells from fetal and adult mice. BER reactions contained the 30 bp DNA duplex with a UG mismatch, [α-³²P]dCTP, and nuclear extracts. Replacement by BER of an unpaired U with a paired C [α-³²P]dCTP produces a radioactive 30 bp band. The corresponding “normal” duplex containing the CG base pair served as a control. The radiolabeled 30 bp fragment was visualized and quantified with a Typhoon PhosphoImager using ImageQuant software. B. Western blot analysis of DNA polymerase β in extracts of fetal and adult cells.