Hironori Yoshino

Individual Radiation Exposure Dose Due to Support Activities at Safe Shelters in Fukushima Prefecture

Immediately after the accidents in the nuclear power stations in Fukushima on March 11, the Japanese Government ordered the evacuation of the residents within a 20-km radius from the station on March 12, and asked various institutions to monitor the contamination levels of the residents. Hirosaki University, which is located 355 km north of Fukushima City, decided to send support staff to Fukushima. This report summarizes the results of the exposure of 13 individual teams from March 15 to June 20. The support teams surveyed more than 5,000 people during this period. Almost all subjects had external contamination levels of less than 13 kcpm on Geiger-Müller (GM) survey meter, which is categorized as “no contamination level.” The 1st team showed the highest external exposure dose, but the 4th team onward showed no significant change. Subsequently, the internal radiation exposure was measured using a whole body counter that indicated undetectable levels in all staff members. Although the measured external radiation exposure dose cannot have serious biological effects on the health of an individual, a follow-up study of the residents in Fukushima and other regions where the radioactive material has spread will be required for a long time.

Characteristics of Myeloid Differentiation and Maturation Pathway Derived from Human Hematopoietic Stem Cells Exposed to Different Linear Energy Transfer Radiation Types

Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34+ cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34+ cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D 0 = 0.65) than to X-rays (D 0 = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar. In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a+ erythroid-related fraction, whereas carbon-ion beams increased the CD34+CD38− primitive cell fraction and the CD13+CD14+/−CD15− fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface antigen expression by mature myeloid cells derived from HSPCs exposed to each type of radiation was similar to that by controls.

Mitigative Effects of a Combination of Multiple Pharmaceutical Drugs on the Survival of Mice Exposed to Lethal Ionizing Radiation

It is important to establish an easy-to-use therapeutic protocol for the emergency medical care of patients involved in radiation accidents to reduce the radiation-related casualties. The present study aimed to establish an optimum therapeutic protocol using currently approved pharmaceutical drugs to increase the survival of victims exposed to lethal radiation. Different combinations of four drugs-recombinant human erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), c-mpl receptor agonist romiplostim (RP) and nandrolone decanoate (ND)-were administered to mice within 2 h after exposure to a lethal 7 Gy dose of γ-irradiation. On day 30 after irradiation, the condition of the mice was analyzed using various hematological parameters, such as the number of peripheral blood cells, bone marrow cells, hematopoietic progenitor cells and the expression of cell surface antigens. Approximately 10% of the untreated irradiated control mice survived for 21 days, but all of the control mice died by day 30. The combined administration of G-CSF, EPO and RP for five days immediately after irradiation led to a complete survival of the irradiated mice until day 30. However, the treatment with G-CSF, EPO and RP with ND led to only 75% survival at day 30. The hematological analyses showed that the numbers of almost all of hematopoietic cells in the surviving mice treated with effective medications recovered to the levels of non-irradiated mice. The present findings show that the combination of G-CSF, EPO and RP may be a useful countermeasure for victims exposed to accidental lethal irradiation.

Effect of ascorbic acid and X-irradiation on HL-60 human leukemia cells: The kinetics of reactive oxygen species

Ascorbic acid (AsA) treatment is expected to be a potential cancer therapy strategy with few side-effects that can be used alone or in combination with chemotherapy. However, the combination of AsA, a free radical scavenger, with radiation is not clearly understood; conflicting data are reported for cancer cell death. We conducted this study to determine the effect of AsA treatment combined with X-irradiation and the role of reactive oxygen species (ROS) in HL-60 human promyelocytic leukemia cells. Additive cytotoxic effects were observed when the cells were exposed to 2 Gy X-irradiation after 2.5 mM AsA treatment. When catalase was added to the culture with AsA alone, the cytotoxic effects of AsA disappeared. X-irradiation increased intercellular ROS levels and mitochondrial superoxide levels. By contrast, AsA alone and in combination with X-irradiation decreased ROS levels. However, in the presence of catalase neutralizing H2O2, AsA alone or in combination with X-irradiation only slightly decreased the intercellular ROS. Moreover, AsA decreased the mitochondrial membrane potential, which is commonly associated with apoptosis. These results suggest that the reduction of ROS did not result from ROS scavenging by AsA, and AsA induced apoptosis through a ROS-independent pathway. This study reports that a combination of AsA with radiation treatment is effective in cancer therapy when considering ROS in cancer cells.

Effects of X‑ray irradiation in combination with ascorbic acid on tumor control

Our previous studies demonstrated that the combination of treatment with ascorbic acid (AsA) and X‑ray irradiation results in increased apoptosis in HL60 cells. The present study was performed to investigate the effects of the combined use of AsA and X‑ray irradiation on epithelial cancer and sarcoma cells, and its potential use in future clinical treatment. X‑ray irradiation combined with AsA treatment resulted in increased suppression of cell growth of HT1080, SAS and A549 cells in vitro compared with X‑ray irradiation alone. The combined treatment also suppressed tumor growth in implanted HT‑1080 cells in vivo. Using annexin V/propidium iodide staining and the detection of activated caspase 3, it was found that X‑ray irradiation increased the apoptotic rate of HT1080 cells and resulted in G2/M arrest. However, apoptosis in the HT1080 cells treated with 5 mM AsA remained unchanged, and no changes were observed in the G2/M fraction. By contrast, AsA treatment caused increased suppression of proliferation compared with X‑ray irradiation. These results suggested that 5 mM AsA slowed the cell cycle and reduced tumor growth. Therefore, X‑ray irradiation combined with AsA treatment may be effective against epithelial cancer and sarcoma cells.

Synthesis and anticancer properties of phenyl benzoate derivatives possessing a terminal hydroxyl group

To assess the cytotoxic effects on A549 human lung cancer cells, we investigated a liquid-crystalline compound possessing a terminal hydroxyl group at concentrations of 0.1-20 μM. The compound, 4-butylphenyl 4-(6-hydroxyhexyloxy)benzoate (2), showed marked cell-growth inhibition at concentrations higher than 5 μM. Cell accumulation in the Sub-G1 phase indicating apoptosis was observed only at the highest concentration. Dynamic light scattering measurements show that the molecules form a spherical nanoparticle with a diameter of 130-170 nm at concentrations of 5-20 μM. We prepared the corresponding dimeric compounds and investigated their anticancer activity. The 1,2-benzene derivative, 1,2-bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (4), exhibited cell-growth inhibition without affecting the cell cycle. However, the 1,3-benzene derivative, 1,3-bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (5), was found to induce marked cell accumulation in the Sub-G1 phase. Furthermore, we assessed the cytotoxic effects of compounds 2, 4 and 5 on SW480 colon cancer cells and THP1 leukemic cells, as well as on WI-38 normal fibroblast cells. Both compounds 2 and 5 suppressed the growth of the solid cancer cells (A549 and SW480) more strongly compared with that of the hematological cancer cells (THP1). Unexpectedly, they also exhibited a strong cytotoxicity against the normal cells. We discuss the structure-property relationship in the anticancer activity of the mesogenic compounds.

Terminal Maturation of Megakaryocytes and Platelet Production by Hematopoietic Stem Cells Irradiated with Heavy-Ion Beams

Abstract Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to high-linear energy transfer (LET) radiations such as heavy-ion beams that have greater biological effects than low-LET radiation. This study examined the terminal maturation of megakaryocytes and platelet production derived from hematopoietic stem cells irradiated with heavy-ion beams. CD34+ cells derived from human placental/umbilical cord blood were exposed to monoenergetic carbon-ion beams (LET  =  50 keV/µm) and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. There was no significant difference in megakaryocyte-specific markers between nonirradiated control and irradiated cells. Expression of Tie-2, a receptor that acts in early hematopoiesis, showed a significant 1.31-fold increase after 2 Gy irradiation compared to control cells on day 7. There was a significant increase in Tie-2 mRNA expression. In addition, the expression of other mRNAs, such as PECAM1, SELP and CD44, was also significantly increased in cells irradiated with heavy-ion beams. However, the adherent function of platelets derived from the irradiated cells showed no difference from that in the controls. These results clarify that the functions of megakaryocytopoiesis and thrombopoiesis derived from hematopoietic stem/progenitor cells irradiated with heavy-ion beams are similar to those in the unirradiated cells, although heavy-ion beams affect the expression of genes associated with cellular adhesion.

A liquid crystal-related compound induces cell cycle arrest at the G2/M phase and apoptosis in the A549 human non-small cell lung cancer cell line

Liquid crystals are the state of matter existing between liquid and crystalline phases, and recently there has been increasing interest in their biological effects. Following our recently reported work, we investigated the cell suppressive effects of liquid crystal-related compounds (LCRCs), which are precursors of liquid crystals, in the human non-small lung cancer cell line A549. We found that 2-(4-butoxyphenyl)-5-(4-hydroxyphenyl)pyrimidine (LCRC-1) dramatically suppressed cell growth. Treatment with 12 µM LCRC-1 for 12 h induced cell cycle arrest at the G2/M phase. Furthermore, LCRC-1 increased the sub-G1 fraction and Annexin V-positive cells and activated caspase-3 in A549 cells, which showed that it can induce apoptosis in these cells. Furthermore, because the induction of apoptosis by LCRC-1 was partly inhibited by treatment with pan-caspase inhibitor, it appeared that LCRC-1 induced apoptosis by a caspase-dependent pathway. The ability of LCRC-1 to cause DNA damage was assessed, but LCRC-1 did not induce expression of γ-H2AX, which is a marker of DNA damage. Treatment with LCRC-1 did not inhibit the proliferation of WI-38 normal fibroblast cells, which makes the tumor-specific suppressive effect of LCRC-1 attractive for its application as a new antitumor drug.

Impairment of Mature Dendritic Cells Derived from X-Irradiated Human Monocytes Depends on the Type of Maturation Stimulus Used

Dendritic cells play an essential role in the immune system. We have previously reported that X-irradiated monocytes, precursors of dendritic cells, can differentiate into dendritic cells and then mature in terms of surface antigen expression after tumor necrosis factor-α stimulation, but show reduced functionality. Dendritic cells can mature in response to various types of maturation stimuli. Therefore, this study investigated whether dendritic cells from monocytes exposed to ionizing radiation can adequately respond to pathogen-derived components and proinflammatory cytokines. Human monocytes separated from buffy coats were exposed to X rays, and were then differentiated into immature dendritic cells. Immature dendritic cells were stimulated by lipopolysaccharide (LPS) or proinflammatory cytokine mixture. The dendritic cells from nonirradiated and X-irradiated monocytes showed maturation after LPS and proinflammatory cytokine mixture stimulation as confirmed by findings of surface antigen expression. Upon LPS stimulation, however, the expression levels of CD80 and CD83 on dendritic cells from X-irradiated monocytes were lower than those of dendritic cells from nonirradiated monocytes. Such reductions were not observed upon proinflammatory cytokine mixture stimulation. Similarly, an impairment of matrix metalloproteinase-9 and cytokine production was observed in LPS-stimulated dendritic cells from X-irradiated monocytes, whereas these impairments were not observed upon proinflammatory cytokine mixture stimulation. The ability of dendritic cells to stimulate T cells was lower in the irradiated group compared with the nonirradiated group despite the type of maturation stimulus. Thus, the present study suggests that the influence of X irradiation on the maturation of dendritic cells depends on the type of maturation stimulus used and that X irradiation impairs the response of dendritic cells to LPS.

Involvement of reactive oxygen species in ionizing radiation–induced upregulation of cell surface Toll-like receptor 2 and 4 expression in human monocytic cells

Abstract Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns and are indispensable for antibacterial and antiviral immunity. Our previous report showed that ionizing radiation increases the cell surface expressions of TLR2 and TLR4 and enhances their responses to agonists in human monocytic THP1 cells. The present study investigated how ionizing radiation increases the cell surface expressions of TLR2 and TLR4 in THP1 cells. The THP1 cells treated or not treated with pharmaceutical agents such as cycloheximide and N-acetyl-L-cysteine (NAC) were exposed to X-ray irradiation, following which the expressions of TLRs and mitogen-activated protein kinase were analyzed. X-ray irradiation increased the mRNA expressions of TLR2 and TLR4, and treatment with a protein synthesis inhibitor cycloheximide abolished the radiation-induced upregulation of their cell surface expressions. These results indicate that radiation increased those receptors through de novo protein synthesis. Furthermore, treatment with an antioxidant NAC suppressed not only the radiation-induced upregulation of cell surface expressions of TLR2 and TLR4, but also the radiation-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Since it has been shown that the inhibitor for JNK can suppress the radiation-induced upregulation of TLR expression, the present results suggest that ionizing radiation increased the cell surface expressions of TLR2 and TLR4 through reactive oxygen species–mediated JNK activation.