Keywords

1 Background

1.1 Why Have Low-Dose Radiation Issues Not Been Resolved, Despite the Passage of More Than 90 Years?

A study report by Muller in 1927 using Drosophila illustrated the concerns of the effect of low-dose (LD) radiation in humans on the next generation [1, 2]. Then, in the 1950s these concerns became more pressing with the expected increase in environmental radiation (global fallout) caused by nuclear tests at Bikini Atoll and the rise of pronuclear power advocates [3]. And it had been proposed to establish United Nations Scientific Committee on the effects of Atomic Radiation (UNSCEAR) at the UN General Assembly in 1955.

Recessive lethal mutations generated by genetic damage caused by radiation-induced DNA double-strand breaks create latent lethal actions in offspring. In particular, recessive lethal mutations on the X chromosome exert a more lethal effect in males, who have only one X chromosome, than in females, who have two X chromosomes. For this reason, the male population was expected to decrease, presenting an urgent problem for humankind, and there was heated discussion on the establishment of safe ranges for public exposure [4].

There is an increased incidence of cancer in the atomic bomb survivors of Hiroshima and Nagasaki (Hibakusha) [5,6,7,8]. Gene mutations are considered as the main cause of carcinogenesis [9]. These further increased the concern over radiation-induced mutagenesis [10,11,12,13] and carcinogenesis [14]. Fortunately, there was no reduction in the male population due the effect of radiation even among Hibakusha [15, 16], but concerns over the genetic and the carcinogenic effect of low-dose (LD) radiation exposure on the next generation has remained the central proposition of the discussion [17,18,19,20,21,22,23].

However, even in 2018, more than 90 years since these concerns were first raised, there are no firm conclusions on the safe range of LD radiation. The scientific opinion on the effect of LD radiation has been divided extremely as seen in the debate on the effect of LD radiation on the prevalence of childhood thyroid cancer attributed to the Fukushima Daiichi Nuclear Power Plant (FNPP) accident [24, 25]. Dispute of so-called scientists on this topic in front of the general public created blunders resulting in a massive loss of faith in scientists by the general public. Unfortunately, some people misunderstand the dose limit of radiation management guidelines as the dangerous border. Furthermore, the baseless testimony by some scientists adversely affects the consensus formation between the administration and the public, which could be the field of trans-science [26]. The job of scientists is not to decide whether LD radiation is safe or dangerous but to provide evidence-based quantitative data for risk assessment [27] and publicize the data to the public in a fair and easy-to-understand manner.

Why have these issues not been resolved, despite the passage of more than 70 years? However, most of the scientists have virtually focused their research on the use of radiation as a tool, but not mainly implemented on the presence or absence of a safety range of LD radiation. There are a number of reasons for this question such as:

  1. 1.

    Large-scale samples and long-term observations are required to obtain data on the effect of LD radiation.

  2. 2.

    Physical dose measurement is rather easy, but the evaluation of absorbed dose is difficult.

  3. 3.

    The impact of the results could be low in spite of the laborious experiments under limited conditions, which makes scientists have little incentive for this research.

  4. 4.

    Difficulty to quantify confounding factors and to evaluate exposure dose in epidemiological studies.

  5. 5.

    Difficult to verify negative data and, as a result, to acquire research funding constantly.

It is also envisioned that there are a large volume of buried data on the effect of LD radiation that have not been disclosed to the world among research, which is because positive data are publishable but negative ones are not even well-planned and precise as the research is (particularly with animal experiments).

To resolve these issues, it is essential to motivate scientists to acquire data as a legacy for future generations, develop methods able to quantitatively evaluate radiation with a high level of sensitivity even in areas of LD radiation, and accumulate basic data that will form evidence for risk evaluation.

1.2 Investigating the Effect of Multigenerational Low-Dose (LD) Internal Exposure on Offspring

Concern of LD radiation exposure as a social problem immediately after the Chernobyl nuclear power plant (CNPP) accident (1986) put emphasis on the carcinogenicity of the radiation-exposed generation and the genetic impact on the next generation. Previous research on the effect of radiation exposure have been reported through the life span study (LSS) and adult health study (AHS) of Hibakusha, industrial radiation exposure [28,29,30,31,32,33], medical radiation exposure [34,35,36], high background exposure [37,38,39] and various animal experiments [40,41,42,43]. Recently studies have attempted to establish highly sensitive quantitative detection of biological reactants after exposure to LD radiation [44,45,46]. However, almost all of these studies focused on carcinogenesis and mutation of the first or the second generation only. It can be said that there has been no research on analyzing the effect of LD and LD-rate (LDR) internal exposure on descendants of multiple generations. Minor variations of each generation need to be accumulated over consecutive generations until the effect of radiation becomes evident, which would take more than several hundred years in humans. Therefore, the author attempted to evaluate the transgenerational effect of LD radiation within a short timeframe using inbred mice with more rapid intergenerational changes than humans. The author believes that a variety of compelling results on biological effects of LD radiation have been obtained from animal experiments in the author’s laboratory. In this chapter, the author would like to introduce those results in the hope of helping readers consider the effect of persistent internal LD radiation.

2 Experimental Methods and Results

2.1 Effects of Radioactive Cesium-Containing Water on Mice

Two littermates were selected from a litter of A/J mouse strain. One group was reared with free drinking of radioactive cesium chloride (137CsCl) containing water (100 Bq/ml) as the LD internal exposure group (137Cs group), and the other group was reared with 137Cs free intake of water as control without radiation. The offspring of these mice produced more than 15 generational changes through sibling mating (equates to approximately 300 years of generational changes in humans), and the following 1–8 experiments were carried out by selected mice from the 137Cs and the control groups with the same ancestor (origin). Since manuscripts of experiments 3 to 8 are in preparation, detailed data of these experiments are not shown.

  1. 1.

    Organ distribution of 137Cs in Mice

    Figure 18.1 shows the distribution of 137Cs in the organs of small wild animals collected in a moderately contaminated region of Belarus (Babchin Village) 11 years after the CNPP accident [47, 48].

    Fig. 18.1
    figure 1

    Distribution of 137Cs in the various organs of wild animals living in the middle-level contaminated areas (Babchin Village) of Belarus in 1997

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    Figure 18.2 shows the difference of 137Cs concentration in organs of A/J mice that continuously drank 137CsCl water (100 Bq/ml) as drinking water for 8 months in the laboratory [49]. As shown in Figs. 18.1 and 18.2, there was a large accumulation of 137Cs in muscles. Also, despite drinking a constant daily amount of 137Cs, the amount of 137Cs in the organs was constant, reaching a plateau corresponding to the consumption amount and flattening out thereafter.

    Fig. 18.2
    figure 2

    (a) The accumulation of 137Cs over time in each organ in male mice that had ingested water supplemented with 137CsCl (100 Bq/ml). The estimated intake was 16 Bq/g body weight/day and 440 Bq/mouse/day. (b) Body distribution of 137Cs after 8 months of ad libitum 137Cs water (10 Bq/ml or 100 Bq/ml) (error bar represents 95% CI)

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    Figure 18.3 shows the overview of the experiment on the transgenerational effect by internal exposure to 137 Cs water. Figure 18.4 shows the chronic internal exposure and 137Cs concentration in the liver at all stages of mouse development in this experiment.

    Fig. 18.3
    figure 3

    The overview of experiments on transgenerational effects by internal exposure of 137Cs

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    Fig. 18.4
    figure 4

    Chronic internal exposure and 137 Cs concentration at all stages of mouse development. The graph shows the change of internal 137Cs concentration in the liver of mouse from fetus to 15 months age

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    Figure 18.5 presents the 18th-generation (8 months old) mice and shows that the amount of 137Cs in all the organs rapidly attenuated after 137Cs was ceased from drinking. 137Cs concentration in muscles declined more slowly than that in other organs [49].

    Fig. 18.5
    figure 5

    The decrease of the 137Cs level in 8-month-old female mice in the 18th generation after the mice had started drinking nonradioactive water

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  2. 2.

    Effect on Intergenerational Litter Size and Sex Ratio

    Mean sex ratio (Fig. 18.6a) and mean litter size (Fig. 18.6b) were compared between the 137Cs group and the control group over generations 1–18 [49]. If the amount of intake was converted to drinking water in humans, 100 Bq/ml 137Cs water for mice would equate to 100,000 Bq/L for humans. There was no effect on the mouse sex ratio and litter size even after 18 continuous generations of mice continued to drink 100 Bq/ml 137Cs water [49]. In this experiment, it will be the proof that there was no decrease in the male population due to radiation effects of the fallout of nuclear tests.

    Fig. 18.6
    figure 6

    (a) The mean sex ratio of all generations (F1–F18), (b) the mean litter size of all generations (F1–F18) (error bar represents 95% CI)

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  3. 3.

    Induction of DNA Double-Strand Breaks in Hepatocytes

    Measurement of the number of γ-H2AX foci, which is the indicator of the first process for repair of DNA double-strand breaks generated in cells [50,51,52,53,54], was performed with hepatocytes. There were significantly more DNA double-strand breaks per generation (100 days) with approximately 50 mGy (2313 Bq per mouse, mean 93.54 Bq/g body weight, 59.5 Bq per liver) of 137Cs internal exposure compared to the control group.

  4. 4.

    Chromosomal Abnormalities and the Micronucleus Test in Bone Marrow Cells

    Chromosomal abnormalities common to all cells were detected using the multicolor Fluorescence In Situ Hybridization (FISH) technique, and the micronucleus test was conducted in the tenth-generation mice. No common chromosomal abnormalities were found in any of bone marrow cells in the 137Cs group. There was increased incidence of DNA double-strand breaks, as described in 3 (above), but common chromosomal abnormalities carried over to the next generation were not found after the tenth generation of mice. Similarly, the micronucleus test revealed no significant increase in the 137Cs group.

  5. 5.

    Effect of Low-Dose (LD) Radiation on Lung Carcinogenesis

    The incidence of lung tumors and the mean tumor mass in 10-month-old mice were assessed. There was no difference in the incidence of spontaneous onset and urethane-induced lung tumors between the 137Cs group and the control group. Interestingly, the growth rate of tumors was significantly inhibited in the 137Cs group.

  6. 6.

    Oxidative Stress in Blood Plasma

    The balance of oxidative stress and antioxidant capacity in the body was studied. In the 137Cs group, there was a significant elevation of plasma 8-oxodihydroguanine, which is an indicator of oxidative stress, but there was no significant difference from the control group in terms of antioxidant capacity. The generated oxidative stress was considered to be within an allowable biological range.

  7. 7.

    Metabolome Analysis in the Heart

    The glycolytic pathway in the 137Cs group was inhibited, and there was also a reduction in antioxidants such as glutathione (GSH) and cysteine.

  8. 8.

    Quantitative Analysis of DNA Base Mutations by Whole Genome Sequencing in the Liver

    Detection of base sequence mutations in noncoding regions, where base mutations generated in germ cells do not affect life support but are expected to accumulate in each subsequent generation, was attempted in male mice of F20 and F23 origin. There was a higher incidence of single-nucleotide variant (SNV) and insertion/deletion (Indel) per 3 billion bases at intron and intergenic sites than at exon sites by comparing whole genome sequence in each generation. However, even with repeated generations, there was not a large difference in the total base mutation rate between the 137Cs group and the control group.

2.2 Detecting Carcinogenesis and Mutations Caused by Oxidative Stress

The fatal effect of radiation is the DNA double-strand break. In the high-dose range of high linear energy transfer (LET) radiation, dominant process of the breaks is caused by direct action, while in low LET radiation such as X-rays, about two-thirds of the biologic damages are caused by indirect action through radicals generated by reaction with water in the cell [55]. In case of LD and LDR radiation, the action exerting the greatest effect is assumed to be oxidative stress caused by reactive oxygen species attributed to radiation exposure. Reports indicate that using genetically modified mice (DNA mismatch repair-deficient mice) that are unable to repair the DNA damage enables highly sensitive detection of DNA damage caused by oxidative stress agents and radiation [56, 57]. Also, these mice show a high incidence of small intestine tumors through oral consumption of an oxidative stress agent potassium bromide (KBr) aqueous solution [58, 59]. The author has started quantitative research on carcinogenesis and mutations caused by oxidative stress using this mouse strain.

2.3 Comparison of Internal Exposure and External Exposure

The effect of exposure in humans is based on the absorbed radiation dose in Gy (J/kg). However, this unit is obtained from the energy absorbed per kg of tissue. Internal exposure experiments using mice are different from external exposure ones, so the absorbed radiation dose from the radioactive substance must be considered in terms of the amount accumulated in a body that weighs less than 1 kg (around 0.025 kg). However, most of the γ-rays from a mouse body measuring approximately 3 cm in diameter completely pass out of the body without transferring the energy. In these instances, is it a good idea to evaluate with Gy, which is an absorption dose to the order of kg? This same question can be applied to humans. It is vital to confirm if the effect of internal exposure can be evaluated with a dose conversion factor from Bq to Gy in same way in infants and adults where there are differences in the distribution and size of organs. The dose conversion factor from Bq to Gy for rats is shown by ICRP (pub108), but no information for mice. If the actual internal exposure dose is not accurately evaluated in the mouse experiment, it significantly affects the quality of outcome of the experiment. To determine the dose conversion factor of the mice in these experimental conditions, it can be considered three evaluation methods. Previous evaluation methods for the internal exposure dose include the dose estimated from physical calculations (3.14 μGy.g/Bq.day) and evaluation methods using Monte Carlo analysis such as the EGS5 code system (Electron Gamma Shower Version 5) (3.00 μGy.g/Bq.day, evaluated by Endo D, Rakuno Gakuen Univ.) and the PHITS (Particle and Heavy Ion Transport Code System) (3.40 μGy.g/Bq.day: evaluated by Endo S, Hiroshima Univ.). We are continuing research to establish methods for evaluating internal exposure dose with an attempt to adopt new techniques such as quantifying noncoding RNA (ncRNA) specific to the reaction against radiation exposure [45]. If it is possible to detect a dose-dependent reaction with highly sensitive and precise quantification of the biological reactions caused by radiation, then it would be a great help to verify equivalent dose of internal and external exposure.

There are strategy gap between in vivo and in vitro experiments (Fig.18.7). “In vivo” is like a black box, and “in vitro” is like a complex electronic circuit. We want to know what is going on inside the black box, and we want to know the pathway of circuit from the beginning to the end. Particularly in studies of LD radiation effects, bridging the gap between in vivo and in vitro experiments can effectively lead to clear results.

Fig. 18.7
figure 7

The strategy gap in vivo and in vitro

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3 Conclusion

The experiments with Drosophila made people aware of the effect of radiation on human genetics. The mouse is a closer experimental animal to humans. However, the data obtained from inbred mouse strains are the same as those obtained from monozygotic twins, as all the mice have the same genetic background, so the obtained data reflect genetic bias of one person, irrespective of the number of mice used. Therefore, inbred mice may not be suitable for regulatory science research for the general public, where a variety of exposure disorders are envisioned. However, mice have almost the same spontaneous mutation rate per generation, base substitution rate per nucleotide per generation, and number of genes as humans (Table 18.1) [60,61,62,63,64,65,66,67,68,69,70,71]. Therefore, the inbred mouse experiment is effective for removing confounding factors and for detecting fundamental biological effects, which would be inconceivable in humans.

Table 18.1 Homology between mouse and human. Spontaneous mutation rate per generation, base substitution rate per nucleotide per generation and the number of genes in mice are almost the same as humans. This is the reason that the mice can be used as the experimental animal to know hereditary influence in humans. bAverage of base substitution rate in references of human [58,59,60,61,62,63,64,65,66,67,68]
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Obtaining data that contribute to the establishment of the radiation safety range has not necessarily been vigorously implemented for more than 70 years since the nuclear test at the Bikini Atoll. Those data will also be extremely effective to determine if an unpredictable radiation exposure occurs. We hope that many scholars will undertake research on the effect of LD radiation throughout the world.