Involvement of H2A variants in DNA damage response of zygotes.

Phosphorylated H2AX, known as γH2AX, forms in response to genotoxic insults in somatic cells. Despite the high abundance of H2AX in zygotes, the level of irradiation-induced γH2AX is low at this stage. Another H2A variant, TH2A, is present at a high level in zygotes and can also be phosphorylated at its carboxyl end. We constructed H2AX- or TH2A-deleted mice using CRISPR Cas9 and investigated the role of these H2A variants in the DNA damage response (DDR) of zygotes exposed to γ-ray irradiation at the G2 phase. Our results showed that compared to irradiated wild-type zygotes, irradiation significantly reduced the developmental rates to the blastocyst stage in H2AX-deleted zygotes but not in TH2A-deleted ones. Furthermore, live cell imaging revealed that the G2 checkpoint was activated in H2AX-deleted zygotes, but the duration of arrest was significantly shorter than in wild-type and TH2A-deleted zygotes. The number of micronuclei was significantly higher in H2AX-deleted embryos after the first cleavage, possibly due to the shortened cell cycle arrest of damaged embryos and, consequently, the insufficient time for DNA repair. Notably, FRAP analysis suggested the involvement of H2AX in chromatin relaxation. Moreover, phosphorylated CHK2 foci were found in irradiated wild-type zygotes but not in H2AX-deleted ones, suggesting a critical role of these foci in maintaining cell cycle arrest for DNA repair. In conclusion, H2AX, but not TH2A, is involved in the DDR of zygotes, likely by creating a relaxed chromatin structure with enhanced accessibility for DNA repair proteins and by facilitating the formation of pCHK2 foci to prevent premature cleavage.

DNA repair is efficient in irradiated M phase zygotes.

In somatic cells, DNA repair is attenuated during mitosis to prevent the formation of anaphase bridges and facilitate the proper segregation of sister chromatids. Irradiation-induced γH2AX foci persist for hours in M phase somatic cells. However, we observed that anaphase bridges formed in a significant fraction of mouse zygotes irradiated during mitosis. Additionally, γH2AX signals in M phase zygotes peaked 30 min after irradiation and subsequently reduced with a half-life within 1-2 h. These results suggest that the DNA repair system may operate efficiently in M phase zygotes following irradiation, leading to the frequent formation of anaphase bridges. The absence of H2AX promoted the successful segregation of sister chromatids and enhanced the development of embryos to the blastocyst stage. The DNA repair system may be differentially regulated during the M phase of the first cell cycle to ensure the immediate elimination of damaged zygotes, thereby efficiently preventing transmission of mutations to subsequent generations.

Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes.

PURPOSE: Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina's inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. METHODS: We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. RESULTS: One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. CONCLUSIONS: Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.

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.

Acquirement of the autonomic nervous system modulation evaluated by heart rate variability in medaka (Oryzias latipes).

Small teleosts have recently been established as models of human diseases. However, measuring heart rate by electrocardiography is highly invasive for small fish and not widely used. The physiological nature and function of vertebrate autonomic nervous system (ANS) modulation of the heart has traditionally been investigated in larvae, transparent but with an immature ANS, or in anesthetized adults, whose ANS activity may possibly be disturbed under anesthesia. Here, we defined the frequency characteristics of heart rate variability (HRV) modulated by the ANS from observations of heart movement in high-speed movie images and changes in ANS regulation under environmental stimulation in unanesthetized adult medaka (Oryzias latipes). The HRV was significantly reduced by atropine (1 mM) in the 0.25-0.65 Hz and by propranolol (100 µM) at 0.65-1.25 Hz range, suggesting that HRV in adult medaka is modulated by both the parasympathetic and sympathetic nervous systems within these frequency ranges. Such modulations of HRV by the ANS in adult medaka were remarkably suppressed under anesthesia and continuous exposure to light suppressed HRV only in the 0.25-0.65 Hz range, indicating parasympathetic withdrawal. Furthermore, pre-hatching embryos did not show HRV and the power of HRV developed as fish grew. These results strongly suggest that ANS modulation of the heart in adult medaka is frequency-dependent phenomenon, and that the impact of long-term environmental stimuli on ANS activities, in addition to development of ANS activities, can be precisely evaluated in medaka using the presented method.

Cell cycle-dependent radiosensitivity in mouse zygotes.

Mammalian zygotes are hypersensitive to radiation exposure compared with later-stage embryos and somatic cells, which may be due to an unusual DNA damage response (DDR). DNA damage checkpoints are an essential part of the DDR, allowing for faithful replication of cells. Although the DDR and radiosensitivity of somatic cells are dependent on the cell cycle phase, it remains largely unclear how the irradiation of zygotes at different phases affects cell cycle progression and preimplantation development. Here, mouse zygotes were irradiated with 10 Gy γ-rays at all four cell cycle phases. DNA damage checkpoints were activated by γ-irradiation at the G2 phase, but not at the G1, S, and M phases. The absence of DNA damage checkpoints at the G1 and M phases seems to be due to the low abundance of phosphorylated CHK2, which plays a key role in checkpoint activation in response to ionizing radiation. The cause of the inoperative S phase checkpoint may lie downstream of CHK2 activation. The inactive DNA damage checkpoints at the G1 and S phases contributed to micronucleus formation in the subsequent 2-cell stage, whereas irradiation at the M phase led to the highest incidence of chromatin bridges. The low developmental rates of embryos irradiated at the G1, S, and M phases suggest that embryos with these two types of chromatin abnormalities are prone to developmental failure. Taken together, these results suggest that the radiosensitivity of zygotes can be ascribed to a defective DDR at the G1, S, and M phases.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

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.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos.

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

Orthologous Divergence and Paralogous Anticonvergence in Molecular Evolution of Triplicated Green Opsin Genes in Medaka Fish, Genus Oryzias.

Gene duplication of green (RH2) opsin genes and their spectral differentiation are well documented in many teleost fish. However, their evolutionary divergence or conservation patterns among phylogenetically close but ecologically diverse species is not well explored. Medaka fish (genus Oryzias) are broadly distributed in fresh and brackish waters of Asia, with many species being laboratory-housed and feasible for genetic studies. We previously showed that a Japan strain (HNI) of medaka (Oryzias latipes) possessed three RH2 opsin genes (RH2-A, RH2-B, and RH2-C) encoding spectrally divergent photopigments. Here, we examined the three RH2 opsin genes from six Oryzias species representing three species groups: the latipes, the celebensis, and the javanicus. Photopigment reconstitution revealed that the peak absorption spectra (λmax) of RH2-A were divergent among the species (447-469 nm), whereas those of RH2-B and RH2-C were conservative (516-519 and 486-493 nm, respectively). For the RH2-A opsins, the largest spectral shift was detected in the phylogenetic branch leading to the latipes group. A single amino acid replacement T94C explained most of the spectral shift. For RH2-B and -C opsins, we detected tracts of gene conversion between the two genes homogenizing them. Nevertheless, several amino acid differences were maintained. We showed that the spectral difference between the two opsins was attributed to largely the E/Q amino acid difference at the site 122 and to several sites with individually small spectral effects. These results depict dynamism of spectral divergence of orthologous and paralogous green opsin genes in phylogenetically close but ecologically diverse species exemplified by medaka.

Comparative histology of wild-type and p53-deficient medaka (Oryzias latipes): nephrotoxic effect of ultraviolet A radiation.

Ultraviolet radiation is an ecological factor that directly affects terrestrial organisms through suppression of immunity or damage to internal organs. The present study assessed the effects of ultraviolet A (UVA) radiation on the kidneys of both wild-type (WT) and p53-deficient medaka (Oryzias latipes) and evaluated which strain was more resistant to the effects of UVA. Fish were divided into four groups: control group 1 (Cwt and Cp53), kept for 3 days without UVA exposure; group 2 (1wt and 1p53), fish exposed daily to UVA for 1 h day-1 for 3 days; group 3 (2wt and 2p53), fish exposed daily to UVA for 2 h day-1 for 3 days; and group 4 (3wt and 3p53), fish exposed daily to UVA for 3 h day-1 for 3 days. Samples of tissues were obtained 24 h after UVA exposure. The most obvious histopathological changes induced by UVA radiation in kidney tissues of both strains of medaka (WT and p53-deficient) were high levels of vacuolation of tubular cells followed by necrosis. The tubular segments lost their normal shape which appeared like a network structure and their cells with clear cytoplasm. Necrosis of lymphoid tissues and spots of brown pigmentation (possibly melanomacrophages) were sporadically seen in interstitial lymphoid tissues, while shrinkage of glomeruli, diminution of periodic acid-Schiff staining, and increased amount of collagenous fibers were observed. Our results confirmed the harmful effects of UVA radiation on kidney tissues of both WT and p53-deficient medaka. However, WT medaka was affected more than p53-deficient medaka.

γ-H2AX foci as indication for the DNA damage in erythrocytes of medaka (Oryzias latipes) intoxicated with 4-nonylphenol.

The present study aimed to investigate the genotoxicity in erythrocytes induced after exposure of medaka (Oryzias latipes) to 4-nonylphenol (4-NP). Adult female medaka fish were exposed to 4-NP at three sublethal concentrations for 15 days to compare their sensitivity with that of catfish as an aquatic model. Comet assay and γ-H2AX were used as biomarkers to detect DNA damage in erythrocytes. Exposure to 4-NP resulted in an increase in the tail moment in a dose-dependent manner. The highest level of DNA damage was recorded after exposure to 100 µg/l 4-NP. The number of foci was increased after exposure to 4-NP, indicating damage to DNA. The present results confirmed the high level of morphological alterations and apoptosis of erythrocytes detected in the first part of this study. 4-NP induced genotoxic effects in medaka, which were found to be more sensitive than catfish after exposure to 4-nonylphenol. Graphical abstract.

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.

Cytotoxic and genotoxic effects of arsenic on erythrocytes of Oryzias latipes: Bioremediation using Spirulina platensis.

BACKGROUND: Exposure to the environmental pollutants poses a serious threat to aquatic organism. The arsenic exposure in fish increases the risk of developing serious alterations from embryo to adult. OBJECTIVES: The present investigation was done to study the toxic effects of heavy metal arsenic [As(III)] on medaka (Oryzias latipes). Morphological alterations, apoptosis, nuclear abnormalities, and genotoxic biomarkers in erythrocytes were used to determine the stress caused by arsenic (As) exposure. METHODS: Medaka was exposed to As for 15 days at two toxic sublethal concentrations (7 ppm and 10 ppm) in combination with Spirulina platensis (SP) treatment as antioxidant algae at 200 mg/L. RESULTS: Results were consistent with a previous study results on tilapia. Exposure of medaka to As resulted in a dose-dependent increase in most the biomarkers used in the current study. Fish exposed to10 ppm As showed highest level of DNA damage. For the first time to our knowledge, using SP to counter the As toxicity in medaka, DNA damage restored to control levels. CONCLUSION: Accordingly, those results suggests that SP can protect medaka in aquaculture against As-induced damage by its ability as reactive oxygen species (ROS) reducer, antioxidant role, and DNA damage scavenger.

UVA-induced neurotoxicity in Japanese medaka (Oryzias latipes).

Ultraviolet radiation-induced neurodegeneration has been studied in the early stages of development in fish, but not extensively in the adult stage. The present study aimed at investigating the effects of ultraviolet radiation-A (UVA) in adult Japanese medaka (Oryzias latipes). The brain, spinal cord, and retina were examined histopathologically as nervous system target organs. Japanese medaka fish were exposed to 15, 30, and 60 min day-1 UVA for 3 days, and samples were obtained 24 h and 14 days after UVA exposure. Neurohistopathological alterations in brain tissue included vacuoles, blood congestion, degeneration of neuropils, and pyknotic nuclei in neurons. Alterations in the spinal cord included neuronal cell degeneration, reduction in the spinal cord area, and degeneration of Mauthner cells. Retinal tissue showed vacuolation in the nerve fiber layer (NFL), pyknotic nuclei in the ganglion cell layer (GCL), and decreased cell populations particularly in the inner nuclear layer (INL) and GCL. The degree of degeneration was dependent on the duration of UVA exposure. The signs of degeneration decreased gradually and disappeared completely after the 14-day recovery period. In addition, p53-deficient medaka fish were more tolerant than were wild-type (Hd-rR) Japanese medaka. In conclusion, UV radiation induced neurodegeneration in the brain, spinal cord, and retina of adult Japanese medaka (Oryzias latipes) but their normal histological architecture reappeared in these tissues after 14 days.

Medaka Population Genome Structure and Demographic History Described via Genotyping-by-Sequencing.

Medaka is a model organism in medicine, genetics, developmental biology and population genetics. Lab stocks composed of more than 100 local wild populations are available for research in these fields. Thus, medaka represents a potentially excellent bioresource for screening disease-risk- and adaptation-related genes in genome-wide association studies. Although the genetic population structure should be known before performing such an analysis, a comprehensive study on the genome-wide diversity of wild medaka populations has not been performed. Here, we performed genotyping-by-sequencing (GBS) for 81 and 12 medakas captured from a bioresource and the wild, respectively. Based on the GBS data, we evaluated the genetic population structure and estimated the demographic parameters using an approximate Bayesian computation (ABC) framework. The genome-wide data confirmed that there were substantial differences between local populations and supported our previously proposed hypothesis on medaka dispersal based on mitochondrial genome (mtDNA) data. A new finding was that a local group that was thought to be a hybrid between the northern and the southern Japanese groups was actually an origin of the northern Japanese group. Thus, this paper presents the first population-genomic study of medaka and reveals its population structure and history based on chromosomal genetic diversity.

Radical change of apoptotic strategy following irradiation during later period of embryogenesis in medaka (Oryzias latipes).

Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.

Oocyte atresia in WT (HdrR) and P53 (-/-) medaka (Oryzias latipes) exposed to UVA.

The negative effects of ambient ultraviolet (UVA) on the water environment have been recently highlighted; UVA can create deleterious effects by stimulating stress on pelagic organisms. Little is known about UVA effects on oocyte characteristics of female fish. In the present study we explored the effects of exposure to ecologically relevant levels of simulated UVA radiation on ovaries of two major strains WT (HdrR) and P53 (-/-) of medaka (Oryzias latipes) mature female. Fish were assigned to control and three UVA-exposed groups as (15 min, 30 min, and 60 min/day) for three days and sample selection was 24 h and 14 days after exposure. Histological alterations and oocyte atresia percentage were analyzed in the UVA-exposed fish compared to control. Alteration comprised hyperthrophied follicular cells with increased thickness, breakdown of egg chorion (zona radiata), damage of cortical alveoli, and distorted nucleus and cytoplasm. The atresia percentages significantly increased with higher UVA exposure dose and time for both the wild type and the p53 deficient fish. The wild type displayed significantly higher oocyte atresia percentage than the p53 mutant. These results suggested that UVA exposure provoked histological alterations in both p53 and WT medaka oocytes leading to follicular atresia, which reduce female reproductive ability and larval production. UVA oocyte response showed p53 dependent and independent histological alteration, however, the p53 mutant was less sensitive to UVA than the wild type in medaka fish.

Sensitivity of medaka (Oryzias latipes) to 4-nonylphenol subacute exposure; erythrocyte alterations and apoptosis.

The present study was undertaken to assess the effects of the endocrine-disrupting compound; 4-nonylphenol (4-NP) in medaka (Oryzias latipes). The frequencies of erythrocyte alterations, apoptosis, and micronuclei were used as biological indicators of damage. Medaka were exposed 15 days to 4-NP at three sublethal concentrations (50, 80, and 100 µg/l 4-NP) and results compared with those of a previous study using catfish as an animal model. Exposure of medaka resulted in a dose-dependent increase in the frequency of erythrocyte alterations, apoptosis and micronucleus (MN). Many morphological alterations and nuclear abnormalities were observed, including acanthocytes, lobed nucleus, eccentric nucleus, fragmented nucleus, blebbed nucleus, binuclei, deformed nucleus, notched nucleus, hemolysed cells, crenated cells, teardrop-like cells, and schistocytes. Mortality was recorded after treatment with 80 and 100 µg/l 4-NP, indicating that medaka are more sensitive than catfish to 4-NP exposure. We concluded that, 4-NP causes several malformations in the shape and number of erythrocytes in medaka, indicating its genotoxicity.

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.