High-energy SR-XRF Imaging of Cesium and Trace Elements in Mouse Kidneys: Short Communication.

For understanding trace element dynamics in tissues, methods for analyzing elemental distribution and localization without destroying tissue structures and cell arrangements are desired. Synchrotron radiation X-ray fluorescence (SR-XRF) analysis is one of the non-destructive and multi-element simultaneous analyses. The kidney is the major excretion pathway of cesium (Cs) taken into the body, and an understanding of cesium distribution in the kidney would be useful for establishing technology to facilitate the excretion of radioactive Cs from the body due to nuclear disasters. In the present study, the distribution of cesium and trace elements, such as iron (Fe) and zinc (Zn), corresponding to the kidney structure was examined in Cs-administered mice by SR-XRF imaging with high-energy excitation X-rays (40 keV). By beam scanning with a 200-µm square beam, clear Cs images corresponding to the renal layer structure were obtained for the renal specimen at the early phase after Cs administration with the mean renal Cs concentration of 24.1 ± 3.2 µg/g. Cs was distributed mainly in the medulla and the outer stripe of the outer medulla located in the center area of the kidney. Unlike the Cs distribution, endogenous Fe and Zn tended to be lower in the medulla than in the outer stripe of the outer medulla and the cortex. This method is effective for analyzing Cs distribution because it can simultaneously analyze the distribution of endogenous trace elements.

Uranium chelating ability of decorporation agents in serum evaluated by X-ray absorption spectroscopy.

Internal exposure to actinides such as uranium and plutonium has been reduced using chelating agents for decorporation because of their potential to induce both radiological and chemical toxicities. This study measures uranium chemical forms in serum in the presence and absence of chelating agents based on X-ray absorption spectroscopy (XAS). The chelating agents used were 1-hydroxyethane 1,1-bisphosphonate (EHBP), inositol hexaphosphate (IP6), deferoxamine B (DFO), and diethylenetriaminepentaacetate (DTPA). Percentages of uranium-chelating agents and uranium-bioligands (bioligands: inorganic and organic ligands coordinating with uranium) dissolving in the serum were successfully evaluated based on principal component analysis of XAS spectra. The main ligands forming complexes with uranium in the serum were estimated as follows: IP6 > EHBP > bioligands > DFO ≫ DTPA when the concentration ratio of the chelating agent to uranium was 10. Measurements of uranium chemical forms and their concentrations in the serum would be useful for the appropriate treatment using chelating agents for the decorporation of uranium.

Comparison of Absorbents and Drugs for Internal Decorporation of Radiocesium: Advances of Polyvinyl Alcohol Hydrogel Microsphere Preparations Containing Magnetite and Prussian Blue.

Radiocesium nuclides, used as a gamma ray source in various types of industrial equipments and found in nuclear waste, are strictly controlled to avoid their leakage into the environment. When large amounts of radiocesium are accidentally incorporated into the human body, decorporation therapy should be considered. Although standard decorporation methods have been studied since the 1960s and were established in the 1970s with the drug Radiogardase(®) (a Prussian blue preparation), application of recent advances in pharmacokinetics and ethical standards could improve these methods. Here we designed a modern dosage form of hydrogel containing cesium-absorbents to alleviate intestinal mucosa irritation due to the cesium-binding capacity of the absorbents. The effectiveness of the dosage form on fecal excretion was confirmed by quantitative mouse experiments. The total cesium excretion rate of the crystal form (1.37±0.09) was improved by the hydrogel form (1.52±0.10) at the same dose of Prussian blue, with a longer gastrointestinal tract transit time. Using a mouse model, we compared the effects of several drugs on fecal and urinary excretion of internal cesium, without the use of absorbents. Only phenylephrine hydrochloride significantly enhanced cesium excretion (excretion rate of 1.17±0.08) via the urinary pathway, whereas none of the diuretic drugs tested had this effect. These findings indicate that modifying the dosage form of cesium absorbents is important for the decorporation of internal radiocesium contamination.

Quantification of damage due to low-dose radiation exposure in mice: construction and application of a biodosimetric model using mRNA indicators in circulating white blood cells.

Biodosimetry, the measurement of radiation damage in a biologic sample, is a reliable tool for increasing the accuracy of dose estimation. Although established chromosome analyses are suitable for estimating the absorbed dose after high-dose irradiation, biodosimetric methodology to measure damage following low-dose exposure is underdeveloped. RNA analysis of circulating blood containing radiation-sensitive cells is a candidate biodosimetry method. Here we quantified RNA from a small amount of blood isolated from mice following low-dose body irradiation (<0.5 Gy) aimed at developing biodosimetric tools for situations that are difficult to study in humans. By focusing on radiation-sensitive undifferentiated cells in the blood based on Myc RNA expression, we quantified the relative levels of RNA for DNA damage-induced (DDI) genes, such as Bax, Bbc3 and Cdkn1a. The RNA ratios of DDI genes/Myc in the blood increased in a dose-dependent manner 4 h after whole-body irradiation at doses ranging from 0.1 to 0.5 Gy (air-kerma) of X-rays, regardless of whether the mice were in an active or resting state. The RNA ratios were significantly increased after 0.014 Gy (air-kerma) of single X-ray irradiation. The RNA ratios were directly proportional to the absorbed doses in water ranging from 0.1 to 0.5 Gy, based on gamma-irradiation from (137)Cs. Four hours after continuous irradiation with gamma-rays or by internal contamination with a beta-emitter, the increased RNA ratios resembled those following single irradiation. These findings indicate that the RNA status can be utilized as a biodosimetric tool to estimate low-dose radiation when focusing on undifferentiated cells in blood.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Pharmaceutical drugs supporting regeneration of small-intestinal mucosa severely damaged by ionizing radiation in mice.

Accidental exposure of the abdomen to high-dose radiation leads to severe consequences initiated by disruption of the mucosa in the small intestine. Therapeutic options are limited, even though various treatments have been investigated, particularly in the field of regenerative therapy. In order to identify readily available treatment methods, we included several current pharmaceutical drugs, for which the clinical trials have already been completed, in tests on mice that had undergone severe mucosal damage by radiation. The drugs were injected into mice 24 h after exposure to 15.7 Gy X-rays. The effects of the drugs on the damaged mucosa of the small intestine were evaluated using early regeneration indices [the expression of c-myb mRNA, and proliferation of epithelial cells in the form of microcolonies (MCs) by Days 4 and 5 post-irradiation] and the survival rate of the mice. Enhancement of mucosal regeneration at Day 4 (c-myb: P < 0.01, MC: P < 0.05) and improvement of the survival rate (P < 0.05) were observed when a clinical dose of gonadotropin, a stimulator of androgen, was injected. Similarly, a clinical dose of thiamazole (which prevents secretion of thyroid hormone) stimulated mucosal growth by Day 5 (c-myb: P < 0.01, MC: P < 0.05) and also improved the survival rate (P < 0.05). The nonclinical drugs histamine and high-dose octreotide (a growth hormone antagonist) also gave significant survival-enhancing benefits (P < 0.01 and P < 0.05, respectively). These results can be used to construct therapeutic programs and applied in various experimental studies to control the regeneration of damaged mucosa.

Acceleration of regeneration of mucosa in small intestine damaged by ionizing radiation using anabolic steroids.

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification.

Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time RT-PCR and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per GAPDH also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation.

Modification of nitroxyl contrast agents with multiple spins and their proton T(1) relaxivity.

The purpose of this study is to test the performance of multispin nitroxyl contrast agents in improving the sensitivity of MR detection for nitroxyl contrast agents. The relation between T(1) relaxivity and the number of paramagnetic centers in a molecule was investigated. Compound 1 is a single molecule of methoxycarbonyl-PROXYL (MC-PROXYL). Two and three MC-PROXYL molecules were chemically coupled to obtain Compounds 2 and 3, which have two and three nitroxyl spins in the molecule, respectively. A good linear relation, the slope of which increased depending on the number of nitroxyl spins in the molecule, was obtained between T(1)-weighted (fast low-angle shot) MR image contrast enhancement at 7 T and the concentration of nitroxyl contrast agents. T(1)-weighted MR image contrast enhancement and T(1) relaxivity levels of nitroxyl contrast agents were increased depending on the number of nitroxyl spins in the molecule. Multicoupling nitroxyl molecules can enhance the T(1)-weighted contrast effect while maintaining the quantitative behavior of the molecule for up to three spins.

Nitroxyl radicals: electrochemical redox behaviour and structure-activity relationships.

Comparative study of electrochemical redox behaviour of five different nitroxyl radicals leads to the direct correlation between one-electron redox potentials and group electronegativity of the beta-substituent on the ring. Beta-substituents with an electron-donating effect caused a negative shift in the one-electron oxidation and one-electron reduction potentials of the nitroxyl radicals. In a similar aspect, beta-substituents with an electron-withdrawing effect behaved oppositely.

Drastic effect of several caffeic acid derivatives on the induction of heme oxygenase-1 expression revealed by quantitative real-time RT-PCR.

Among antioxidative polyphenols, caffeic acid esters such as caffeic acid phenethyl ester (CAPE) and chlorogenic acid are contained in propolis, vegetables and coffee. In this study, we compared the efficacy of some polyphenols on the activation level of a cytoprotective heme oxygenase-1 (HO-1) gene in RAW264.7 mouse macrophage cells using quantitative real-time RT-PCR. The quantitative study revealed a variety of activation level of HO-1 gene by the chemicals. CAPE and caffeic acid ethyl ester (CAEE) at the final concentration of 2 muM drastically activated the HO-1 gene to 39.2-fold and 20.1-fold, respectively. Curcumin, structurally related with caffeic acid and an element of turmeric, induced the HO-1 gene to 5.8-fold. In contrast, no activation was observed by other caffeic acid esters such as chlorogenic acid and rosmarinic acid. Higher concentrations were necessary for the activation by an antioxidant cysteamine and the electrophile diethyl maleate. Although the inducible activities of CAPE and chlorogenic acid were distinctly different, they showed similar reductive capacities when determined by cyclic voltammetry. These results show that the drastic activation of HO-1 gene by CAPE and CAEE is dependent upon their chemical structures, rather than the reductive activity of polyphenols, possibly reflecting the physiological effects of the nutritional elements.

Efficient radical scavenging ability of artepillin C, a major component of Brazilian propolis, and the mechanism.

Hydrogen transfer from artepillin C to cumylperoxyl radical proceeds via one-step hydrogen atom transfer rather than via electron transfer, the rate constant of which is comparable to that of (+)-catechin, indicating that artepillin C can act as an efficient antioxidant.