Toshiaki Kokubo

Uranium dynamics and developmental sensitivity in rat kidney

Renal toxicity is the principal health concern after uranium exposure. Children are particularly vulnerable to uranium exposure; with contact with depleted uranium in war zones or groundwater contamination the most likely exposure scenarios. To investigate renal sensitivity to uranium exposure during development, we examined uranium distribution and uranium‐induced apoptosis in the kidneys of neonate (7‐day‐old), prepubertal (25‐day‐old) and adult (70‐day‐old) male Wistar rats. Mean renal uranium concentrations increased with both age‐at‐exposure and exposure level after subcutaneous administration of uranium acetate (UA) (0.1–2 mg kg–1 body weight). Although less of the injected uranium was deposited in the kidneys of the two younger rat groups, the proportion of the peak uranium content remaining in the kidneys after 2 weeks declined with age‐at‐exposure, suggesting reduced clearance in younger animals. In situ high‐energy synchrotron radiation X‐ray fluorescence analysis revealed site‐specific accumulation of uranium in the S3 segment of the proximal tubules, distributed in the inner cortex and outer stripe of the outer medulla. Apoptosis and cell loss in the proximal tubules increased with age‐at‐exposure to 0.5 mg kg–1 UA. Surprisingly, prepubertal rats were uniquely sensitive to uranium‐induced lethality from the higher exposure levels. Observations of increased apoptosis in generating/re‐generating tubules particularly in prepubertal rats could help to explain their high mortality rate. Together, our findings suggest that age‐at‐exposure and exposure level are important parameters for uranium toxicity; uranium tends to persist in developing kidneys after low‐level exposures, although renal toxicity is more pronounced in adults. Copyright

Functional analysis of lysosomes during mouse preimplantation embryo development.

Abstract Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

Disruption of Aspm causes microcephaly with abnormal neuronal differentiation

AIMS A number of ASPM mutations have been detected in primary microcephaly patients. In order to evaluate the function of ASPM in brain development, we generated model animals of human autosomal recessive primary microcephaly-5 (MCPH5). METHODS In the Aspm knock-out mice, the exon 2-3 of the Aspm gene was encompassed by a pair of loxP signals so that cre-recombinase activity switched the allele from wild-type to null zygotes as frequently, as expected from the Mendelian inheritance. We precisely analyzed the brains of adults and fetuses using immunohistochemistry and morphometry. RESULTS The adult brains of the Aspm(-/-) mice were smaller, especially in the cerebrum. In the barrel field of the somatosensory cortex, layer I was significantly thicker, whereas layer VI was significantly thinner in Aspm(-/-) mice, compared with Aspm(+/+) mice. The total number of cells and the thickness of the cortical plate at embryonic day 16.5 was significantly decreased in Aspm(-/-) mice, compared with Aspm(+/+) mice. Furthermore, the expression of transcription factors, such as Tbr1 and Satb2, was significantly increased in the subplate of the Aspm(-/-) mice. CONCLUSIONS The results suggested that Aspm is essential to the proliferation and differentiation of neural stem/progenitor cells. The Aspm gene loss model provided a novel pathogenetic insight into acquired microcephaly, which can be caused by in utero exposure to both known and unknown teratogens.

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.

Cancer prevention by adult-onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice

Children are especially sensitive to ionizing radiation and chemical carcinogens, and limiting their cancer risk is of great public concern. Calorie restriction (CR) is a potent intervention for suppressing cancer. However, CR is generally not appropriate for children. This study, therefore, examined to see if adult‐onset CR influences the lifetime cancer risk in mice after early‐life exposure to ionizing radiation. Infant male mice (1‐week‐old) were exposed to 3.8 Gy X‐rays, fed a control 95 kcal/week or CR 65 kcal/week diet from 7 weeks of age (adult stage), and their lifespan and tumor development were assessed. Irrespective of CR, X‐rays shortened lifespan by 38%, and irrespective of irradiation CR extended lifespan by 20%. Thymic lymphoma (TL) and early‐occurring non‐TL were induced by radiation. The liver and Harderian gland were more susceptible to radiation‐induced tumors than the lungs and non‐thymic lymphoid tissues (late occurring). CR reduced the risk of hepatocellular carcinoma, late‐occurring non‐TL, lung tumor, Harderian tumor, and hemangioma but had less impact on TL and early‐occurring non‐TL. Most notably, the effects of X‐rays on induction of lung tumors, late‐occurring non‐TL and hemangioma were essentially canceled by CR. The ability of CR to prevent late‐occurring tumors was the same for non‐irradiated and irradiated mice, indicating that the mechanism by which CR influences cancer is independent of irradiation. Our results indicate that adult‐onset CR significantly inhibits late‐occurring tumors in a tissue‐dependent manner regardless of infant radiation exposure.

Influence of Age on the Relative Biological Effectiveness of Carbon Ion Radiation for Induction of Rat Mammary Carcinoma

PURPOSE The risk of developing secondary cancer after radiotherapy, especially after treatment of childhood cancers, remains a matter of concern. The high biological effects of carbon-ion radiation have enabled powerful radiotherapy, yet the approach is commonly restricted to the treatment of adults. Susceptibility of the fetus to particle radiation-induced cancer is also unclear. The present study is aimed to investigate the effect of carbon-ion irradiation in childhood on breast carcinogenesis. METHODS AND MATERIALS We irradiated female Sprague-Dawley rats of various ages (embryonic days 3, 13, and 17 and 1, 3, 7, and 15 weeks after birth) with (137)Cs γ rays or a 290-MeV/u monoenergetic carbonion beam (linear energy transfer, 13 keV/μm). All animals were screened weekly for mammary carcinoma by palpation until they were 90 weeks old. RESULTS Irradiation of fetal and mature (15-week-old) rats with either radiation source at a dose of 0.2 or 1 Gy did not substantially increase the hazard ratio compared with the nonirradiated group. Dose responses (0.2-2.0 Gy) to γ rays were similar among the groups of rats irradiated 1, 3, and 7 weeks after birth. The effect of carbon ions increased along with the age at the time of irradiation, indicating relative biological effectiveness values of 0.2 (-0.3, 0.7), 1.3 (1.0, 1.6), and 2.8 (1.8, 3.9) (mean and 95% confidence interval) for animals that were 1, 3, and 7 weeks of age, respectively. CONCLUSIONS Our findings imply that carbonion therapy may be associated with a risk of secondary breast cancer in humans, the extent of which may depend on the age of the patient at the time of irradiation.

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

Abstract 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.

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 β‐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 4ʹ‐[methyl‐11C]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.

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

Abstract 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.