Responses of hematopoietic cells after ionizing-irradiation in anemic adult medaka (Oryzias latipes).

PURPOSE: Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Oryzias latipes), which has been a model animal and a large body of knowledge has been accumulated in radiation biology. METHODS: Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated. RESULTS: When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction. CONCLUSIONS: The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation.

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs' production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

Ultrasonic treatment suppresses ethylene signaling and prolongs the freshness of spinach.

There have been many studies investigating the application of ultrasonic treatment in vegetables and fruits to eliminate surface contaminants including dirt, microbes, and chemicals such as pesticides. Using a jet ultrasonic washer developed by us to wash food materials, we found that ultrasonic treatment prolonged the freshness of spinach. The stomata closed in the ultrasonicated spinach leaves, whereas those in spinach soaked in water remained open during 24-h storage. Transcriptome analysis revealed that the expression of Ethylene-insensitive 3 Binding F-box protein 1 and 2 (EBF1 and EBF2), which inhibit ethylene signaling, was remarkably increased by ultrasonic treatment, suggesting that the suppression of ethylene signaling allowed stomatal closure in response to abscisic acid signals in the ultrasonicated leaves. Although the precise mechanism of the induction of EBF1 and EBF2 expression by ultrasonic treatment needs to be addressed in further studies, our findings suggest that ultrasonic treatment can be applied to revive and prolong the freshness of leaf vegetables, as well as for their cleaning.

Strain difference in transgene-induced tumorigenesis and suppressive effect of ionizing radiation.

Transgenic expression in medaka of the Xiphophorus oncogene xmrk, under a pigment cell specific mitf promoter, induces hyperpigmentation and pigment cell tumors. In this study, we crossed the Hd-rR and HNI inbred strains because complete genome information is readily available for molecular and genetic analysis. We prepared an Hd-rR (p53+/-, p53-/-) and Hd-rR HNI hybrid (p53+/-) fish-based xmrk model system to study the progression of pigment cells from hyperpigmentation to malignant tumors on different genetic backgrounds. In all strains examined, most of the initial hyperpigmentation occurred in the posterior region. On the Hd-rR background, mitf:xmrk-induced tumorigenesis was less frequent in p53+/- fish than in p53-/- fish. The incidence of hyperpigmentation was more frequent in Hd-rR/HNI hybrids than in Hd-rR homozygotes; however, the frequency of malignant tumors was low, which suggested the presence of a tumor suppressor in HNI genetic background fish. The effects on tumorigenesis in xmrk-transgenic immature medaka of a single 1.3 Gy irradiation was assessed by quantifying tumor progression over 4 consecutive months. The results demonstrate that irradiation has a different level of suppressive effect on the frequency of hyperpigmentation in purebred Hd-rR compared with hybrids.

Protective effect of p53 knockout on 4-nonylphenol-induced nephrotoxicity in medaka (Oryzias latipes).

In the past few decades, environmental pollutants have become common because of misused nonionic surfactants and detergents. Nonylphenol ethoxylates (NPs) are one of the most important contaminants of water. Therefore, the present study aimed to investigate the protective blocking effect of apoptosis (deficient P53 gene) on 4-nonylphenol (4-NP)-induced nephrotoxicity of medaka (Oryzias latipes). We divided 36 fish into six groups: two different control groups of wild type (Wt; Hd-rR) control and p53 (-/-) control, and four different treated with 4-nonylphenol (50 µg/L and 100 µg/L) for 15 days. Histology, immunochemistry, and TUNEL assays confirmed that 4-NP causes nephrotoxicity. Our results showed that 4-NP administration significantly disturbed the kidney structure and function and 4-NP-treated fish showed dilated glomerular vessels, had less glomerular cellular content, decreased expression of glomerular proteins, and an increased level of apoptosis compared with a Wt control group (P < 0.05). As p53 is an apoptotic inducer, some protection in p53-deficient medaka was found as nephrotoxic effects of 4-NP were minimized significantly. Our study demonstrated for the first time to our knowledge that 4-NP induces apoptosis, causing nephrotoxicity in medaka. We found that blocking apoptosis blocking was able to protect the kidney from the toxic effects of 4-NP.

Prognosis of Hippocampal Function after Sub-lethal Irradiation Brain Injury in Patients with Nasopharyngeal Carcinoma.

This work emphasizes the value of assessing hippocampal function by making a timely MRI-based prognosis following a minor dose of hippocampal irradiation after nasopharyngeal carcinomas (NPC) radiotherapy. A quasi-experiment with case-control design and functional assessments (e.g., neuroimaging analysis with fMRI) was conducted to assess hippocampal function after radiotherapy. We delivered 70 Gy of irradiation to nasopharyngeal carcinomas by 6MV helical radiotherapy and collected data from twenty NPC patients and 24 healthy age-matched subjects. Inevitably, hippocampi also received an average dose of 6.89 Gy (range, 2.0-14 Gy). Seed-based functional connectivity of the hippocampus was applied to estimate the cognitive alteration by time before, one month, and four months after irradiation. Afterward, longitudinal-and-cross-sessional statistical inference was determined with time-dependent measurement analysis of variance (ANOVA) with controlled covariance. Over time, there were longitudinal changes in the functional connectivity of hippocampal-related cortices, including the right middle frontal lobe, left superior temporal lobe, and left postcentral gyrus. The findings indicate the presence of functional plasticity, demonstrating how minor irradiation affects functional performance during the early delayed phase of irradiation-induced brain injury.

An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations.

Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation.

PURPOSE: Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24 hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24 h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation. MATERIALS AND METHODS: Sexually mature wild-type, WT (Hd-rR) and p53(-/-) adult female medaka (O. latipes) were exposed to 4 Gy dose of γ-IR and sampling were collected after 4, 8 and 24 h and 14 days. RESULTS: Apoptosis and morphological alterations were observed from 4 h after irradiation and remarkably increased 8 h after irradiation in the wild-type. Apoptotic cell death has been observed 8 h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation. CONCLUSIONS: We conclude that apoptosis and malformations caused by 4 Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4-24 h and the initial response until 8 h was p53-dependent.

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model.

Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body, and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

Apoptosis and morphological alterations after UVA irradiation in red blood cells of p53 deficient Japanese medaka (Oryzias latipes).

Morphological alterations in red blood cells were described as hematological bioindicators of UVA exposure to investigate the sensitivity to UVA in wild type Japanese medaka (Oryzias latipes) and a p53 deficient mutant. The fewer abnormal red blood cells were observed in the p53 mutant fish under the control conditions. After exposure to different doses of UVA radiation (15min, 30min and 60min/day for 3days), cellular and nuclear alterations in red blood cells were analyzed in the UVA exposed fish compared with non-exposed controls and those alterations included acanthocytes, cell membrane lysis, swollen cells, teardrop-like cell, hemolyzed cells and sickle cells. Those alterations were increased after the UVA exposure both in wild type and the p53 deficient fish. Moreover, apoptosis analyzed by acridine orange assay showed increased number of apoptosis in red blood cells at the higher UVA exposure dose. No micronuclei but nuclear abnormalities as eccentric nucleus, nuclear budding, deformed nucleus, and bilobed nucleus were observed in each group. These results suggested that UVA exposure induced both p53 dependent and independent apoptosis and morphological alterations in red blood cells but less sensitive to UVA than Wild type in medaka fish.

Acute Toxicity of Gamma Radiation to the Monogonont Rotifer Brachionus koreanus.

We examined the tolerance of the monogonont rotifer Brachionus koreanus in response to gamma radiation. In order to determine the median lethal dose (LD50) of rotifers against gamma radiation, we irradiated B. koreanus with gamma rays from 0 to 7000 grays (Gy). The LD50s were 2900 and 2300 Gy at 24 h (LD50-24 h) and 96 h (LD50-96 h) after irradiation, respectively. In addition, the no observed effect levels (NOEL) were 1500 and 1000 Gy at 24 and 96 h, respectively. This is the first determination of lethal doses of gamma radiation for B. koreanus, which could be useful in ecological assessment of gamma radiation toward aquatic life and could be useful for understanding toxic mechanisms over sublethal doses.

Irradiation-injured brain tissues can self-renew in the absence of the pivotal tumor suppressor p53 in the medaka (Oryzias latipes) embryo.

The tumor suppressor protein, p53, plays pivotal roles in regulating apoptosis and proliferation in the embryonic and adult central nervous system (CNS) following neuronal injuries such as those induced by ionizing radiation. There is increasing evidence that p53 negatively regulates the self-renewal of neural stem cells in the adult murine brain; however, it is still unknown whether p53 is essential for self-renewal in the injured developing CNS. Previously, we demonstrated that the numbers of apoptotic cells in medaka (Oryzias latipes) embryos decreased in the absence of p53 at 12-24 h after irradiation with 10-Gy gamma rays. Here, we used histology to examine the later morphological development of the irradiated medaka brain. In p53-deficient larvae, the embryonic brain possessed similar vacuoles in the brain and retina, although the vacuoles were much smaller and fewer than those found in wild-type embryos. At the time of hatching (6 days after irradiation), no brain abnormality was observed. In contrast, severe disorganized neuronal arrangements were still present in the brain of irradiated wild-type embryos. Our present results demonstrated that self-renewal of the brain tissue completed faster in the absence of p53 than wild type at the time of hatching because p53 reduces the acute severe neural apoptosis induced by irradiation, suggesting that p53 is not essential for tissue self-renewal in developing brain.

Embryonic Medaka Model of Microglia in the Developing CNS Allowing In Vivo Analysis of Their Spatiotemporal Recruitment in Response to Irradiation.

Radiation therapy (RT) is pivotal in the treatment of many central nervous system (CNS) pathologies; however, exposure to RT in children is associated with a higher risk of secondary CNS tumors. Although recent research interest has focused on the reparative and therapeutic role of microglia, their recruitment following RT has not been elucidated, especially in the developing CNS. Here, we investigated the spatiotemporal dynamics of microglia during tissue repair in the irradiated embryonic medaka brain by whole-mount in situ hybridization using a probe for Apolipoprotein E (ApoE), a marker for activated microglia in teleosts. Three-dimensional imaging of the distribution of ApoE-expressing microglia in the irradiated embryonic brain clearly showed that ApoE-expressing microglia were abundant only in the late phase of phagocytosis during tissue repair induced by irradiation, while few microglia expressed ApoE in the initial phase of phagocytosis. This strongly suggests that ApoE has a significant function in the late phase of phagocytosis by microglia in the medaka brain. In addition, the distribution of microglia in p53-deficient embryos at the late phase of phagocytosis was almost the same as in wild-type embryos, despite the low numbers of irradiation-induced apoptotic neurons, suggesting that constant numbers of activated microglia were recruited at the late phase of phagocytosis irrespective of the extent of neuronal injury. This medaka model of microglia demonstrated specific recruitment after irradiation in the developing CNS and could provide a useful potential therapeutic strategy to counteract the detrimental effects of RT.

Nuclear and cytoplasmic changes in erythrocytes of p53-deficient medaka fish (Oryzias latipes) after exposure to gamma-radiation.

Previous studies have examined the effects of gamma-radiation on Japanese fish, in particular medaka (Oryzias latipes). In the present work, alterations in erythrocytes were recorded as haematological bio-indicators of exposure to gamma-radiation. After exposure of medaka fish to two different doses of radiation (2 Gy and 10 Gy), many malformations in red blood cells were observed in the irradiated fish compared with control fish. These malformations included acanthocytes, crenated cells, amoeboid cells, and sickle cells. More malformations were seen at the higher radiation dose. No micronuclei were seen in any group, but nuclear abnormalities were observed. We conclude that gamma-radiation causes morphological malformations of erythrocytes and is harmful to medaka fish.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells.

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation in RIC1 cells. It is known that 53BP1 is involved in both DNA-PK dependent and independent NHEJ. Therefore we suggest that DNA-PK independent NHEJ repair DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency.

Neurocytotoxic effects of iron-ions on the developing brain measured in vivo using medaka (Oryzias latipes), a vertebrate model.

PURPOSE: Exposure to heavy-ion radiation is considered a critical health risk on long-term space missions. The developing central nervous system (CNS) is a highly radiosensitive tissue; however, the biological effects of heavy-ion radiation, which are greater than those of low-linear energy transfer (LET) radiation, are not well studied, especially in vivo in intact organisms. Here, we examined the effects of iron-ions on the developing CNS using vertebrate organism, fish embryos of medaka (Oryzias latipes). MATERIALS AND METHODS: Medaka embryos at developmental stage 28 were irradiated with iron-ions at various doses of 0-1.5 Gy. At 24 h after irradiation, radiation-induced apoptosis was examined using an acridine orange (AO) assay and histologically. To estimate the relative biological effectiveness (RBE), we quantified only characteristic AO-stained rosette-shaped apoptosis in the developing optic tectum (OT). At the time of hatching, morphological abnormalities in the irradiated brain were examined histologically. RESULTS: The dose-response curve utilizing an apoptotic index for the iron-ion irradiated embryos was much steeper than that for X-ray irradiated embryos, with RBE values of 3.7-4.2. Histological examinations of irradiated medaka brain at 24 h after irradiation showed AO-positive rosette-shaped clusters as aggregates of condensed nuclei, exhibiting a circular hole, mainly in the marginal area of the OT and in the retina. However, all of the irradiated embryos hatched normally without apparent histological abnormalities in their brains. CONCLUSION: Our present study indicates that the medaka embryo is a useful model for evaluating neurocytotoxic effects on the developing CNS induced by exposure to heavy iron-ions relevant to the aerospace radiation environment.

Effects of dioxin isomers on induction of AhRs and CYP1A1 in early developmental stage embryos of medaka (Oryzias latipes).

The aryl hydrocarbon receptor (AhR) binds to polyaromatic compounds, including dioxins, and enhances the expression of several target genes, including drug-metabolizing cytochrome P450s (CYP1As). Four AhR genes (AhR1b-1, AhR1b-2, AhR2a, and AhR2b) were identified in the medaka genome. The molecular machinery involved in the dioxin response has been clarified chiefly in mammals, although fish models, such as zebrafish (Danio rerio), medaka (Oryzias latipes), and Fundulus, are excellent candidates for examining the mechanisms of developmental dioxin toxicity. Using these fish models, several experimental studies investigating the induced expression of CYP1A1 and AhRs, including functional evaluations by 2378T4CDD exposure, have been performed. However, few studies have examined the exposure to other dioxin isomers and it is not certain whether similar induced expressions patterns and toxicity-mediating functions of CYP1A1, AhRs, and AhR repressor (AhRR) compare with 2378T4CDD exposure. In this study, we investigated the toxicity of 13 dioxin isomers, including 2378T4CDD, and the induced expression of AhRs, AhRR, and CYP1A1 (CYP1A1_ORYLA) in the early life stages of medaka embryos. After exposure to dioxin isomers for 24-48h, the expression of AhR2a and CYP1A1_ORYLA correlated to the dioxin toxicity, and AhRR mRNA was widely expressed indicating it modulates AhR activity during the early stages of medaka embryos.

Cell lines derived from a medaka radiation-sensitive mutant have defects in DNA double-strand break responses.

It was reported that the radiation-sensitive Medaka mutant "ric1" has a defect in the repair of DNA double-strand breaks (DSBs) induced by gamma-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to gamma-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after gamma-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (gammaH2AX) foci were formed after gamma-irradiation, however, the gammaH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of gammaH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro.

Live imaging of radiation-induced apoptosis by yolk injection of Acridine Orange in the developing optic tectum of medaka.

To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) solution into the yolk of embryo and visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT, were irradiated with 5 Gy X-rays which is a non-lethal dose for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase up to 24 h after irradiation and completely disappeared up to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.