Shin Ohtani

The in vivo Pig-a gene mutation assay is useful for evaluating the genotoxicity of ionizing radiation in mice

The in vivo Pig‐a mutation assay has been adapted for measuring mutation in rats, mice, monkeys, and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals that are known to be powerful point mutagens. The assay has not been used to measure the biological effects associated with ionizing radiation. In this study, we modified the Pig‐a gene mutation assay (Kimoto et al. [2011b]: Mutat Res 723:36‐42) and used 3‐color staining with fluorescently labeled anti‐CD24, anti‐TER‐119, and anti‐CD71 to detect the Pig‐a mutant frequencies in total red blood cells (RBCs) and in reticulocytes (RETs) from X‐irradiated mice. Single exposures to X‐irradiation resulted in dose‐ and time‐dependent increases in Pig‐a mutant frequencies, and these subsequently declined over time returning to background frequencies. The same total amount of radiation, delivered either as a single dose or as four repeat doses at weekly intervals, increased Pig‐a mutant frequencies to comparable levels, reaching maxima 2–3 weeks after the single dose or 2–3 weeks after the last of the repeat doses. These increased frequencies subsequently returned to background levels. Our results indicated that the 3‐color Pig‐a assay was useful for evaluating the in vivo genotoxicity of radiation. Environ. Mol. Mutagen., 2012.

Effects of 21-kHz intermediate frequency magnetic fields on blood properties and immune systems of juvenile rats

Abstract Purpose: Due to a lack of science-based evidence, we explored the effects of exposure to intermediate frequency magnetic fields (IF-MF) on experimental animals. We assessed several immunological parameters to determine the effect of exposure of the whole body to IF-MF. Materials and methods: Male Sprague-Dawley rats (4–5 weeks old) were divided into three groups: Cage-control, sham, and 3.8-mT (rms) exposure groups. The animals were exposed to IF-MF at 21 kHz under fixed conditions in an acrylic holder. Exposure was performed for 1 h/day for 14 consecutive days. On the 15th day following the exposure, biochemical and hematological parameters in blood were analyzed. The effects of the exposure on immunological functions such as the cytotoxic activity of lymphocytes, chemotactic and phagocytic activity of granulocytes, and T (cluster of differentiation 4 [CD4] and cluster of differentiation 8 [CD8])-cell frequency were also examined. Results: Hematological parameters were not affected by IF-MF exposure. Other immune functions such as the cytotoxic activity and phagocytic activity were not affected. Populations of T cells after exposure also did not show any significant differences. In blood biochemistry, there was significant difference in inorganic phosphorus level between sham and exposure group. However, this will not induce any pathophysiological status, because they were still within physiological range. Overall, no significant effect by exposure of IF-MF was observed under our experimental conditions. Conclusions: Our results suggest that exposure to 21-kHz sinusoidal IF-MF at 3.8 mT for 1 h/day for 14 days did not affect immune function in juvenile rats.

Early Exposure to Intermediate-Frequency Magnetic Fields Alters Brain Biomarkers without Histopathological Changes in Adult Mice

Recently we have reported that intermediate-frequency magnetic field (IF-MF) exposure transiently altered the mRNA expression levels of memory function-related genes in the hippocampi of adult male mice. However, the effects of IF-MF exposure during brain development on neurological biomarkers have not yet been clarified. In the present study, we investigated the effect of IF-MF exposure during development on neurological and immunological markers in the mouse hippocampus in 3- and 7-week-old male mice. Pregnant C57BL/6J mice were exposed to IF-MF (21 kHz, 3.8 mT) for one hour per day from organogenesis period day 7 to 17. At adolescence, some IF-MF-exposed mice were further divided into exposure, recovery, and sham-exposure groups. The adolescent-exposure groups were exposed again to IF-MF from postnatal day 27 to 48. The expression of mRNA in the hippocampi was examined using a real-time RT-PCR method, and microglia activation was examined by immunohistochemical analysis. The expression levels of NR1 and NR2B as well as transcription factors (CaMKIV, CREB1), inflammatory mediators (COX2, IL-1 β,TNF-α), and the oxidative stress marker heme-oxygenase (HO)-1 were significantly increased in the IF-MF-exposed mice, compared with the control group, in the 7-week-old mice, but not in the 3-week-old mice. Microglia activation was not different between the control and other groups. This study provides the first evidence that early exposure to IF-MF reversibly affects the NMDA receptor, its related signaling pathways, and inflammatory mediators in the hippocampus of young adult mice; these changes are transient and recover after termination of exposure without histopathological changes.

The effects of radio-frequency electromagnetic fields on T cell function during development

With the widespread use of radio-frequency devices, it is increasingly important to understand the biological effects of the associated electromagnetic fields. Thus, we investigated the effects of radio-frequency electromagnetic fields (RF-EMF) on T cell responses during development due to the lack of science-based evidence for RF-EMF effects on developmental immune systems. Sprague Dawley (SD) rats were exposed to 2.14-GHz wideband code division multiple-access (W-CDMA) RF signals at a whole-body specific absorption rate (SAR) of 0.2 W/kg. Exposures were performed for a total of 9 weeks spanning in utero development, lactation and the juvenile period. Rats were continuously exposed to RF-EMF for 20 h/day, 7 days/week. Comparisons of control and exposed rats using flow cytometry revealed no changes in the numbers of CD4/CD8 T cells, activated T cells or regulatory T cells among peripheral blood cells, splenocytes and thymocytes. Expression levels of 16 genes that regulate the immunological Th1/Th2 paradigm were analyzed using real-time PCR in the spleen and thymus tissues of control and RF-EMF–exposed rats. Although only the Il5 gene was significantly regulated in spleen tissues, Il4, Il5 and Il23a genes were significantly upregulated in thymus tissues following exposure to RF-EMF. However, ELISAs showed no changes in serum IL-4 protein concentrations. These data indicate no adverse effects of long-term RF-EMF exposure on immune-like T cell populations, T cell activation, or Th1/Th2 balance in developing rats, although significant transcriptional effects were observed.

Exposure time-dependent thermal effects of radiofrequency electromagnetic field exposure on the whole body of rats

We investigated the thermal effects of radiofrequency electromagnetic fields (RF-EMFs) on the variation in core temperature and gene expression of some stress markers in rats. Sprague-Dawley rats were exposed to 2.14 GHz wideband code division multiple access (W-CDMA) RF signals at a whole-body averaged specific absorption rate (WBA-SAR) of 4 W/kg, which causes behavioral disruption in laboratory animals, and 0.4 W/kg, which is the limit for the occupational exposure set by the International Commission on Non-Ionizing Radiation Protection guideline. It is important to understand the possible in vivo effects derived from RF-EMF exposures at these intensities. Because of inadequate data on real-time core temperature analyses using free-moving animal and the association between stress and thermal effects of RF-EMF exposure, we analyzed the core body temperature under nonanesthetic condition during RF-EMF exposure. The results revealed that the core temperature increased by approximately 1.5°C compared with the baseline and reached a plateau till the end of RF-EMF exposure. Furthermore, we analyzed the gene expression of heat-shock proteins (Hsp) and heat-shock transcription factors (Hsf) family after RF-EMF exposure. At WBA-SAR of 4 W/kg, some Hsp and Hsf gene expression levels were significantly upregulated in the cerebral cortex and cerebellum following exposure for 6 hr/day but were not upregulated after exposure for 3 hr/day. On the other hand, there was no significant change in the core temperature and gene expression at WBA-SAR of 0.4 W/kg. Thus, 2.14-GHz RF-EMF exposure at WBA-SAR of 4 W/kg induced increases in the core temperature and upregulation of some stress markers, particularly in the cerebellum.

Effects of intermediate frequency magnetic field exposure at 85 kHz on oxidative stress in mice

To explore the possible non-thermal biological effects of intermediate frequency magnetic field (IF-MF) on mice, we developed exposure apparatus using 85 kHz time-varying magnetic field and examined the effects on oxidative stress by subacute and acute exposure conditions. Our data showed no effect on oxidative stress under the exposure condition, which approximately corresponds to the basic restriction level of ICNIRP guidelines.

Development of the exposure apparatus of intermediate frequency magnetic field for mice and biological effects on blood properties

To explore the possible non-thermal biological effects of the intermediate frequency magnetic field, we have developed the exposure apparatus for 85 kHz time varying magnetic field. When the current of 30Arms is applied to the solenoidal coil, effective value of alternating magnetic flux density 6.5 mT is obtained. It is found that, induced electric field of 7.8V/m, that is whole body average, will be achieved by this exposure system. As preliminary experiment, we exposed this magnetic field to mice for 1 hour and examined the effect on blood properties.

Effects of the exposure of 2.14GHz W-CDMA radiofrequency electromagnetic fields to rats on body temperature increase and heat shock proteins expression

The radio frequency-electromagnetic field (RF-EMF) exposure and heat shock protein (Hsp) expression has been discussed, but there is no data on the relationship between thermal increase by RF-EMF and Hsp expression. Rats were exposed to RF-EMFs [2.14 GHz, wideband-code division multiple access (W-CDMA) signals] for 2 h/day for 3 days or 1 h/day for 10 days with a whole body average-specific absorption rate (WBA-SAR) of 4 W/kg or 0.4 W/kg, respectively. It appeared the intraperitoneal temperature was increased during RF-EMFs exposure and the Hsp27 and Hsp110 genes were significantly upregulated in the cerebral cortex and cerebellum at 4 W/kg, but protein upregulation was not detected. In contrast, there was no significant change in any of the genes at 0.4 W/kg. These results indicated that the thermal increase of the body by RF-EMF exposure plays critical role on Hsp expression.

In vivo assessment of Pig-a gene mutation of peripheral red blood cells in mice exposed to X-irradiation

It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytomery. Recently, the Pig-a mutation assay has been developed to evaluate in vivo genotoxicity in laboratory animals and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals known to be powerful point mutagens. In this study, we modified the Pig-a gene mutation assay described by Kimoto et al. (Mutat Res 723:36-42, 2011) and used threecolor staining with fluorescently labeled anti-CD24, anti-TER-119, and anti-CD71 to detect the Pig-a mutant frequency in total red blood cells (RBCs) and in reticulocytes (RETs) from X-irradiated mice. Evaluating RETs enabled us to determine Pig-a mutant frequency more quickly than using RBCs; however, measuring Pig-a mutant frequencies in total RBCs increased the accuracy and reliability of the assessment. Single exposures to X-irradiation resulted in doseand timedependent increases in Pig-a mutant frequency that subsequently were reduced with time until background frequencies were reached. The same total amount of radiation delivered as a single dose or as four repeat doses given at weekly intervals both increased Pig-a mutant frequencies to comparable levels three weeks after the single dose or the last dose of the repeat irradiation; these elevated frequencies subsequently decreased to background levels. Our results indicate that the three-color Pig-a assay will be useful for evaluating the in vivo genotoxicity of radiation. AP-193 Beclin1-induced autophagy abrogates radioresistance

Short-term whole body exposure of intermediate frequency magnetic fields to rats does not affect blood properties and immune systems

Due to the lack of the science based evidences, we explored short term exposure effects of intermediate frequency of magnetic fields (IF-MF) to the experimental animals. Male Sprague-Dawley rats (6–7 week old) were divided into 4 groups; cage-control, sham, 2 mT and 3.8 mT exposure group, respectively. IF-MF at 21 kHz was exposed to the animals under fixed conditions in an acrylic holder. Exposure was done at 1hour/day for 3 consecutive days. On the 4th day following the exposure, biochemical and hematological parameters in the blood were analyzed. We also examined the effects to the immunological functions such as cytotoxic activity and phagocytotic activity. Results indicate that there is no effects to these parameters, even high magnetic flux density (3.8 mT; 141 times higher than the reference level to general public of ICNIRP guideline 2010) was exposed to the animals.