Nobuhiko Takai

Involvement of caspase 3-like protease in methylmercury-induced apoptosis of primary cultured rat cerebral microglia.

Methylmercury (MeHg) has been implicated to induce massive neurodegeneration by disruption of neuron-glia interactions besides a direct potent neurotoxicity. In the present study, we examined potential cytotoxic effects of MeHg on primary cultured rat microglia. Following treatment with a relatively low concentration (0.5 microM) of MeHg, microglia had induced cell death accompanied by DNA fragmentation and an activation of caspase-3-like protease. MeHg-induced microglial death was significantly suppressed by the caspase-3-like protease inhibitor benzyloxycarbonyl-Try-Val-Ala-Asp-fluoromethyl-ketone indicating the occurrence of caspase-3-like protease-executed apoptosis. The aspartic protease inhibitor pepstatin A had a partial but significant inhibitory effect on MeHg-induced microglial apoptosis. These results indicate that a relatively low concentration of MeHg predominantly induces caspase-3-like protease-executed apoptosis of microglia, while the endosomal/lysosomal system is also partially involved in the cell death pathway.

Comparison of Biological Effectiveness of Carbon-Ion Beams in Japan and Germany

PURPOSE To compare the biological effectiveness of 290 MeV/amu carbon-ion beams in Chiba, Japan and in Darmstadt, Germany, given that different methods for beam delivery are used for each. METHODS AND MATERIALS Murine small intestine and human salivary gland tumor (HSG) cells exponentially growing in vitro were irradiated with 6-cm width of spread-out Bragg peaks (SOBPs) adjusted to achieve nearly identical beam depth-dose profiles at the Heavy-Ion Medical Accelerator in Chiba, and the SchwerIonen Synchrotron in Darmstadt. Cell kill efficiencies of carbon ions were measured by colony formation for HSG cells and jejunum crypts survival in mice. Cobalt-60 gamma rays were used as the reference radiation. Isoeffective doses at given survivals were used for relative biological effectiveness (RBE) calculations and interinstitutional comparisons. RESULTS Isoeffective D(10) doses (mean +/- standard deviation) of HSG cells ranged from 2.37 +/- 0.14 Gy to 3.47 +/- 0.19 Gy for Chiba and from 2.31 +/- 0.11 Gy to 3.66 +/- 0.17 Gy for Darmstadt. Isoeffective D(10) doses of gut crypts after single doses ranged from 8.25 +/- 0.17 Gy to 10.32 +/- 0.14 Gy for Chiba and from 8.27 +/- 0.10 Gy to 10.27 +/- 0.27 Gy for Darmstadt, whereas isoeffective D(30) doses after three fractionated doses were 9.89 +/- 0.17 Gy through 13.70 +/- 0.54 Gy and 10.14 +/- 0.20 Gy through 13.30 +/- 0.41 Gy for Chiba and Darmstadt, respectively. Overall difference of RBE between the two facilities was 0-5% or 3-7% for gut crypt survival or HSG cell kill, respectively. CONCLUSION The carbon-ion beams at the National Institute of Radiological Sciences in Chiba, Japan and the Gesellschaft für Schwerionenforschung in Darmstadt, Germany are biologically identical after single and daily fractionated irradiation.

Dose–response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions

Purpose : To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (13 keV/ μ m, 80keV/ μ m), silicon (55 keV/ μ m) and iron (140 keV/ μ m, 185keV/ μ m, 440keV/ μ m) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. Results : The dose-response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80 keV/ μ m and decreasing at higher LET. The dose-response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for γ-rays and 13 keV/ μ m carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13 keV/ μ m (about 7) and 80 keV/ μ m carbon (about 71), and decreased gradually until 440keV/ μ m iron ions (about 66). Conclusions : High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.

Hypoxia-ischemic insult in neonatal rats induced slowly progressive brain damage related to memory impairment

The present study was designed to determine potential associations between the brain damage induced by hypoxic-ischemic (HI) insult and spatial learning impairment in an eight-arm radial maze task. We first determined the pathological outcomes after 2, 5, 9, and 17 weeks of recovery following the HI insult. The results show that the brain damage progressed from 2 up to 17 weeks of recovery. To clarify the time course of the brain damage changes, we investigated the histological changes of the same individual with magnetic resonance imaging (MRI) after 5, 9, and 57 weeks of recovery following the HI insult. The MRI changes were similar to the histological changes, and the brain damages were exacerbated in the contralateral hemisphere after 57 weeks of recovery following the HI insult. To investigate whether alteration in brain function was correlated with MRI and histological changes, the rats were made to find their way through an eight-arm radial maze was performed at either 7th or 16th weeks of recovery. According to the results, the spatial learning impairments of rats in the maze starting at 16 weeks of recovery were more severe than those at 7 weeks of recovery, indicating that the impairments were progressive and depended on the degree of brain damage. The results of the present study are the first demonstration that the evolutional and specific brain damage following the HI insult is slowly and progressively exacerbated to the contralateral hemisphere and rats who experience the HI are at risk for showing a late impairment of brain function.

Hyperexcitability of amygdala neurons of Senescence-Accelerated Mouse revealed by electrical and optical recordings in an in vitro slice preparation

In the amygdala (AMG) slices obtained from both the young (4-7 months old) and aged (17-20 months old) groups of Senescence-Accelerated Mouse (SAM) P10, spontaneous bursts were recorded in the medial, central and basolateral nuclei. The spontaneous bursts were also observed in the slices from the young group of SAMR1, whereas the mean frequency was significantly lower than that from the young group of SAMP10. The spontaneous burst was barely detectable in slices from the aged group of SAMR1 during perfusing with the standard solution, while bicuculline methiodide (10 microM), a GABAA receptor antagonist, or Mg2+-free solution induced a similar bursting activity observed in the young group. The burst response was also evoked in the medial, central and basolateral AMG following stimulation of the stria terminalis (ST). Both spontaneous and evoked bursts were completely suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 4 microM), an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, but not by (+)-5-methyl-10, 11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801, 30 microM), an N-methyl-d-aspartate receptor antagonist. The hyperexcitability of the AMG neurons was further substantiated by optical recordings. Following stimulation of the ST, the optical signals reflected postsynaptic responses spread into the medial and central AMG areas at 2-5 ms and faded out at 20-30 ms after stimulation. The intensity of the optical signal recorded in the slice from the young SAMP10 was significantly higher than that from SAMR1 or ddY mice. These observations indicate that bursts mediated by AMPA/kainate receptors were transiently generated in the AMG of SAMR1 at the young age, while the bursts with higher frequency were continuously generated in the AMG of SAMP10. The chronic neuronal hyperactivity in the AMG may be partially responsible for the age-related deterioration of memory and learning abilities observed in SAMP10.

An increase in [3H]QNB binding by proton-beam irradiation in intact rat brain: an apparent positive cooperativity of binding.

After focal irradiation of rat brains with a beam of proton (dose, 30 Gy), [3H]quinuclidinyl benzilate (QNB) binding, both in vitro and in vivo, was measured, using either autoradiographic or tissue-dissection methods. No changes in in vitro [3H]QNB binding were seen in autoradiograms of brain slices from irradiated rat. The irradiated side of the brain showed a significant increase in [3H]QNB binding in vivo in the striatum and cerebral cortex 24 h after irradiation. This increase in binding was transient, and had almost disappeared 2 weeks after irradiation. These results indicate that early changes in receptor function as a results of radiation damage are only detected in in-vivo conditions. In the striatum of the intact rat brain, an apparent positive cooperativity of binding was observed, which was more pronounced on the irradiated side.

Localized radiation necrosis model in mouse brain using proton ion beams

Brain radiation necrosis is the most serious late adverse event that occurs after 6 months following radiation therapy. Effective treatment for this irreversible brain necrosis has not been established yet. This study tries to establish brain radiation necrosis mouse model using proton or helium beam. The right cerebral hemispheres of C57BL/6J mouse brains were irradiated at doses of 40, 50, 60 Gy with charged particles. In 60 Gy group, brain necrosis that recapitulates human disease was detected after 8 months.

Distinct different intra-tumor distribution of FDG between early phase and late phase in mouse fibrosarcoma

An early image of intra-tumor distribution of14C-labeled fluorodeoxy glucose (14C-FDG) was compared with a late image of18F-labeled FDG (18F-FDG) using mouse fibrosarcoma. Heterogeneous intra-tumor distribution of14C-FDG was observed 1 minute post injection of the tracer, whereas relatively homogeneous distribution of18F-FDG was seen 30 minutes later.14C-FDG was particularly taken up in the peripheral part of the tumor immediately after the tracer injection. A gradual and significant increase in18F-FDG accumulation with time was seen in the central part of tumor, which indicated an enhancement of anaerobic glycolysis. An initial uptake of18F-FDG was also compared with distribution of14C-iodoantipyrine and14C-thymidine uptake. Intratumoral distribution of initial uptake of18F-FDG showed almost the same regional distribution of14C-iodoantipyrine. A similar distribution of14C-thymidine as the initial uptake of18F-FDG was also observed. These results indicated that a high initial FDG uptake area seemed to be highly proliferative. A significant difference in the intratumoral distribution of FDG between early phase and late phase seemed to be related to heterogeneous biological characteristics of tumor cells.

Radioprotective activities of beer administration for radiation-induced acute toxicity in mice

BACKGROUND AND PURPOSE We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collected and irradiated in vitro. In this study, we investigated the radioprotective activities of beer-administration for bone marrow and intestine in mice. METHODS C3H/He female mice received an oral administration of beer, ethanol or saline at a dose of 1 ml/mouse 30 min before whole body irradiation with 137Cs gamma rays or LET 50 keV/microm carbon ions. Radioprotective activities were estimated using a LD(50/30) (The dose required to kill 50% of the mice within 30 days) and a microcolony technique for intestine. RESULTS The LD(50/30) for the beer-administered mice was significantly increased in comparison with saline administered mice. The LD(50/30) of gamma-ray was 7.8 Gy (p < 0.05), 7.6 Gy and 7.3 Gy for beer-, ethanol- and saline-administered group, respectively. The LD(50/30) of carbon ions was 6.6 Gy (p < 0.05), 6.2 Gy and 5.9 Gy for the beer-, ethanol- and saline-administered groups, respectively. The crypt survivals that were semi-logarithmically plotted against dose were well fitted to a linear regression line. The dose reduction factor (DRF) (D10) of beer- and ethanol-administered mice for gamma rays was 1.09 and 1.08, respectively. The DRF (D10) of beer- and ethanol-administered mice for carbon ions was 1.08 and 1.07, respectively. CONCLUSIONS The radioprotection by beer-administration is due to not only OH radical-scavenge action by the ethanol contained in beer.

Intracellular reactions affecting 2-amino-4-([11C]methylthio)butyric acid ([11C]methionine) response to carbon ion radiotherapy in C10 glioma cells

PURPOSE The response of 2-amino-4-([(14)C]methylthio)butyric acid ([(14)C]Met) uptake and [(125)I]3-iodo-alpha-methyl-l-tyrosine ([(125)I]IMT) uptake to radiotherapy of C10 glioma cells was compared to elucidate the intracellular reactions that affect the response of 2-amino-4-([(11)C]methylthio)butyric acid ([(11)C]Met) uptake to radiotherapy. METHODS After irradiation of cultured (3 Gy) or xenografted C10 glioma cells (25 Gy) using a carbon ion beam, the accumulation of [(14)C]Met and [(125)I]IMT in the tumors was investigated. The radiometabolites in xenografted tumors after radiotherapy were analyzed by size-exclusion HPLC. RESULTS [(14)C]Met provided earlier responses to the carbon ion beam irradiation than [(125)I]IMT in both cultured and xenografted tumors. While [(125)I]IMT remained intact in xenografted tumor before and after irradiation, the radioactivity derived from [(14)C]Met was observed both in high molecular fractions and intact fractions, and the former decreased after irradiation. CONCLUSION The earlier response of [(11)C]Met uptake to tumor radiotherapy could be attributable to the decline in the intracellular energy-dependent reactions of tumors due to radiotherapy.