RERF Report No. 6-14§

Paper published in Radiation Research“Mutagenic Effects of Ionizing Radiation on Immature Rat Oocytes”
Jun-ichi Asakawa, Yujiroh Kamiguchi, Kenji Kamiya, and Nori Nakamura
Radiat Res 2014 (October); 182 (4):430–4(doi: 10.1667/RR13776.1)
Study Findings
This study, using rats, examined deletion mutations*, which serve as a marker for
determining genetic effects of radiation. We screened more than one million DNA
fragments from the offspring (F1) of female rats whose immature oocytes had received
2.5 Gy of radiation and a similar number from the offspring of non-irradiated controls.
We did not find any deletion mutations that were derived from the irradiated female rats.
These results suggest that immature oocytes are less sensitive than mature oocytes tomutation induction.
*This type of mutation occurs when a portion of chromosome is lost. It is considered the major
type of mutation induced by radiation.

Explanation
Risk assessment of genetic effects of radiation in humans is mainly based on theresults of mouse experiments. In male mice, much data are available after radiation
exposure of their spermatogonial cells. In female mice, the immature oocytes, which are
the analogous female target of study, are highly sensitive to radiation and die in aprocess called apoptosis, leaving the F1 offspring derived from the surviving immature
oocytes essentially mutation-free. In the case of human females, however, such highsensitivity to radiation-induced apoptosis is not observed in immature oocytes. Given
that apoptotic death eliminates damaged oocytes in mice and prevents viable offspringwith mutations, the possibility exists that mutations may occur at a high frequency insituations where such a clearance mechanism does not function (e.g., in humans). To
explain the results of RERF’s genetic studies of A-bomb survivors and their offspring(F1), experimental data from female animals are indispensable, and hence we havesearched over many years for appropriate experimental animals that can be used as amodel for radiation exposure in human females. Since our investigation into this issue
revealed that rat immature oocytes are not as sensitive to apoptosis as mouse immature
oocytes and can therefore serve as an animal model, we conducted a genetic effects

study on F1 populations derived from irradiated female rats and nonirradiated female
rats utilizing two-dimensional electrophoresis (2DE method).**This method cuts genes (DNA) into segments with a given restriction enzyme and performs
an electrophoresis to separate the DNA segments by size (restriction enzymes cut DNA at given
sequences of 4-8 base pairs). The second separation, again by size, occurs with a different
restriction enzyme. Thus, if there are deletions in autosomal DNA (the numbered chromosomepairs [22] as opposed to the sex chromosome pair [1]) caused by radiation, the copy number ofcorresponding DNA is reduced by one-half, as only one chromosomal DNA of the autosomal
DNA pair is affected).

1. Objectives
To determine the mutation rates induced by radiation in immature oocytes in rats
irradiated with 2.5 Gy and to characterize the mutations observed.
2. Methods(1) Materials: Female Sprague-Dawley (SD) rats* were irradiated with 2.5 Gy ofgamma radiation. To exclude offspring derived from mature or maturing oocytes at
the time of irradiation, irradiated females were mated with nonirradiated Brown
Norway (BN)* males starting at 80 days following irradiation. These F1 rats
comprised the irradiated group, while the F1 rats born to non-irradiated pairs wereconsidered controls. At three weeks of age, the spleen, kidney, and liver werecollected from the F1 rats, quickly frozen with liquid nitrogen, and stored at -80℃.
*SD is a laboratory rat strain established long ago and has been used in many
experiments. Due to the rich store of data regarding these rats, they represent aneasy-to-use strain. On the other hand, BN is a pure laboratory rat strain used in recent
genome analyses. Since large numbers of genomic differences exist between SD and BNrats, these differences are useful in determining the parental origin of any mutations
observed in F1 rats.

(2) Two-dimensional electrophoresis: DNA isolated from the spleen of F1 rats wasdigested with Notl and EcoRV restriction enzymes, and the breakpoints wereisotope labeled. Two types of gels containing Notl-EcoRV fragments of 1–4 kb and4–10 kb were prepared. We conducted two-dimensional electrophoresis of thesegels, dried them, and visualized the DNA fragments on X-ray film byautoradiography. Computer-based analysis of the obtained electrophoresis images
was conducted to detect aberrant fragments that would indicate a deletion mutation.

3. Results(1) Selection of DNA fragments to be examined: Detection of a total of about 3,000
DNA fragments (spots) is possible with use of the aforementioned two types ofautoradiograms. From among the spots, 162 from female SD rats, 179 from male
BN rats, and 1,387 shared by both SD and BN strains were found to be suitable formutation screening (equivalent to testing of 1,549 and 1,566 gene loci, respectively,for SD and BN rats). With regard to a total of about 3,000 images obtained from750 rats each in the F1 group (which was derived from 2.5 Gy-irradiated femalerats) and the control group, we examined DNA fragments by means of computer
image analysis. Radiation emitted from the isotope contained in DNA fragments
hitting silver particles turned them black on the film, and hence we determined as
possible mutation cases those that had lost spot intensity by one-half as well ascases that had lost DNA fragments unique to SD or BN.
(2) Detected mutations: About 1.13 million spots were screened in each of the irradiatedgroup and the control group. As a result, a total of 50 germ cell mutations, 18 and
32 mutations in the irradiated group and control group, respectively, were detected.
A majority of the 50 mutations were caused by changes in number of repeats of 2–8
core sequences that constitute genetically unstable loci called microsatellites, which
our previous research suggest were not caused by radiation. A total of threemutations, two in the control group and one in the irradiated group, werebase-change mutations* at restriction enzyme recognition sites. This type ofmutation is also less sensitive to radiation, and hence radiation exposure is unlikely
to have been involved.
*This type of mutation occurs when one DNA base pair is replaced by another.

With regard to deletion mutations, which serve as a marker for determining genetic
effects of radiation, we detected one and three such mutations, respectively, in thecontrol group and the 2.5 Gy-irradiated group, none of which could be established asbeing derived from the irradiated females (two of the three mutations in the irradiatedgroup were derived from males, and the parental origin of the remaining mutation could
not be determined). The aforementioned results suggest that the paucity of mutations in“mouse” immature oocytes is not attributable to apoptotic elimination ofradiation-damaged cells, because rat immature oocytes that are more resistant did notindicate a higher radiation sensitivity for mutation induction. Therefore, immatureoocytes may be refractory (in other words, resistant) by nature to mutation induction.
Due to the limited number of mutations observed, however, we plan to conduct researchin the future using a different method that would enable screening of a larger number ofgenomic sites.
The Radiation Effects Research Foundation has studied A-bomb survivors and their offspring in
Hiroshima and Nagasaki for more than 60 years. RERF’s research achievements are considered theprincipal scientific basis for radiation risk assessment by the United Nations Scientific Committee on theEffects of Atomic Radiation (UNSCEAR) and for recommendations regarding radiation protection
standards by the International Commission on Radiological Protection (ICRP). RERF expresses its
profound gratitude to the A-bomb survivors and survivors’ offspring for their cooperation in our studies.

§Radiation Research, which is an official monthly journal of the Radiation Research Society, publishes

original, peer-reviewed papers and review articles on radiation effects and related issues in the fields ofphysics, chemistry, biology, and medicine. (Impact factor in 2013: 2.445)