The effects of in utero irradiation on mutation induction and transgenerational instability in mice

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Abstract

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F1) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F1 offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F1 stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.

Introduction

The developing embryo is especially sensitive to ionising radiation, exposure to which results in foetal mortality and increases the risk of leukaemia and solid cancers after birth [1], [2]. However, to date the mechanisms underlying the health risks associated with prenatal irradiation remain poorly understood. Given that the majority of embryonic cells are actively proliferating, exposure during the early stages of development could lead to a substantial accumulation of radiation-induced mutations across all tissues, thus contributing to the risk of cancer after birth. It is therefore clear that the analysis of mutation induction in utero can provide important insights into the long-term effects of foetal exposure to mutagens, including ionising radiation. It should however be stressed that at present the genetic effects of prenatal irradiation remain poorly characterised. To date, the analysis of both gene mutations and chromosome aberrations in the somatic tissues of adult mice irradiated in utero has resulted in a highly controversial set of data. A number of studies have shown that the frequency of chromosome aberrations and gene mutations in haemopoietic cells remained elevated during adulthood following foetal irradiation [3], [4], [5], while in others no measurable increases in translocation frequencies were found in the lymphocytes of in utero exposed adults [6], [7]. Although relatively few studies have analysed the impact of in utero exposure on the developing germline, preliminary evidence suggests a low efficiency for radiation-induced mutations in foetal gonads [8], [9]. One major reason for the apparently conflicting results describing the effects of foetal irradiation is due to the lack of a sensitive in vivo technique that efficiently detects spontaneous and radiation-induced mutations in both the germline and somatic tissues. Further analysis of in utero mutation induction, using an appropriate detection system, therefore offers an opportunity to gain novel insights into the effects of prenatal exposure to mutagenic agents. One such system, that we have used extensively, utilises mouse expanded simple tandem repeat (ESTR) loci as a biomarker of mutation induction in germline and somatic cells following adult exposure to many mutagenic agents, including ionising radiation [10], [11], [12], [13]. These studies have shown that ESTR loci provide a sensitive experimental system for monitoring germline mutation in mice, permitting evaluation of mutation induction at low doses of exposure and in very small population samples.

In addition to the studies of mutation induction in the germline and somatic tissues of directly exposed organisms, considerable progress has been made in the analysis of the transgenerational effects of parental irradiation, which are manifested in the offspring of treated parents (reviewed in Refs. [14], [15], [16], [17]). It was established that mutation rates in the germline and somatic tissues of non-treated offspring of adult male exposed to ionising radiation or chemical mutagens remain highly elevated [17], [18], [19], [20], [21]. These data suggest that transgenerational genomic instability may be a contributory factor in the elevated cancer risk and enhanced tumour progression observed in the offspring of exposed fathers. They also indicate that a transgenerational genomic destabilisation of the genome can be attributed to an as yet unknown radiation-induced signal in the paternal genome which is inherited through sperm in an epigenetic fashion. In previous studies we have evaluated the transgenerational effects of irradiation of different stages of mouse spermatogenesis and found that mutation rates remained equally elevated in the offspring conceived from 1 to 8 weeks after paternal exposure [18], [19], [20]. These results imply that a radiation-induced instability signal is retained during spermatogenesis, even after several rounds of DNA replication. The persistence of such a signal in the adult germline raises questions regarding the effects of in utero exposure on transgenerational instability. It should however be stressed that the massive epigenetic reprogramming occurring in the developing germline [22] could potentially erase all epigenetic marks of foetal radiation exposure, thus preventing the manifestation of genomic instability in subsequent generations.

Using a combination of approaches to assess ESTR mutation rate, here we have studied the effects of in utero irradiation on mutation induction and transgenerational instability in mice.

Section snippets

Animals

BALB/c mice were obtained from Harlan (Bicester, UK) and housed at the Division of Biomedical Services, University of Leicester. Eight-week-old pregnant females (12 days of gestation) and 7-week-old adult males were given whole-body acute irradiation of 1 Gy of X-rays delivered at 0.5 Gy min−1 (250 kV constant potential, HLV 1.5 mm Cu, Pantak industrial X-ray machine, Connecticut, USA). Eight-week-old in utero irradiated and sham-treated male and female mice were mated to non-irradiated BALB/c

Experimental design

As in a number of our previous studies [18], [19], [20], [21], we have used BALB/c inbred mice, to evaluate the effects of in utero irradiation on ESTR mutation induction and transgenerational instability. BALB/c pregnant mice (Theiler stage 20, 12 days of gestation) were exposed to 1 Gy of acute X-rays. At this stage of mouse development, primordial male and female germ cells are already in the genital ridge area and undergo active mitotic proliferation before entering meiotic or mitotic arrest

Discussion

The analysis of the long-term effects of in utero irradiation has established that (i) ESTR mutation frequencies in the adult males and females irradiated in utero are similarly elevated in the germline and somatic tissues; (ii) the efficiency of in utero irradiation on ESTR mutation induction in the male germline is close to that following adult exposure; (iii) paternal in utero irradiation results in transgenerational instability manifested in the germline and somatic tissues of their F1

Conflict of interest statement

None.

Acknowledgments

We thank the Division of Biomedical Services, University of Leicester for their expert animal care. This work was supported by grants from the European Commission (NOTE, Contract Number 036465), the Wellcome Trust (VIP award 0786607/Z/05/Z), Medical Research Council (G0300477/66802) and U.S. Department of Energy (DE-FG02-03ER63631).

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