Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Comparison of biological measurement and physical estimates of space radiation in the International Space Station.

Nowadays, ordinary people can travel in space, and the possibility of extended durations in an environment such as moon of the Earth and Mars with higher space radiation exposures compared to past missions, is increasing. Until now, the physical doses of space radiation have been measured, but measurement of direct biological effects has been hampered by its low dose and low dose-rate effect. To assess the biological effects of space radiation, we launched and kept frozen mouse embryonic stem (ES) cells in minus eighty degree Celsius freezer in ISS (MELFI) on the International Space Station (ISS) for a maximum of 1,584 days. The passive dosimeter for life science experiments in space (PADLES) was attached on the surface of the sample case of the ES cells. The physical dosimeter measured the absorbed dose in water. After return, the frozen cells were thawed and cultured and their chromosome aberrations were analyzed. Comparative experiments with proton and iron ion irradiation were performed at particle accelerators on Earth. The wild-type ES cells showed no differences in chromosomal aberrations between the ground control and ISS exposures. However, we detected an increase of chromosome aberrations in radio-sensitized histone H2AX heterozygous-deficient mouse ES cells and found that the rate of increase against the absorbed dose was 1.54-fold of proton irradiation at an accelerator. On the other hand, we estimated the quality factor of space radiation as 1.48 ± 0.2. using formulas of International Commission of Radiation Protection (ICRP) 60. The relative biological effectiveness (RBE) observed from our experiments (1.54-fold of proton) was almost equal (1.04-fold) to the physical estimation (1.48 ± 0.2). It should be important to clarify the relation between biological effect and physical estimates of space radiation. This comparative study paves a way to reveal the complex radiation environments to reduce the uncertainty for risk assessment of human stay in space.

DOSE-RATE EFFECT OF RADIATION ON RAT MAMMARY CARCINOGENESIS AND AN EMERGING ROLE FOR STEM CELL BIOLOGY.

The uncertain cancer risk of protracted radiation exposure at low dose rates is an important issue in radiological protection. Tissue stem/progenitor cells are a supposed origin of cancer and may contribute to the dose-rate effect on carcinogenesis. The authors have shown that female rats subjected to continuous whole body γ irradiation as juveniles or young adults have a notably reduced incidence of mammary cancer as compared with those irradiated acutely. Experiments using the mammosphere formation assay suggested the presence of radioresistant progenitor cells. Cell sorting indicated that basal progenitor cells in rat mammary gland were more resistant than luminal progenitors to killing by acute radiation, especially at high doses. Thus, the evidence indicates a cell-type-dependent inactivation of mammary cells that manifests only at high acute doses, implying a link to the observed dose-rate effect on carcinogenesis.

Brca1L63X /+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer.

Women who are heterozygous for deleterious BRCA1 germline mutations harbor a high risk of hereditary breast cancer. Previous Brca1-heterozygous animal models do not recapitulate the breast cancer phenotype, and thus all currently used knockout models adopt conditional, mammary-specific homozygous Brca1 loss or addition of Trp53 deficiency. Herein, we report the creation and characterization of a novel Brca1 mutant rat model harboring the germline L63X mutation, which mimics a founder mutation in Japan, through CRISPR-Cas9-based genome editing. Homozygotes (Brca1L63X/L63X ) were embryonic lethal, whereas heterozygotes (Brca1L63X/+ ) showed apparently normal development. Without carcinogen exposure, heterozygotes developed mammary carcinoma at a comparable incidence rate with their wild-type (WT) littermates during their lifetime. Intraperitoneal injection of 1-methyl-1-nitrosourea (25 or 50 mg/kg) at 7 weeks of age induced mammary carcinogenesis at comparable levels among the heterozygotes and their littermates. After exposure to ionizing radiation (0.1-2 Gy) at 7 weeks of age, the heterozygotes, but not WT littermates, displayed dose-dependent mammary carcinogenesis with 0.8 Gy-1 excess in hazard ratio during their middle age; the relative susceptibility of the heterozygotes was more prominent when rats were irradiated at 3 weeks of age. The heterozygotes had tumors with a lower estrogen receptor α immunopositivity and no evidence of somatic mutations of the WT allele. The Brca1L63X/+ rats thus offer the first single-mutation, heterozygous model of BRCA1-associated breast cancer, especially with exposure to a DNA break-inducing carcinogen. This implies that such carcinogens are causative and a key to breast cancer prevention in individuals who carry high-risk BRCA1 mutations.

Copenhagen Rats Display Dominantly Inherited Yet Non-uniform Resistance to Spontaneous, Radiation-induced, and Chemically-induced Mammary Carcinogenesis.

BACKGROUND/AIM: Genetic and environmental factors interact to dictate the risk of cancer, and animal models are expected to provide avenues for identifying such interactions. The aim of the study was to clarify the genetic susceptibility of Copenhagen rats to spontaneous, radiation-induced, and chemically-induced mammary carcinogenesis. MATERIALS AND METHODS: Female Copenhagen and Sprague- Dawley rats and their F1 hybrids were subjected at age 7 weeks to γ-irradiation or intraperitoneal injection with 1-methyl-1-nitrosourea or were not treated, and palpable mammary tumours were diagnosed histologically. Data were pooled with previous data acquired for both nontreated and irradiated Sprague-Dawley rats. RESULTS: Radiation and 1-methyl-1-nitrosourea both significantly increased the incidence of mammary cancer in all strains. Copenhagen and F1 rats displayed a significantly lower incidence than Sprague-Dawley rats in all groups, with relatively higher incidence after irradiation. F1 rats exhibited significantly higher mammary cancer incidence than Copenhagen rats in the nontreated, but not the treated, groups. The interaction of the strain and exposure effects was suggested to be quasi-multiplicative. CONCLUSION: Copenhagen rats display non-uniform resistance to spontaneous, radiation-induced, and chemically-induced mammary carcinogenesis with dominant inheritance over Sprague-Dawley rats.

Development of mammary cancer in γ-irradiated F1 hybrids of susceptible Sprague-Dawley and resistant Copenhagen rats, with copy-number losses that pinpoint potential tumor suppressors.

Copenhagen rats are highly resistant to mammary carcinogenesis, even after treatment with chemical carcinogens and hormones; most studies indicate that this is a dominant genetic trait. To test whether this trait is also dominant after radiation exposure, we characterized the susceptibility of irradiated Copenhagen rats to mammary carcinogenesis, as well as its inheritance, and identified tumor-suppressor genes that, when inactivated or mutated, may contribute to carcinogenesis. To this end, mammary cancer-susceptible Sprague-Dawley rats, resistant Copenhagen rats, and their F1 hybrids were irradiated with 4 Gy of γ-rays, and tumor development was monitored. Copy-number variations and allelic imbalances of genomic DNA were studied using microarrays and PCR analysis of polymorphic markers. Gene expression was assessed by quantitative PCR in normal tissues and induced mammary cancers of F1 rats. Irradiated Copenhagen rats exhibited a very low incidence of mammary cancer. Unexpectedly, this resistance trait did not show dominant inheritance in F1 rats; rather, they exhibited intermediate susceptibility levels (i.e., between those of their parent strains). The susceptibility of irradiated F1 rats to the development of benign mammary tumors (i.e., fibroadenoma and adenoma) was also intermediate. Copy-number losses were frequently observed in chromosome regions 1q52-54 (24%), 2q12-15 (33%), and 3q31-42 (24%), as were focal (38%) and whole (29%) losses of chromosome 5. Some of these chromosomal regions exhibited allelic imbalances. Many cancer-related genes within these regions were downregulated in mammary tumors as compared with normal mammary tissue. Some of the chromosomal losses identified have not been reported previously in chemically induced models, implying a novel mechanism inherent to the irradiated model. Based on these findings, Sprague-Dawley × Copenhagen F1 rats offer a useful model for exploring genes responsible for radiation-induced mammary cancer, which apparently are mainly located in specific regions of chromosomes 1, 2, 3 and 5.

Novel method for evaluating the health condition of mice in space through a video downlink.

Clarification of the criteria for managing animal health is essential to increase the reliability of experiments and ensure transparency in animal welfare. For experiments performed in space, there is no consensus on how to care for animals owing to technical issues, launch mass limitation, and human resources. Some biological processes in mammals, such as musculoskeletal or immune processes, are altered in the space environment, and mice in space can be used to simulate morbid states, such as senescence acceleration. Thus, there is a need to establish a novel evaluation method and evaluation criteria to monitor animal health. Here, we report a novel method to evaluate the health of mice in space through a video downlink in a series of space experiments using the Multiple Artificial-gravity Research System (MARS). This method was found to be more useful in evaluating animal health in space than observations and body weight changes of the same live mice following their return to Earth. We also developed criteria to evaluate health status via a video downlink. These criteria, with "Fur condition" and "Respiratory" as key items, provided information on the daily changes in the health status of mice and helped to identify malfunctions at an early stage. Our method and criteria led to the success of our missions, and they will help establish appropriate rules for space experiments in the future.

Interstitial chromosomal deletion of the tuberous sclerosis complex 2 locus is a signature for radiation-associated renal tumors in Eker rats.

Ionizing radiation can damage DNA and, therefore, is a risk factor for cancer. Eker rats, which carry a heterozygous germline mutation in the tumor-suppressor gene tuberous sclerosis complex 2 (Tsc2), are susceptible to radiation-induced renal carcinogenesis. However, the molecular mechanisms involved in Tsc2 inactivation are unclear. We subjected Fischer 344 × Eker (Long Evans Tsc2+/- ) F1 hybrid rats to gamma-irradiation (2 Gy) at gestational day 19 (GD19) or postnatal day 5 (PND5) and investigated the patterns of genomic alterations in the Tsc2 allele of renal tumors that developed at 1 year after irradiation (N = 24 tumors for GD19, N = 10 for PND5), in comparison with spontaneously developed tumors (N = 8 tumors). Gamma-irradiation significantly increased the multiplicity of renal tumors. The frequency of LOH at the chromosome 10q12 region, including the Tsc2 locus, was 38%, 29% and 60% in renal carcinomas developed from the nonirradiated, GD19 and PND5 groups, respectively. Array comparative genomic hybridization analysis revealed that the LOH patterns on chromosome 10 in renal carcinomas were classified into chromosomal missegregation, mitotic recombination and chromosomal deletion types. LOH of the interstitial chromosomal deletion type was observed only in radiation-associated carcinomas. Sequence analysis for the wild-type Tsc2 allele in the LOH-negative carcinomas identified deletions (nonirradiated: 26%; GD19: 21%) and base-substitution mutations (GD19: 4%). Reduced expression of Tsc2 was also observed in the majority of the LOH-negative carcinomas. Our results suggest that interstitial chromosomal deletion is a characteristic mutagenic event caused by ionizing radiation, and it may contribute to the assessment of radiation-induced cancer risk.

Synthesis and maintenance of lipid droplets are essential for mouse preimplantation embryonic development.

Lipid droplets (LDs), which are ubiquitous organelles consisting of a neutral lipid core coated with a phospholipid monolayer, play key roles in the regulation of cellular lipid metabolism. Although it is well known that mammalian oocytes and embryos contain LDs and that the amount of LDs varies among animal species, their physiological functions remain unclear. In this study, we have developed a method based on two-step centrifugation for efficient removal of almost all LDs from mouse MII oocytes (delipidation). We found that delipidated MII oocytes could be fertilized in vitro, and developed normally to the blastocyst stage even when the embryos were cultured in the absence of a fatty acid supply. LDs were newly synthesized and accumulated soon after delipidation, but chemical inhibition of long chain acyl-CoA synthetases (ACSLs) blocked this process, resulting in severe impairment of early embryonic development. Furthermore, we found that overabundance of LDs is detrimental to early embryonic development. Our findings demonstrate the importance of synthesis and maintenance of LDs, mediated in part by ACSL activity, during preimplantation embryonic development.

Phosphorus Localization and Its Involvement in the Formation of Concentrated Uranium in the Renal Proximal Tubules of Rats Exposed to Uranyl Acetate.

Although the kidneys comprise a critical target of uranium exposure, the dynamics of renal uranium distribution have remained obscure. Uranium is considered to function physiologically in the form of uranyl ions that have high affinity for phosphate groups. The present study applied microbeam-based elemental analysis to precisely determine the distribution of phosphorus and uranium in the kidneys of male Wistar rats exposed to uranium. One day after a single subcutaneous injection of uranyl acetate (2 mg/kg), areas of concentrated phosphorus were scattered in the S3 segments of the proximal tubule of the kidneys, whereas the S3 segments in control rats and in rats given a lower dose of uranium (0.5 mg/kg) contained phosphorus without concentrated phosphorus. Areas with concentrated phosphorus contained uranium 4- to 14-fold more than the mean uranium concentration (126-472 vs. 33.1 ± 4.6 µg/g). The chemical form of uranium in the concentrated phosphorus examined by XAFS was uranium (VI), suggesting that the interaction of uranyl ions with the phosphate groups of biomolecules could be involved in the formation of uranium concentration in the proximal tubules of kidneys in rats exposed to uranium.

Prominent Dose-Rate Effect and Its Age Dependence of Rat Mammary Carcinogenesis Induced by Continuous Gamma-Ray Exposure.

Although the risk of breast cancer after high-dose-rate irradiation has been firmly established, however, the risk incurred for low-dose-rate irradiation is not well understood. Here we provide experimental evidence for dose rate and age dependencies induced by continuous γ-ray irradiation on mammary carcinogenesis. Female rats received continuous whole-body irradiation at one of the following time points: at 7 weeks of age (denoted adults) at a dose rate of 3-60 mGy/h (4 Gy total); or at either 3 weeks (denoted juveniles) or 7 weeks of age at a dose rate of 6 mGy/h (1-8 Gy total). Additional rats were acutely irradiated at 13 weeks of age at a dose rate of 30 Gy/h (0.5-4 Gy total). We observed the incidence of mammary tumors by weekly palpation until they were 90 weeks old and after pathological inspection upon autopsy. The tumor incidence rate for each group was characterized by Cox regression analysis. When adult rats were irradiated at 60 mGy/h for a total of 4 Gy, their hazard ratio for mammary carcinoma significantly increased relative to nonirradiated controls; however, for adult rats irradiated at 3-24 mGy/h, even though they also received a total of 4 Gy, their hazard ratio for carcinoma incidence did not significantly increase. A larger increase in the incidence rate of carcinoma per dose was found for the juveniles than for the adults irradiated at 6 mGy/h, whereas age did not influence the effect of acute irradiation at 30 Gy/h; a threshold-like dose response was observed for irradiation at 6 mGy/h (threshold, ∼2.5 and ∼4 Gy for juveniles and adults, respectively). Regarding benign tumors of the mammary gland, a significant increase in their incidence was observed for irradiation down to 6 mGy/h, but not at 3 mGy/h and there was no evidence of age-dependent induction. Thus, induction of female rat mammary carcinogenesis by continuous γ-ray exposure was age dependent and drastically increased for adult rats that received between 24 and 60 mGy/h irradiation.

Differential effect of parity on rat mammary carcinogenesis after pre- or post-pubertal exposure to radiation.

Radiation exposure during the peri-pubertal period is a proven risk factor for breast cancer, whereas parity is an established protective factor. The present study investigated whether parity imposes differential protective effects against radiation-induced rat mammary carcinoma depending on the age at exposure. Pre- and post-pubertal female rats, irradiated or left unirradiated, were mated and allowed to nurse until weaning or left unmated. Appearance of mammary tumors was monitored, and serum concentrations of estradiol and progesterone were measured following weaning. Carcinomas were evaluated by immunohistochemistry for estrogen receptor, progesterone receptor, and the cell proliferation marker Ki-67. Parity reduced the risk of carcinoma in unirradiated and pre-pubertally irradiated rats but not post-pubertally irradiated rats. Although radiation exposure increased serum progesterone level, parity after pre-pubertal exposure significantly decreased the elevated progesterone to a normal level, reflecting a protective effect. Moreover, parity significantly decreased the proportion of hormone receptor-positive carcinomas after pre-pubertal exposure. Parity was also related to the observed positive association between progesterone receptor and Ki-67 indices in cancer tissue, implying progesterone receptor-dependent cell proliferation. Thus, parity protects against radiation-induced rat mammary carcinogenesis depending on the age at exposure; the mechanisms may involve changes in hormone levels and cancer tissue.

Age Modifies the Effect of 2-MeV Fast Neutrons on Rat Mammary Carcinogenesis.

The relative biological effectiveness (RBE) of neutrons depends on their physical nature (e.g., energy) and the biological context (e.g., end points, materials). From the perspective of radiological protection, age is an important biological context that influences radiation-related cancer risk, but very few studies have addressed its potential impact on neutron effects. We therefore investigated the influence of age on the effect of accelerator-generated fast neutrons (mean energy, ∼2 MeV) in an animal model of breast carcinogenesis. Female Sprague-Dawley rats at 1, 3 and 7 weeks of age were irradiated with fast neutrons at absorbed doses of 0.0485-0.97 Gy. All animals were kept under specific pathogen-free conditions and screened weekly for mammary tumors by palpation until they were 90 weeks old. Tumors were diagnosed based on histology. Mathematical modeling was used to analyze mammary cancer incidence, collectively using data from this study and a previously reported experiment on 137Cs gamma rays. The results indicate that neutron irradiation elevated the risk of palpable mammary carcinoma with a linear dose response, the slope of which depended on age at time of irradiation. The RBE of neutron radiation was 7.5 ± 3.4, 9.3 ± 3.5 and 26.1 ± 8.9 (mean ± SE) for animals exposed at 1, 3 and 7 weeks of age, respectively. Our results indicate that age of the animal is an important factor influencing the effect of fast neutrons on breast cancer risk.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis.

Biological measures to minimize the risk of radiotherapy-associated second cancer: A research perspective.

Purpose Second cancers are among the most serious sequelae for cancer survivors who receive radiotherapy. This article aims to review current knowledge regarding how the risk of radiotherapy-associated second cancer can be minimized by biological measures and to discuss relevant research needs. Results The risk of second cancer can be reduced not only by physical measures to decrease the radiation dose to normal tissues but also by biological means that interfere with the critical determinants of radiation-induced carcinogenesis. Requirements for such biological means include the targeting of tumor types relevant to radiotherapy-associated risk, concrete safety and efficacy evidence and feasibility and minimal invasiveness. Mechanistic insights into the process of radiation carcinogenesis provide rational approaches to minimize the risk. Five mechanism-based strategies are proposed herein based on the current state of knowledge. Epidemiological studies on the joint effects of radiation and lifestyle or other factors can provide evidence for factors that modify radiation-associated risks if deliberately controlled. Conclusions Mechanistic and epidemiological evidence indicates that it is possible to develop interventional measures to minimize the second cancer risk associated with radiotherapy. Research is needed regarding the critical determinants of radiation-induced carcinogenesis available for intervention and joint effects of radiation and controllable factors.

Evaluation of X-ray doses and their corresponding biological effects on experimental animals in cone-beam micro-CT scans (R-mCT2).

Studies show that the radiation dose received during a micro-CT examination may have adverse effects on living subjects. However, the correlations between the biological effects and the radiation doses have never been thoroughly evaluated in the majority of cases. In this study, we evaluated the biological radiation effects of measured radiation doses in ICR mice using cone-beam micro-CT scans. Long-term in vivo whole-body micro-CT scans of ICR mice were performed for a duration of 4 weeks. Although a scanning frequency of three scans per week is higher than that necessary for conventional studies, this study represents particular cases where the subjects may undergo an extreme number of examinations. The average X-ray dose of a CT scan measures 16.19 mGy at the center of a phantom and 16.24 mGy at an offset position of 7.5 mm from the center of the phantom. The total average dose at the center of the phantom during the 4-week scanning period was 194.3 mGy. No significant radiation effects were observed in the weight gain curves, organ weights, blood analyses, litter sizes, reared offspring sizes, and the histopathologic results. Therefore, it is unlikely that the measured doses for the CT scans caused any radiation damage in the mice.

Filobacterium rodentium gen. nov., sp. nov., a member of Filobacteriaceae fam. nov. within the phylum Bacteroidetes; includes a microaerobic filamentous bacterium isolated from specimens from diseased rodent respiratory tracts.

Strain SMR-CT, which was originally isolated from rats as the SMR strain, had been named 'cilia-associated respiratory bacillus' ('CAR bacillus'). 'CAR bacillus' was a Gram-stain-negative, filamentous argentophilic bacterium without flagella. SMR-CT grew at 37 °C under microaerobic conditions, showed gliding activity, hydrolysed urea and induced chronic respiratory diseases in rodents. The dominant cellular fatty acids detected were iso-C15 : 0 and anteiso-C15 : 0. The DNA G+C content was 47.7 mol%. 16S rRNA gene sequence analysis revealed SMR-CT and other strains of 'CAR bacillus' isolated from rodents all belonged to the phylum Bacteroidetes. The nearest known type strain, with 86 % 16S rRNA gene sequence similarity, was Chitinophaga pinensis DSM 2588T in the family Chitinophagaceae. Strain SMR-CT and closely related strains of 'CAR bacillus' rodent-isolates formed a novel family-level clade in the phylum Bacteroidetes with high bootstrap support (98-100 %). Based on these results, we propose a novel family, Filobacteriaceae fam. nov., in the order Sphingobacteriales as well as a novel genus and species, Filobacterium rodentium gen. nov., sp. nov., for strain SMR-CT. The type strain is SMR-CT ( = JCM 19453T = DSM 100392T).

Ionizing radiation, inflammation, and their interactions in colon carcinogenesis in Mlh1-deficient mice.

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and ß-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency.

Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging.

Radiation-induced thymic lymphoma (RITL) in mice is induced by fractionated whole-body X-irradiation (FX) and has served as a useful model for studying radiation carcinogenesis. In this model, the initial postirradiation cellular events in the thymus and bone marrow (BM) are critically important for tumorigenesis, and BM transplantation (BMT) prevents RITL. However, direct assessment of these events is so far restricted by the lack of noninvasive monitoring techniques. Here, we have developed positron emission tomography (PET) and magnetic resonance imaging (MRI) methods to quantify the events critical for RITL development and the effects of BMT in living animals. Apparent diffusion coefficients (ADCs) were calculated from diffusion-weighted MRI to evaluate the changes in the BM of mice receiving FX. ADC values dramatically changed in the irradiated BM, corresponding to pathological findings of the irradiated BM, returning to normal levels following BMT sooner than with spontaneous recovery. PET with 4'-[methyl-(11)C]thiothymidine, a novel tracer for cell proliferation, revealed that the irradiated thymus showed significantly higher tracer uptake than the unirradiated thymus 1 week after FX. Interestingly, its increased uptake was completely abolished by BMT, even with very few donor-derived cells in the thymus. Thereafter, the thymus receiving BMT had significantly increased tracer uptake. These findings suggest that BMT first suppresses FX-induced aberrant thymocyte proliferation and then accelerates thymic regeneration. This study demonstrates the feasibility of using PET and MRI for noninvasive monitoring of tumorigenic cellular processes in an animal model of radiation-induced cancer.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability.

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.