Spontaneous p53 activation in middle-aged C57BL/6 mice mitigates the lifespan-extending adaptive response induced by low-dose ionizing radiation.

Understanding the biological effects of low-dose (<100 mGy) ionizing radiation (LDR) is technically challenging. We investigated age-dependent LDR effects using adaptive response experiments in young (7-to 12-week-old) and middle-aged (40-to 62-week-old) C57BL/6 mice. Compared with 3 Gy irradiation, 0.02 Gy preirradiation followed by 3 Gy irradiation prolonged life in young mice but not middle-aged mice. Preirradiation also suppressed irradiation-induced 53BP1 repair foci in the small intestines, splenic apoptosis, and p53 activity in young mice but not middle-aged mice. Young p53+/- C57BL/6 mice did not show these adaptive responses, indicating that insufficient p53 function in young mice mitigated the adaptive responses. Interestingly, p53 activation in middle-aged mice spontaneously became approximately 4.5-fold greater than that in young mice, possibly masking LDR stresses. Furthermore, adaptive responses in young mice, but not in middle-aged mice, suppressed some senescence-associated secretory phenotype (SASP) factors (IL-6, CCL2, CCL5, CXCL1). Thus, LDR-induced adaptive responses associated with specific SASP factors may be attenuated by a combination of reduced DNA damage sensor/transducer function and chronic p53 activation in middle-aged mice.

Relationship between haematological data and radiation doses of TEPCO workers before and after the FDNNP accident.

We evaluated the correlation between radiation dose and the medical examination data of Tokyo Electric Power Company Holdings, Inc (TEPCO) employees working during the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in 2011. This study included 2164 male TEPCO workers who received periodic medical examinations from March 2006 to January 2013. First, we conducted log-linear regression analyses using the haematological data of 585 emergency workers and confounding factors to examine the effect of internal radiation exposure in March 2011. Since external radiation exposure was a major influence, we then evaluated the correlation between both internal and external radiation dose and the haematological data of 1801 emergency workers and confounding factors before and after the accident. Among 585 workers, internal radiation exposure in March 2011 alone was mainly due to thyroid doses (0.1-10 Gy) but not to bone marrow (BM) doses (0.01-1 mGy). Compared to before and after the accident, we found that the levels of monocytes, eosinophils (Eos) and basophils increased slightly, whereas the frequency of smoking and alcohol consumption decreased substantially. External dose exposure was positively correlated with haemoglobin (Hb), red blood cell and Eos but negatively correlated with age, haematocrit and frequency of alcohol consumption. Among these variables, Hb exhibited the strongest correlation with external dose. Regarding the correlation with Hb, although there is a possibility that confounding factors other than exposure were not evaluated, our findings on emergency workers can serve as a reference for the evaluation of health conditions during the emergency period of future nuclear-related accidents.

Long Bones Exhibit Adaptive Responses to Chronic Low-Dose-Rate Ionizing Radiation despite its Lifespan-Shortening and Carcinogenic Effects on C57BL/6 Mice.

Ionizing radiation (IR) is a well-known carcinogen. High-dose-rate (HDR) IR is known to damage long bones (in terms of mass and structure), but the relationships among dose rates (particularly low-dose-rate [LDR] IR), long-bone condition, cancer incidence, and lifespan shortening remain elusive. The aim of this study was to elucidate the effects of LDR-IR on long-bone condition by comparing the long-term consequences of HDR- and LDR-IR exposure in mice. We utilized micro-computed tomography (µCT) scans of the long bones at similar ages (mean 77-96 weeks) to compare mice receiving approximately equivalent total doses of internal LDR-IR or external HDR-IR starting at 4 weeks of age, and their respective control groups. The lifespan-shortening effects of LDR-IR and HDR-IR were similar in these mixed-sex cohorts. Notably, compared to HDR-IR mice, mice internally exposed to chronic LDR-IR with continuous hypohematopoiesis showed a significantly higher trabecular bone connective density [femur: 247% (p = 0.0042), tibia: 169% (p = 0.0005)] and midshaft cortical bone thickness/area (femur: 130% [p = 0.0079]/120% [p = 0.021], tibia: 148% [p = 0.0004]/129% [p = 0.002]). Consistent with this result, when comparing 26-32 weeks post-IR with 2-8 weeks post-IR, compared to HDR-IR-treated mice, LDR-IR-treated mice exhibited higher levels of an osteoblast marker (OPG; p = 0.67 for HDR-IR, p = 0.068 for LDR-IR) and lower levels of an osteoclast marker (CTX-I; p = 0.0079 for HDR-IR, p = 0.72 for LDR-IR) despite significantly higher levels of inflammatory markers (CCL2 and CXCL1; p = 0.36-0.8 for HDR-IR, p = 0.013-0.041 for LDR-IR). These results suggest that long bones under chronic LDR-IR stress may exhibit an adaptive response to stresses such as chronic inflammation associated with IR-induced lifespan shortening. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Mammalian Resilience Revealed by a Comparison of Human Diseases and Mouse Models Associated With DNA Helicase Deficiencies.

Maintaining genomic integrity is critical for sustaining individual animals and passing on the genome to subsequent generations. Several enzymes, such as DNA helicases and DNA polymerases, are involved in maintaining genomic integrity by unwinding and synthesizing the genome, respectively. Indeed, several human diseases that arise caused by deficiencies in these enzymes have long been known. In this review, the author presents the DNA helicases associated with human diseases discovered to date using recent analyses, including exome sequences. Since several mouse models that reflect these human diseases have been developed and reported, this study also summarizes the current knowledge regarding the outcomes of DNA helicase deficiencies in humans and mice and discusses possible mechanisms by which DNA helicases maintain genomic integrity in mammals. It also highlights specific diseases that demonstrate mammalian resilience, in which, despite the presence of genomic instability, patients and mouse models have lifespans comparable to those of the general population if they do not develop cancers; finally, this study discusses future directions for therapeutic applications in humans that can be explored using these mouse models.

Contribution of radiation education to anxiety reduction among Fukushima Daiichi Nuclear Power Plant workers: a cross sectional study using a text mining method.

The purpose of this study is to investigate the frequency of education, knowledge of radiation and workplace anxiety of Fukushima Daiichi Nuclear Power Plant (FDNPP) workers and to analyze what type of words are used for anxiety with a text mining method. An original questionnaire survey was given to FDNPP workers, and a text mining method was used to extract information from free-entry fields. The questionnaires were collected from 1135 workers (response rate: 70.8%). It was found that when workers receive education on radiation, the increased knowledge helps to reduce their anxiety. Among the 1135 workers, 92 of 127 completed the free-entry field with valid entries. Seventy-one words were extracted by the text mining method. The words used differed depending on the degree of anxiety. The text mining method revealed information about the presence or absence of radiation anxiety and the subjects' working environment and background.

Comparison of the fertility of tumor suppressor gene-deficient C57BL/6 mouse strains reveals stable reproductive aging and novel pleiotropic gene.

Tumor suppressor genes are involved in maintaining genome integrity during reproduction (e.g., meiosis). Thus, deleterious alleles in tumor suppressor-deficient mice would exhibit higher mortality during the perinatal period. A recent aging model proposes that perinatal mortality and age-related deleterious changes might define lifespan. This study aimed to quantitatively understand the relationship between reproduction and lifespan using three established tumor suppressor gene (p53, APC, and RECQL4)-deficient mouse strains with the same C57BL/6 background. Transgenic mice delivered slightly reduced numbers of 1st pups than wild-type mice [ratio: 0.81-0.93 (p = 0.1-0.61)] during a similar delivery period, which suggest that the tumor suppressor gene-deficient mice undergo relatively stable reproduction. However, the transgenic 1st pups died within a few days after birth, resulting in a further reduction in litter size at 3 weeks after delivery compared with that of wild-type mice [ratio: 0.35-0.68 (p = 0.034-0.24)] without sex differences, although the lifespan was variable. Unexpectedly, the significance of reproductive reduction in transgenic mice was decreased at the 2nd or later delivery. Because mice are easily affected by environmental factors, our data underscore the importance of defining reproductive ability through experiments on aging-related reproduction that can reveal a trade-off between fecundity and aging and identify RECQL4 as a novel pleiotropic gene.

[Questionnaire Survey of Fukushima Daiichi Nuclear Power Plant Workers in 2016 on Knowledge and Anxiety About Radiation].

The results of a survey of radiation workers suggest that they are worried about the effects of radiation exposure on health, and approximately 30% of Fukushima Daiichi Nuclear Power Plant (FDNPP) workers have anxiety. This questionnaire survey reveals that the higher the frequency of radiation education, the higher the knowledge of radiation the workers will have, and that the higher the level of knowledge, the lower the anxiety. To reduce anxiety, it is important to increase knowledge about radiation through radiation education. However, even those workers who had radiation education several times still had anxiety. According to the Ordinance on the Prevention of Ionizing Radiation Hazards, the time spent on education about the effects of radiation on the human body is only about 30 minutes. This education is not enough to reduce anxiety. FDNPP workers needed more effective education to increase their knowledge and to reduce their anxiety.

Human RECQL4 represses the RAD52-mediated single-strand annealing pathway after ionizing radiation or cisplatin treatment.

Ionizing radiation (IR) and cisplatin are frequently used cancer treatments, although the mechanisms of error-prone DNA repair-mediated genomic instability after anticancer treatment are not fully clarified yet. RECQL4 mutations mainly in the C-terminal region of the RECQL4 gene lead to the cancer-predisposing Rothmund-Thomson syndrome, but the function of RECQL4ΔC (C-terminus deleted) in error-prone DNA repair remains unclear. We established several RECQL4ΔC cell lines and found that RECQL4ΔC cancer cells, but not RECQL4ΔC nontumorigenic cells, exhibited IR/cisplatin hypersensitivity. Notably, RECQL4ΔC cancer cells presented increased RPA2/RAD52 foci after cancer treatments. RECQL4ΔC HCT116 cells exhibited increased error-prone single-strand annealing (SSA) activity and decreased alternative end-joining activities, suggesting that RECQL4 regulates the DNA repair pathway choice at double-strand breaks. RAD52 depletion by siRNA or RAD52 inhibitors (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside [AICAR], (-)-epigallocatechin [EGC]) or a RAD52-phenylalanine 79 aptamer significantly restrained the growth of RAD52-upregulated RECQL4ΔC HCT116 cells in vitro and in mouse xenografts. Remarkably, compared to single-agent cisplatin or EGC treatment, cisplatin followed by low-concentration EGC had a significant suppressive effect on RECQL4ΔC HCT116 cell growth in vivo. Together, the regimens targeting the RAD52-mediated SSA pathway after anticancer treatment may be applicable for cancer patients with RECQL4 gene mutations.

[A Survey about the Radiation Effects and A Health Survey of Fukushima Inhabitants after the Fukushima Daiichi Nuclear Power Plant Accident].

According to questionnaire surveys in 2011 and 2013 about the health effects of radiation after the Fukushima Daiichi Nuclear Power Plant Accident, the guardians of child patients were more anxious than doctors and medical students. Also, according to the thyroid examinations in a Fukushima health survey, 190 cases of thyroid cancer were reported, and anxiety about radiation effects remained. This study is based on a survey about the guardians of child patients anxiety about radiation effects six years after the nuclear power plant accident, and includes a questionnaire survey about radiation effects and thyroid examinations in a Fukushima health survey. Anonymous question sheets with 20 questions were sent to pediatric medical facilities in Fukushima, and the parents of children who consulted the pediatric and medical staff answered the questionnaire. Thirty percent of the guardians of child patients had never been educated about radiation and 67% had never been educated about the effects of radiation on humans. The guardians of child patients were more anxious than the medical staff about thyroid cancer, health effects on children and genetic effects. Our results indicate that the guardians of child patients think that the increase in the incidence of thyroid cancer is due to radiation effects after the nuclear power plant accident and they desire continued thyroid examinations.

What have we learned from a questionnaire survey of citizens and doctors both inside and outside Fukushima?: survey comparison between 2011 and 2013.

The disaster at the Fukushima Daiichi Nuclear Power Plant (FDNPP) remains unresolved because the estimated time to decommission a nuclear reactor appears to be approximately 40 years. The number of workers exposed to radiation doses ranging from 1 to 100 mSv continues to increase. To understand the accident progression at Fukushima and to anticipate what we should do in the future for occupational and environmental health, we performed a survey of citizens and doctors who lived inside and outside Fukushima in 2011 and 2013. In a comparison of these 2 years, the citizens inside Fukushima continue to suffer anxiety, although those living outside Fukushima tended to feel less anxious. Medical students who had recently studied radiation biology showed much less ongoing anxiety compared with other groups, suggesting that learning about the effects of radiation is essential to understanding one's own circumstances objectively and correctly. The lack of trust in the government and in the Tokyo Electric Power Company (TEPCO) in 2013 remains high in all groups. Therefore, long-term forthright explanations from the government, TEPCO, and radiation experts are indispensable not only to establish trust with people but also to alleviate psychological stress.

The helicase domain and C-terminus of human RecQL4 facilitate replication elongation on DNA templates damaged by ionizing radiation.

The vertebrate RECQL4 (RECQ4) gene is thought to be the ortholog of budding yeast SLD2. However, RecQL4 contains within its C-terminus a RecQ-like helicase domain, which is absent in Sld2. We established human pre-B lymphocyte Nalm-6 cells, in which the endogenous RECQL4 gene was homozygously targeted such that the entire C-terminus would not be expressed. The RECQL4(ΔC/ΔC) cells behaved like the parental cells during unperturbed DNA replication or after treatment with agents that induce stalling of DNA replication forks, such as hydroxyurea (HU). However, after exposure to ionizing radiation (IR), the RECQL4(ΔC/ΔC) cells exhibited hypersensitivity, inability to complete S phase and prematurely terminated or paused DNA replication forks. Deletion of BLM, a gene that also encodes a RecQ helicase, had the opposite phenotype; an almost wild-type response to IR, but hypersensitivity to HU. Targeting both RECQL4 and BLM resulted in viable cells, which exhibited mostly additive phenotypes compared with those exhibited by the RECQL4(ΔC/ΔC) and the BLM(-/-) cells. We propose that RecQL4 facilitates DNA replication in cells that have been exposed to IR.

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint.

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double-strand breaks (DSBs), but only in the G1-phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1-2 and 7-8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1-2 and 4-5. The BRCT domains 4-5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S-phase, we observed a checkpoint defect after siRNA-mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.

DNA polymerases nu and theta are required for efficient immunoglobulin V gene diversification in chicken.

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis-dependent point mutations (Ig hypermutation) and homologous recombination (HR)-dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polnu and Pol led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN(-/-)/POLQ(-/-) cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Poleta has also been previously implicated in Ig gene conversion. We show that a POLH(-/-)/POLN(-/-)/POLQ(-/-) triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polnu and Pol in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.

Cooperative roles of vertebrate Fbh1 and Blm DNA helicases in avoidance of crossovers during recombination initiated by replication fork collapse.

Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1(-)(/)(-) DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1(-)(/)(-) cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM(-)(/)(-) cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1(-)(/)(-) cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.

Vertebrate POLQ and POLbeta cooperate in base excision repair of oxidative DNA damage.

Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H(2)O(2), but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLbeta. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLbeta, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLbeta share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER.

Multiple roles of vertebrate REV genes in DNA repair and recombination.

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1-/- REV3-/- REV7-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polzeta (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.