|Year : 2017 | Volume
| Issue : 3 | Page : 123-134
Transgenerational effects of radiation on cancer and other disorders in mice and humans
Taisei Nomura1, Larisa S Baleva2, Haruko Ryo3, Shigeki Adachi3, Alla E Sipyagina2, Natalya M Karakhan2
1 Nomura Project, National Institutes of Biomedical Innovation, Health and Nutrition; Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan
2 Children's Center of Antiradiation Protection, Research Institute for Pediatrics, Pirogov Russian National Research Medical University, Moscow, Russian Federation
3 Nomura Project, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka University, Osaka, Japan
|Date of Web Publication||17-Oct-2017|
Nomura Project, National Institutes of Biomedical Innovation, Health and Nutrition; Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Osaka
Source of Support: None, Conflict of Interest: None
Parental exposure of mice to radiation and chemicals causes a variety of adverse effects in the progeny, and the tumor-susceptibility phenotype is transmissible beyond the first postradiation generation. The induced rates of tumors were 100-fold higher than those known for mouse specific locus mutations. There were clear strain differences in the types of naturally-occurring and induced tumors and most of the latter were malignant. Another important finding was that germ-line exposure elicited very weak tumorigenic responses, but caused persistent hypersensitivity in the offspring for the subsequent development of cancer by the postnatal environment. Various disorders were induced in the offspring of mice exposed to radiation. Microsatellite mutations increase dose-dependently and accumulated for 58 generations in the offspring of male parental mice exposed to single dose of X-rays. Changes in gene expression also transmitted to further generations. Radiation-induced genomic instability in germ cells may enhance cancer and other disorders in next generation. In humans, a higher risk of leukemia and birth defects has been reported in the children of fathers who had been exposed to radionuclides in the nuclear reprocessing plants and to diagnostic radiation. These findings have not been supported in the children of atomic bomb survivors in Hiroshima and Nagasaki, who were exposed to higher doses of atomic radiation. However, long-term monitoring of children by Russian Federation Children's Center of Antiradiation Protection after Chernobyl accident shows higher prevalence of malignant neoplasm, mostly childhood cancer, malformation, and other disorders in the children of residents exposed to contaminated radionuclides (>556 kBq/m2). Persistent accumulation of genomic instability may cause various disorders in a further generation in human. This view will gain support from our mouse experiments, because the induced rate of solid tumors in the offspring of mice exposed to radiation is much higher than that of leukemia.
Keywords: Humans, gene expression, genomic instability, mice, transgenerational effects, radiation
|How to cite this article:|
Nomura T, Baleva LS, Ryo H, Adachi S, Sipyagina AE, Karakhan NM. Transgenerational effects of radiation on cancer and other disorders in mice and humans. J Radiat Cancer Res 2017;8:123-34
|How to cite this URL:|
Nomura T, Baleva LS, Ryo H, Adachi S, Sipyagina AE, Karakhan NM. Transgenerational effects of radiation on cancer and other disorders in mice and humans. J Radiat Cancer Res [serial online] 2017 [cited 2019 Oct 23];8:123-34. Available from: http://www.journalrcr.org/text.asp?2017/8/3/123/216871
| Introduction|| |
Cancer is the leading cause of death in childhood in the developed countries of the world. In our investigations reported in this paper, cancers were induced in mice either by in utero or germ cell exposure to radiation and chemicals. Other adverse effects, for example, abortions and congenital malformations were also seen in the offspring.,,,,,,,,,,,,,,,,,,,,,,,,,, Although it is expected that exposure of germ cells to radiation will cause adverse effects such as cancer, malformation, abortions, and mutations in the offspring,,,,,,,,,,,, only a very limited number of studies had been focused on this question and on the potential causal mechanisms. To fill this gap in knowledge, in 1967, the first and largest series of mouse experiments were launched by Nomura with ICR/Jcl, N5 and other strains in Japan, and it was found that radiation-induced germ cell alterations causing tumors, malformations, and embryonic deaths in the offspring of irradiated parental mice.,,,,,,,,,,,,,,,,, These studies have been referred to under the heading of “transgenerational carcinogenesis and teratogenesis.”,
In humans, higher risk of leukemia and congenital malformations has been reported in the children of fathers who had been exposed to radionuclides in the nuclear reprocessing plants and to diagnostic radiations.,,,,,,, Although no increases in adverse effects (mutations, malformation, cancer, etc.) have been demonstrated in the children of atomic bomb survivors in Hiroshima and Nagasaki, who had been exposed to higher doses of atomic radiations, the most recent study in the Children's Center of Antiradiation Protection, which was founded in 1990 as Russian Federation program for the protection of children's health from Chernobyl catastrophe, suggests higher prevalence of cancer, and various diseases in the children of the residents exposed to radionuclides in highly contaminated area in Russia after Chernobyl catastrophe,,,, as reported in mice.,,,,,,,,,,,,,,,,
In this article, we first present an overview of the results of mouse and human studies and then discuss subsequent studies aimed at gaining insights into the possible mechanisms underlying transgenerational effects of radiation. Using these as a framework, we consider the question of transmissible genetic risk in our future generation.
| Cancer in Mouse Studies|| |
The initial experiments on transgenerational effects of radiation were carried out with mice most extensively from 1969 to 1975, and the data were analyzed subsequently (1975–1981) and confirmed with other streams of mice. Adult male mice were exposed to radiation and then mated with untreated estrous females at various times before conception; vaginal plugs were checked to determine the date of conception and the stages of germ cells exposed. Some mice were euthanized on the 18th day of gestation, and F1 fetuses were examined for embryonic deaths (dominant lethals), congenital malformations, etc., by cesarean operation.,, Other parental mice delivered live offspring and screened for congenital malformations 7 days after birth, and tumors, chronic diseases, and related chromosomal and molecular changes were examined to analyze the mechanism of transgenerational disasters.,,,,,,,,,,,,,, Live offspring were also treated postnatally with tumor-promoting agents with appropriate concurrent controls [Figure 1].,
|Figure 1: Experimental procedures for transgenerational effects of radiation and chemicals in mice|
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Dose responses and stage sensitivities
About 87% of the induced tumors in ICR mice were found in the lung, the remainder being of various types – ovarian tumors, lymphocytic leukemia, stomach tumors, lipoma, granulosa cell tumors, thyroid tumors, liver hemangioma and hepatoma, and majority was malignant.,, Most tumors were transplantable and killed recipient mice., The spectrum of tumor incidence in the offspring of treated animals is essentially the same as that in controls (90% in the lung). The incidence of tumors in F1 offspring increased with X-ray doses to the parents in the range from 0.36 to 5.04 Gy [Figure 2]. Induced frequencies of lung tumors per Gy were 2.27, 1.49 and 3.17 × 10−2 for irradiation of postmeiotic stages (spermatozoa and spermatids), spermatogonia, and oocytes, respectively,, indicating that postmeiotic stages are more sensitive than the spermatogonial stages. Frequencies (per Gy) by protracted irradiation were 2.09 (postmeiotic male germ cells), 0.09 (spermatogonia), and 0.29 × 10−2 (oocytes),,,, No differences were observed in the incidence of tumors between single and protracted doses after postmeiotic exposure., However, with fractionated doses given to spermatogonia, large reductions in tumor incidence in F1 offspring were observed, the figures approaching those seen in controls [Figure 2]., This finding is confirmed more precisely in N5 mice with chronic doses of60 Co-γ-rays at very low dose rate 0.0002 Gy/min against high dose rate 0.57 Gy/min. Oocytes in the mature follicle stages were resistant to single dose of X-rays up to 1 Gy for tumor induction in F1, but large increases were observed at higher doses., Again, low-dose fractionation to mature oocytes showed a large reduction in tumor incidence [Figure 2]. These observations suggest significant repair capabilities of spermatogonia and mature oocytes.
|Figure 2: Parental exposure to X-rays induces heritable tumors in the next generation of mice. Solid line; single and high dose rate, broken line; low fractionated dose|
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The germ cell sensitivity for tumor induction in the offspring is very similar to that of specific locus mutations induced by radiation,,,,, but the tumor frequencies are more than 2 orders of magnitude higher than that of specific locus mutations. Consequently, to examine the generality of our observations, studies were carried out with two other inbred strains of mice (LT and N5). By the postmeiotic treatment of the N5 and LT male mice, induced incidence of lung tumors in N5 and LT was similar to that in ICR mice.,,, However, much higher induced incidence of leukemia was observed in N5 and LT than in ICR., Spermatogonial treatment of N5 mice increased the frequencies of various tumors such as lung tumors, ovarian tumors, leukemia, hepatoma, and multiple embryonic tumors in the F1 offspring. Induced rates of lung and ovarian tumors in N5 were similar to those in ICR., However, N5 mice showed higher incidences of lymphocytic leukemia and hepatoma than ICR and/or LT. Consequently, there are strain differences in the incidence of tumors among these 3 strains, and most induced tumor types were same as those of background incidence in each strain, suggesting that radiation enhanced spontaneous incidences of these tumor types.,,,,
Heritable nature of tumors
Heritability of lung tumors were examined by retrospective analysis in ICR mice,, and showed that significantly higher frequencies of lung tumors were observed in the F2, when their parental F1 had tumors. Again, lung tumor incidence was significantly higher in the F3, when F2 male parent had tumors.,
Inheritance of induced tumors was also examined in the N5 strain,,,, which develops various types of tumors. Mating of preconceptionally-irradiated lung tumor-bearing Fl progeny with their litter mates yields a variety of tumors, such as lung tumors, ovarian tumors, multiple embryonic tumors, myxoma, and thymic lymphomas (lymphocytic leukemias), and congenital anomalies in the progeny of the F2 generation. Observation continued for further generations. F3 progeny of F2 parental mice with multiple tumors and lung tumors developed not only multiple tumors and lung tumors, but also those of other types (ovarian tumor, hepatoma, leukemia, etc.), cataracts, and congenital anomalies [Figure 3].,, Such a tendency has been found to persist (up to F58; Jun, 2014). The pattern is similar to that of Li-Fraumeni syndrome in humans, suggesting the inheritance of tumor susceptibility. We interpret these findings as indicating that germ cell exposure to radiation may have induced transmissible hypersusceptibility to tumor induction in the next generation. Cancer-prone progeny were used for chromosomal and molecular analyses and these studies are shown in later section.
|Figure 3: Transmission of tumor susceptibility to the next generation of N5 male mice exposed to a single dose of X-rays (5.04 Gy)|
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| Further Mouse Studies|| |
After the publication of Gardner's report in 1990 on the higher risk of leukemia in the children of fathers who had been exposed to nuclear radiations, further experimental studies were carried out.,,
A high incidence of hepatomas was observed in the Fl offspring of C3H male mice exposed to 0.5 Gy of252 Cf (66% neutron) and mated with C57BL/6 females., Cattanach et al.,, however, observed no significant increases but reported a seasonal change in the incidence of lung tumors in the offspring of BALB/cJ or C3H/HeH male mice exposed to X-rays following the experimental protocol of Nomura., It is a well-known fact that the incidence of cancer is influenced and/or modified by postnatal environments, including light-dark intervals. Before their reports, we had published that change of light-dark intervals in the mouse room significantly influenced tumor frequencies in mice. A Canadian group carried out experiments with N5 mice provided by Nomura. In these experiments, male N5 mice were irradiated with 5 Gy of X-rays under conditions close to those of Nomura.,, The results showed that the probability of dying from leukemia and overall survival were statistically different between the offspring of X-ray-treated males and of unirradiated controls. Earlier occurrence of leukemia was also observed in the F1 offspring after the treatment of male N5 mice with tritiated water.
A lifespan experiment showed a trend toward a higher incidence of tumors of the hematopoietic system and bronchoalveolar adenocarcinomas in the offspring of male CBA/JNCrj mice exposed to X-rays 1 week before mating (spermatozoal exposure). However, no increase in tumor incidence was observed in the offspring of males irradiated 9 weeks before conception, since the less sensitive spermatogonial stage was treated.,
In mice, there are clear strain differences in the types of induced tumors and tumor incidences in the preconceptionally irradiated offspring, indicating that pre-existing genetic predisposition i.e. strain differences, is a critical component also in transgenerational radiation carcinogenesis.,
Selby et al. argued that preconceptionally irradiated offspring develop tumors in higher incidence, because postmeiotic exposure to high dose exposure at postmeiotic stages induced high incidence of dominant lethals (i.e. decrease of litter size), and heavier mice are born. The above argument, however, is not a valid one. His argument was followed by our report that the number of lung tumor nodules was proportional to the size of the lung (i.e. the number of target cells) irrespective of treatment. Although the litter size was less than half after 5.04 Gy of X-ray exposure at postmeiotoc stage, increase in the body and organ weight is not 2 to 3 folds, in the range of 5%–15%. Furthermore, as noted earlier, very little dominant lethality is induced spermatogonial exposure and yet significant increases were found in tumor frequencies in the offspring descended from irradiated spermatogonia. It is therefore worth stressing thatlitter size effects on carcinogenesis are at the level of a few percentage, whereas strain differences and transgenerational effects on carcinogenicity differ by orders of magnitude.
| Cancer in Human Studies|| |
In 1990, Gardner et al. reported that there was about 6–8 fold higher risk of leukemia (mostly acute lymphocytic leukemia) in the children of fathers who were employed at Sellafield nuclear reprocessing plant in the UK and had been exposed to 10–100 mSv of radiation before conception. In particular, extremely high risk (about 7-fold) of childhood leukemia from the father's exposure to doses as small as 10 mSv during the 6 months before conception suggested that the postmeiotic sperm was more sensitive to radiation than spermatogonia. The general pattern, for example, germ cell stage sensitivity, was very similar to that seen in our mouse experiments,,,,, and also in mutational sensitivity in mice by specific loci method.,,,, However, it should be borne in mind that in the human studies, induced rate of leukemia/mSv was 3–150 fold higher (depending on the mouse strains used) than in the mouse experiments,, and besides, mouse tumors did not show any increases in frequency in the progeny after protracted or chronic spermatogonial irradiation.,,,, Furthermore, numbers of victims were too small for statistical analysis.,,,
Roman et al. also reported on the significantly higher risk of leukemia in the children of fathers who had been exposed to nuclear radiation at atomic weapons establishments in the UK., Again, induced rate of leukemia/mSv in human was much higher than mouse experiments., These findings have not been supported by the epidemiological study on the children of atomic bomb survivors in Hiroshima and Nagasaki who were exposed to an average dose of 435 mSv. Clinical health studies of the children of atomic bomb survivors started from 2002, but significant differences have not yet been confirmed.
In Russian Federation, about 214,000 children (0–17 years old) were living in the radionuclide contaminated area (137 Cs > 185 kBq/m2) after Chernobyl catastrophe (April 26, 1986). To monitor the health status of the child population exposed to radiation after the Chernobyl accident, Russian Federation created a model of allocation of children in the reference cohort that served as the basis of the database of the register of children in the Children Scientific and Practical Center for Radiation Protection [Figure 4]. In connection with the features of the radionuclide spectrum, the release of short-lived radioisotopes131 I and long-lived radioisotopes137 Cs and90 Sr, in different ways of isotope intake (alimentary, inhalation, transplacental, contact), duration of their exposure (acute and chronic irradiation), the reference cohorts of children subjected to long-term dynamic observation were formed in the Children's Scientific and Practical Center for Radiation Protection. For 30 years of the postaccident period, the staffs of the scientific and practical center succeeded in creating the conditions for effective medical monitoring, recovery and rehabilitation to ensure the improvement of medico-psycho-social help and support for a different cohort of children.
|Figure 4: Protocol for long-term monitoring of children (0–17 years old) after Chernobyl accident in Russia|
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During this period, two groups (generations) of children (0–17 years); children born in 1969–1987, who received radiation exposure during the accident and living in contamination regions (F0) and the other group of children (F1) born from the exposed parents. All children were traced using actual data of the specialized annual medical examination of the various cohorts of child population, the long-term monitoring of the health state of children, and the results of scientific research, then compared them with practical public health and the data in the forms of official federal statistical surveys. The results of continuous annual medical examination of the child population living in the areas with soil contamination levels of long-lived radionuclide137 Cs over 556 kBq/m2 (94,875 children) are entered in the form of the special Federal Statistical Observation (form 16) and compared with the form to the entire, pediatric population of the Russian Federation (form 12) [Figure 4].
[Figure 5] shows the high rate of malignant neoplasms in the children of radiation-exposed parents (F1), compared to the level of malignant neoplasms prevalence in the children (0–17 years old) of whole Russian Federation. [Figure 6] indicates the structure of malignant neoplasms in the children (0–17 years) (F0) exposed to radionuclides in comparison with the children (0–17 years) (F1) of radiation exposed parents. In the children (F0) living in the radionuclides contaminated area and directly exposed to radioactive iodines just after the accident, very high prevalence of thyroid cancer developed in comparison with the children (F1) who born from radiation-exposed parents. In the children (F1) of radiation-exposed parents, however, typical malignant childhood neoplasms of the lymphoid and hematopoietic tissue, central nervous system, musculoskeletal system, genitourinary system, and pancreas (pancreatoblastoma) developed. It should be noted that in the zone more than 1660 kBq/m2 the rate of malignancies is almost two times higher than in the areas with a right for relocation.
|Figure 5: Prevalence of malignant neoplasms in the children (0-17 years old) (F1) of residents exposed to radionuclides after Chernobyl accident compared with the children of whole Russia (per 100,000 children)|
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|Figure 6: Structure of malignant neoplasms in the children (F0) exposed to radionuclides and children (F1) of residents exposed to radionuclides after Chernobyl accident. Ordinate shows numbers of cancer children|
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| Possible Mechanisms: Cytogenetic and Molecular Studies|| |
Transgenerational chromosomal changes
We studied radiation-induced translocations cytogenetically in the germ cells of treated adult ICR males as well as in the F1 male progeny sired by them to examine whether there was any correlation between induced translocations and tumors in the F1 progeny., Translocation studies in the F1 males descended from postmeiotic germ cells of X-ray irradiated (5.04 Gy) parental males showed that of a total of 81 F1 males, 10 had characteristic translocation configurations. A further 3 had a deficit of postmeiotic germ cells in the seminiferous tubules (histological analysis). In 70 concurrent control F1 males, there were no translocations. The F1 males were also screened for the presence of tumors and congenital anomalies. However, tumors occurred no more frequently in F1 offspring with translocations than in those without translocations, versus translocations were induced no more frequently in F1 with tumors, i.e. there is no correlation between the induction of translocations and the occurrence of tumors.,
Then, we examined whether any relationship could be discerned between visible chromosomal changes (in bone-marrow preparations using G and CQ-band analysis) in 36 tumor-bearing offspring in the N5 strain (from postradiation generations F1 to F5) and 53 irradiated but nontumor-bearing controls. Again, these results did not provide any evidence for a cytogenetically detectable chromosomal abnormality in these animals., The above sets of data considered together suggest that induced germ cell changes causing tumors are not related to gross chromosomal changes detectable with the cytogenetic techniques employed. However, they do not exclude the possibility that smaller genetic changes such as gene mutations may be involved.,,, These findings were supported by the fact that urethane, an intragenic mutagen,, induces tumors, but neither translocations nor dominant lethals in the offspring.
In humans, significant increase of chromosomal changes was not observed in the children of atomic bomb survivors in Hiroshima and Nagasaki probably due to dose and sample size problem. In Russia, however, the specific features of a manifestation of genomic instability, as shown by the karyological as indicated by the micronucleus test using buccal epithelial cells, were investigated in the children of exposed parents. To assess an immune response to the mutagenic action of ionizing radiation peripheral blood lymphocytes with the phenotypic characteristics of CD95 + subpopulations were quantified using flow-through cytofluorometry, by estimating the possible consequences of transferring the mutagenic effect of ionizing radiation to posterity. Children directly (F0) or pre-conceptionally (F1) exposed to iodines and residing in the areas polluted with radionuclide137 Cs (more than 556 kBq/m2) were examined in the aspect of family. The data obtained in the investigation employing the models for individual families may anticipatorily consider certain cytogenetic indicators in the children as predictors of carcinogenesis and the possibility of their transgenerational transfer in the generations of persons exposed to radiation [Figure 7].,
|Figure 7: Indicators of readiness to apoptosis (the level of CD95 + lymphocytes) in the children directly (F0) or pre-conceptionally (F1) exposed to 131I and residing in the 137Cs contaminated areas (>556 KBg/m2). A total of 112 children and their parents were examined|
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To determine the contribution of oncogenes to multi-generation carcinogenesis, activation of 17 known oncogenes was examined in DNA extracted from transplantable tumors induced in the offspring of X-irradiated N5 parents (undifferentiated multiple tumors, fibrosarcomas, lung tumors, lymphocytic leukemias, etc.).Ras, mos and/or abl genes were amplified in two rare tumors (undifferentiated multiple tumors) [Figure 3] and lymphocytic leukemia., Furthermore, two of three tumor DNAs which did not hybridize with any oncogenes so far tested had transforming ability on cultured cells from golden hamster embryos, and were later found to contain mos and cot oncogenes., (Cot was isolated from human thyroid cancer tissue). These were the first reports of detection of activated mos and cot genes by transfection assay.P53 mutations were also detected in a brain tumor (glioma) of X-irradiated N5 progeny. Thus, known and new oncogenes were activated in the tumors of the descendants of X-irradiated N5 parents. In commonly observed types of tumors, however, we rarely found oncogene activation and p53 mutations. Consequently, the majority of tumors induced in the progeny of irradiated parents were commonly observed types in the respective strains of mice in which oncogene activations were rarely detected, although germ-line alterations could produce some rare types of tumors and molecular changes.
In humans, Radiation Effect Research Foundation failed to detect mutations in the children of atomic bomb survivors in Hiroshima and Nagasaki, simply because base substitution mutations, which are rarely induced by radiation, were examined by electrophoresis analysis of peripheral blood protein. Charles and Pretsch examined electrophoresis mutation of same genes in F1 of parental mice exposed to large doses of radiations and confirmed that radiation never induced electrophoresis base substitution mutations in F1 mice. Radiation is known to induces deletion mutations, as it was in specific locus mutation, cataract mutation, and dominant skeletal mutation. Consequently, enzyme activity mutation (caused by deletion mutation) was examined in mice and humans. Enzyme activity mutation was significantly induced by radiation in F1 mice, and one mutation was induced in the children of atomic bomb survivors in Hiroshima and Nagasaki. Unfortunately, examination in humans deceased after then. Frequency of enzyme activity mutation (per locus per rad) is similar between mice and men, and values are also similar to those of specific locus mutation in mice.
Consequently, negative results of gene mutations in Hiroshima and Nagasaki is caused by erroneous methodology to detect radiation-induced mutations. Gene mutation has to be examined by proper method to detect deletion mutations. Such studies were recommended in Hiroshima and Nagasaki.
If germ-line mutation can lead to cancer in a given organ, all cells composing that organ must be mutated and have an equal likelihood to form tumors. However, only one tumor nodule was induced in the organ. One can hypothesize that the “mutational alteration” transmitted from the irradiated parent to the offspring is presumably weakly carcinogenic by itself, and that its expression can be influenced by aging, naturally existing carcinogenic and promoting agents in the diet and environment.,
The above hypothesis was proven by the findings that unusually large clusters of tumor nodules developed in the lung after postnatal treatment with small amounts of urethane., Further supporting evidence was reported for lung tumors in the offspring of paternally irradiated Outbred SHR mice (Rappolovo Animal Farm of the National Academy of Medical Sciences, Russia) and postnatally treated with urethane, for skin cancer and leukemia in N5 and SHR mice by postnatal treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA),, and for leukemia by preconceptional239 Pu irradiation and postnatal methylnitrosourea treatment. However, such enhancing effects were not observed when CBA/J male mice were irradiated and their offspring were postnatally treated with urethane. The main conclusion that can be drawn from these studies is that germ cell exposure to radiation, although very weakly carcinogenic by itself, presumably induces transmissible hypersensitivity or predisposition to tumor induction in the offspring., This finding by Nomura is recognized as a starting point for the study of radiation-induced genome instability in germline, which may cause tumors in the next generation.
There is a considerable literature on germline mutations induced at the mouse expanded simple tandem repeat (ESTR) (former nomenclature is minisatellite) loci by both low and high linear energy transfer (neutrons from252 Cf) irradiation.,,,,,, In the work of Dubrova et al.,,,,, however, spermatogonial stages were found to be more sensitive than postmeiotic stages and there were no dose rate effects for these mutations induced in spermatogonia. Their results are thus different from those of other investigators, and are also at variance with findings from studies with specific locus mutations,,,,, as well as our mouse studies.,
In human, there are several reports on minisatellite mutations in the children of radiation exposed parents [Table 1].,,,,,,,, Four reports by Dubrova et al., showed that the mutation rates were elevated 1.6–1.8 fold in the children of exposed group compare to those in the nonexposed group.,,, In contrast, 4 opposite results were reported that there were no increase of minisatellite mutations in the children of radiation-exposed groups.,,,, Those contradictory results made inconclusive for the mutation induction in the offspring of radiation exposed parent in human population. That the positive group was inhabitants in radionuclides contaminated area, versus, negative group was liquidator or direct exposure to atomic radiation, might be one of the critical factors for the increment of minisatellite mutation rates. Internal exposure and/or postnatal exposure to radionuclides may cause higher frequency of mutation.
|Table 1: Mini-,,,,,,,, and microsatellite, mutations in the children of radiation-exposed human population|
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Mouse ESTR is far different from human minisatellites in their structure. It would be important to confirm the results by using same kind of loci for the detection of mutation in human and mice, because microsatellites, a repetitive DNA sequence, are found in both human and mice, and also identification of microsatellite mutation is precise and objective, since single repeat unit mutations can be detected using capillary sequencer and analyzing software.,,
Microsatellite mutation in mice and human
Mutations in tandem repeat loci are mostly detected as gain or loss of repeat unit. We examined microsatellite mutations at D12Mit136 (Ref. for Mit136: Mouse Genome Informatics), pul67, p1 and p5 (Ref. for p1 p5, pul67) loci in the mouse germ cell. N5 male mice were exposed to 0.1, 0.2, 0.4, and 0.6 Gy of fission neutrons by the nuclear reactor, UTR-KINKI (Kinki University) at the dose rate 0.2 Gy/h of neutrons and 0.2 Gy/h of gamma-rays. Irradiated males at spermatogonial stage were mated with N5 un-irradiated females and microsatellite mutations were assayed in F1 offspring. Polymerase chain reaction products of tail DNA in the F1 offspring were analyzed by capillary sequencer (3010xl, Applied Biosystems) and software (Gene Mapper, Applied Biosystems Inc., Foster City, CA94404, USA). Microsatellite mutations increased significantly in a dose-dependent manner; significant increase was observed at pul67 locus [Figure 8], but not at other 3 loci. Longer observation of F1 mice yielded higher incidence of lymphocytic leukemia.
|Figure 8: Microsatellite mutation at pul67 locus and leukemia (orange) in the offspring of N5 male mice exposed to fission neutron|
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Elevation of microsatellite mutation rate in the F1 offspring of irradiated male mice suggested the possibility of microsatellite mutation induction in the children of radiation exposed human. In the liquidator families of Chelnobyl accident, 73 children from 70 paternal (61 preconceptional, 9 postconceptional), one maternal and 2 both parents exposed, and 69 children from unexposed parents were examined for microsatellite mutation with 31 autosomal, one X-linked and 40 Y-linked microsatellite loci. However, there was no change in mutation rate between exposed and unexposed groups [Table 1]. Estimated mean radiation dose to liquidatorswas 39 mSv might be one reasonfor no increases in mutation rate.
Children of A-bomb survivors were also examined. Microsatellite mutations were assayed in 66 children with 1.87 Gy of mean paternal dose and 1.27 Gy of mean maternal dose, and 63 control children from parents living 2.5 km outside of hipocenter (estimated external dose <0.01 Gy). Although mean parental exposed dose was high, and 39 autosomal and one X-linked microsatellite loci were examined, no microsatellite mutation rate was increased in the children of exposed group [Table 1]. These two studies were not for the children of the inhabitants of radionuclide contaminated area, but for externally exposed parents like liquidators or atomic bomb survivors. Consequently, all of 2 microsatellite and 4 minisatellite studies in the children of noninhabitant are negative. This may suggest that internal or long term exposure to radionuclides may cause genomic instability in the children of radiation-exposed residents. In case of Hiroshima and Nagasaki, fall-out is ignored, although the killing dose of radionuclides fell out outside of city, i.e. in the control area.
| Persistence and Accumulation of Genomic Instability in the Offspring of Irradiated Male Mice|| |
The important features of the presumed germ-line alterations causing cancer or genomic instability in the progeny of irradiated mice suggest that the accumulation of minor changes or persistent changes in gene expression slightly elevate cancer incidences or fasten the tumor development in mice.
We examined microsatellite mutations and changes in gene expression in cancer-prone descendants of radiation-exposed N5 male mice. Surprisingly, 3 of 4 microsatellite loci examined had been mutated in all offspring of 2 cancer susceptible lines 3566 [Figure 3] and 3513 from 5.04 Gy X-irradiated N5 males, while in concurrent control offspring from untreated N5 males, accumulation never happened in each generation [Table 2].
|Table 2: Accumulation of microsatellite mutations in the two cancer-prone descendants (3566 and 3513) of N5 male mice exposed to 5.04 Gy of X-rays|
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Changes in gene expression have been examined in another descendants of N5 males using microarray technology (GeneChip, Affymetrix Inc., Santa Clara, CA, USA). In these experiments, the affected F1 offspring (with tumors or malformations) of N5 male mice exposed to radiation at spermatogonial stage were used as the starting material. These animals were mated to their litter mates as young adults, and their offspring were examined for the presence or absence of tumors at 12 months of age and classified as to the retrospectively determined type of the parent.,,
In the study, using skin cancer model with TPA, 3 skin cancer lesions and surrounding normal skin area in the F3 offspring of male N5 mice exposed to 2.16 Gy of60 Co-γ-rays were reanalyzed and compared to the normal skin of un-irradiated concurrent control F3. Among 6500 genes, 254 and 75 functional genes (in the skin cancer lesion and the surrounding normal skin area, respectively) showed more than 4-fold differences in the expression level (both increases and decreases). In the non-tumor area, for instance, macrophage inflammatory protein, osteopontin precursor, SV40 induced 24p3 mRNA, and a variety of genes were 50-fold overexpressed or suppressed [Figure 9].
|Figure 9: Changes in gene expression of normal and cancerous tissues of the skin and liver in the offspring of male N5 mice exposed to radiation (2.16 Gy)|
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In similar studies, comparing gene expression in hepatoma and normal liver tissues of the affected offspring and concurrent controls, progeny No. 7 (F1) developed only one hepatoma, and No. 13 (F1) developed 3 hepatoma nodules and lung tumor. No. 35 and No. 43 (F2) born from No. 13 and 12 (F1) developed 2 hepatoma nodules and lung tumor [Figure 10]., Eight hepatoma nodules and 4 surrounding normal liver tissues in the affected F1 and F2 offspring were analyzed and compared to the normal liver tissue of concurrent control F1 and F2, and shown in [Figure 9]. Among 12,000 genes examined, more than 4-fold differences were observed in 30 and 110 functional genes in the normal liver tissue and hepatomas, respectively, of irradiated F1 and F2 offspring. The average number of oncogenes and related genes with abnormal expression was 4.3 and 20.8 per normal liver or hepatoma tissue, respectively.
|Figure 10: Transmission of changes in gene expression of normal and hepatoma tissues in the offspring of N5 male mice exposed to 2.16 Gy of X-rays|
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It is apparent that considerable numbers of genes are abnormally expressed, such altered genes preexist in the nontumorous liver tissue of cancer-prone progeny, and the majority of the abnormally expressed genes are those involved in normal physiological, biochemical and immunological functions. Consequently, changes in gene expression seem to occur in various normal functional genes rather than oncogenes per se in irradiated cancer-prone or tumor-susceptible descendants, and their progressive accumulation may contribute to cancer as we have hypothesized.
Although a direct link between cancer and irradiation of sperm was not confirmed by Sellafield study nor by Hiroshima/Nagasaki study. Survey by Russian Federation in the children of residents exposed to radionuclides after Chernobyl accident is showing an increased prevalence of malignant neoplasms, especially childhood cancer, and other disorders. Persistent accumulation of genomic instability may cause various disorders in further generation in human. This view will gain support from our mouse experiments, because the induced rate of solid tumors in the offspring of mice exposed to radiation is much higher than that of leukemia, as it is in humans.
We would like to thank Dr. Huilgol, Mumbai for his advice and help to edit this review article on transgenerational effects of radiation in mice and humans.
Financial support and sponsorship
The study is supported by Japan Ministry of Education, Science and Culture, Japan Society of Promotion of Science, Princes Takamatsu Fund, Nissan Science Foundation, Heiwa Nakajima Foundation, Osaka Science Prize, Kihara Memorial Prize, Inoue Science Prize, Yasuda Medical Award to Nomura and Japan-Russia Bilateral Joint Research Project (JSPS-RFBR) to Nomura and Baleva.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
[Table 1], [Table 2]