Mouse Model for the Biodosimetry Using Quantification of mRNA for DNA-damage Induced Genes in Peripheral White Blood Cells

Biodosimetry by the measurement of chromosomal damage in lymphocyte has been established. Although the principle is useful to estimate high-dose exposure, expansion of the method to estimate low-dose exposure is difficult. We aimed to develop a biodosimetric method that is suitable for low-dose (around 100 mGy) radiation exosure, and focused on the quantification of RNA in white blood cells. For the future application of the methodology to human blood, basic knowledges were obtained using mouse model. We previously reported increase in the levels of mRNAs for DNA damage-induced (DDI) genes in peripheral blood from whole-body x-irradiated mice (JRR 51, 265-275, 2010). The DDI genes are known to be expressed in radiation-sensitive proliferating cell which is slightly contained in peripheral blood, and it is expected that the relative rates of the level of DDI gene expression per the proliferating cells would show dose-dependency that is necessary for biodosimetry. When bax and puma mRNAs were chose as indexes of DDI gene expression level and c-myc mRNA was used as an index of proliferating cell number, the values of bax/myc and puma/myc rates in peripheral blood were kept low level without significant change among all the mice. When blood were isolated from mice 2h after whole-body x-irradiation in the various doses from 25 to 500mGy, these values of relative rates were increased in exposed dose-dependent manner. Similar dose-dependent increase was also observed in blood samples isolated from 4h after irradiation, but all the values elevated as compared with blood isolated from 2h after irradiation. Thus, records of blood sampling time and predicted irradiation time are essential in this model. No significant change was observed in these values of relative rates among different irradiation experiments using any production batches of mice. This mouse model can be useful not only to study radiation effect in vivo but also to elucidate basic knowledge that is necessary to expand the biodosimetric methodology to human.15th International Congress of Radiation Researc

Hydrogel formulation of Cs-absorbent and laxative drugs for enhancement of decorporation rate of internally contaminated radiocesium

Clinical decorporation procedure using Radiogardase to decrease body-burden of radiocesium caused by to the internal contamination accidents has been established in the last century. We aimed to improve the decorporation treatment to minimize stress of patients as the current tendency. The effects of several drugs were estimated by the measurement of the fecal and urinary excretion rates of radiocesium using mouse model. First, we examined the effect of pharmaceutical modification of the drug formulation of radiocesium-absorbents, because water-insoluble form of absorbents such as Prussian blue is sharp-pointed large crystal and may irritate the mucosa of gastro-intestinal (GI)-tract physically. Crystals of ferrous ferricyanides in the sizes of less than 1 micrometer were embedded in polyvinyl alcohol hydrogel with globular shap in the diameters from 10 to 50 micrometer. They were lyophilized for storage and hydrated just before administration. Though the rude crystal is rapidly excreted within 3h after the intrastomach administration, its hydrogel preparation elongates the retention time in GI-tract up to 6h, suggesting mitigation of irritation in mucosa by the formulation. Simultaneously, the excretion rate of internal radiocesium was increased by the use of the hydrogel formulation. Next, the effects of clinical drugs on the cesium excretion were examined in mouse. No diuretic drugs accelerated urinary excretion rate of internal radiocesium. In contrast, several drugs which has mild laxative action enhance intestinal excretion rate of internal radiocesium. These data show that decorporation rate of intestinal radiocesium can be enhanced by the procedure focusing on internal excretion rate, e.g. the simultaneous use of pharmaceutically formulated absorbents and laxatives.15th International Congress of Radiation Researc

Comparison of substitution velocity of multivalent metallic radionuclide coordinated with serum protein mixture by chelates using in vitro Fe(III) model

Actinoids are transition metallic radionuclides and the most of them are stable in the oxidation state at trivalent and tetravalent. When they penetrate into animal body, they coordinate with endogenous ligand in interstitial (extracellular) fluid, and stagnate in the infiltrated tissue. After the ligand substituted with migrative serum proteins that possess affinity with multivalent metal cation such as transferrin, they are translocated to circulation and dispersed whole body. A chelating agent, diethylenetriaminepentaacetate (DTPA) is used for internal decorporation therapy. The first process of the action mechanism of the chelating agent is that the injected DTPA molecule captures multivalent metal cation from bioorganic ligand. Subsequently, resulted stable chelate coordinated with metal translocates urinary excretion system. However, the practical chelate action is far complicated because the interstitial fluid in the body contains various bioorganic ligands which react with multivalent metal cation and chelate, and the coordination compounds affect on the bind-dissociation reaction each other. The biochemical data on reaction velocity using the interstitial fluid are necessary or more accurate estimation of the effect of chelate treatment on the committed effective dose in organ following internal exposure of tri-/tetravalent radionuclide. We studied metal substitution from serum proteins to chelate using Fe(III) model. 59Fe-bound serum prepared from mouse was treated in physiological condition with DTPA, ethylenediaminetetraacetate, or deferoxamine. The substitution velocity, obtained by the measurement of the amounts of chelate-59Fe and protein-59Fe after the separation by isoelectric focusing electrophoresis, were consistent with decorporation experiments in mice.日本放射線影響学会第63回大

Quantitative comparison of DNA damage by radiation and genotoxic metal compounds using biodosimetric indicators of DNA-damage-induced RNAs and double-strand break in RAW264.7 cells.

We have previously reported that the levels of DNA damage-induced mRNA and lncRNA in blood cells from mouse irradiated with 10mGy class of low-dose ionizing radiation can be used as biodosimetric quantitative indicators of DNA damage, based on conventional molecular biological technique. The principle of the quantification is the measurement of cellular intermediate RNA products which are generated by known and unknown mechanism triggered by various type of DNA damage. The method is suitable for analysis after single and multiple irradiation in relatively short-time. However, method is not suitable for comparison of damage level followed by hours of continuous irradiation, by the low-dose rate effect. We aimed to establish alternative general method to quantitate DNA damage based on different principles that is applicable for low-dose rate irradiation in heterogenous cell population. Several histochemical procedures were compared using a mouse osteoclastoma cell line, RAW264.7. Among them, the quantitative indicator suitable for low dose irradiation was only gamma H2ax, double-strand break-indicator, and we established the universal methodology to quantify damages in the cell population. Several metallic organic compounds have been known to induced DNA damage. However, quantitative study to compare toxicity level between the compounds and ionizing radiation has not well-investigated. We simultaneously quantitated DNA damage-induced RNAs and gamma-H2ax levels in RAW 264.7 cells after treatment with irradiation or metal compounds. The difference in DNA-damage mechanism between radiation and metal compounds reflects the quantitative values of these indicators.日本放射線影響学会第63回大