In vivo genotoxicity of high-intensity intermediate frequency magnetic fields in somatic cells and germ cells.

Intermediate frequency magnetic fields (IF-MFs) at ~85 kHz are one of the components of wireless power transfer (WPT) systems. However, the available data needed for the assessment of the safety of organisms from IF-MF exposure are scarce. Thus, there is an imminent need to accumulate evidence-based assessment data. In particular, if humans are exposed to IF-MF due to an accident or trouble, they are at increased risk of being exposed to high-intensity IF-MF within a short period. The already existing exposure system was improved to a system that could intermittently expose animals at 3 s intervals. This system allows the exposure of a mouse to high-intensity IF-MF (frequency: 82.3 kHz; induced electric field: 87 V/m, which was 3.8 times the basic restriction level for occupational exposure in the ICNIRP guideline), while regulating the heat generated by the coil. In vivo genotoxicity after IF-MF exposure was assessed using micronucleus (MN) test, Pig-a assay, and gpt assay. The results of MN test and Pig-a assay in hematopoietic cells revealed that neither the reticulocytes nor the mature erythrocytes exhibited significant increases in the IF-MF-exposed group compared with that in the sham-exposed group. In germ cells, MN test and gpt assay outcomes showed that IF-MF exposure did not cause any genetic or chromosomal abnormality. Based on these data, there was no genotoxic effect of our set IF-MF exposure on somatic and germ cells. These findings can contribute to the widespread use of WPT systems as effective data of IF-MF safety assessment.

No evidence for genotoxicity in mice due to exposure to intermediate-frequency magnetic fields used for wireless power-transfer systems.

Time varying magnetic fields (MFs) are used for the wireless power-transfer (WPT) technology. Especially, 85 kHz band MFs, which are included in the intermediate frequency (IF) band (300 Hz - 10 MHz), are commonly used WPT system for charging electric vehicles. Those applications of WPT technology have elicited public concern about health effects of IF-MF. However, existing data from health risk assessments are insufficient and additional data are needed. We assessed the genotoxic effects of IF-MF exposure on erythroid differentiation in mice. A high-intensity IF-MF mouse exposure system was constructed to induce an average whole-body electric field of 54.1 V/m. Blood samples were obtained from male mice before and after a 2-week IF-MF exposure (1 h/day, total: 10 h); X-irradiated mice were used as positive controls. We analyzed the blood samples with the micronucleus (MN) test and the Pig-a mutation assay. No significant differences were seen between IF-MF-exposed and sham-exposed mice in the frequencies of either MN or Pig-a mutations in mature erythrocytes and reticulocytes. IF-MF exposure did not induce genotoxicity in vivo under the study conditions (2.36× the basic restriction for occupational exposure, 22.9 V/m, in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines). The absence of significant biological effects due to IF-MF exposure supports the practical application of this technology.

Global Analysis of Transcriptional Expression in Mice Exposed to Intermediate Frequency Magnetic Fields Utilized for Wireless Power Transfer Systems.

BACKGROUND: Intermediate frequency magnetic fields (IF-MFs) at around 85 kHz are a component of wireless power transfer systems used for charging electrical vehicles. However, limited data exist on the potential health effects of IF-MFs. We performed a comprehensive analysis of transcriptional expression in mice after IF-MF exposure. MATERIALS AND METHODS: We developed an IF-MF exposure system to generate a high magnetic flux density (25.3 mT). The system can expose the IF-MF for a mouse whole-body without considering thermal effects. After 10 days (1 h/day) of exposure, a comprehensive expression analysis was performed using microarray data from both the brain and liver. RESULTS: No significant differences in transcriptional expression were detected in the 35,240 probe-sets when controlling the false discovery rate (FDR) under a fold change cutoff >1.5. However, several differential expressions were detected without FDR-adjustment, but these were not confirmed by RT-PCR analysis. CONCLUSIONS: To our knowledge, this is the first in vivo study to evaluate the biological effects of IF-MF exposure with an intense magnetic flux density 253 times higher than the occupational restriction level defined by the International Commission on Non-Ionizing Radiation Protection guidelines. However, our findings indicate that transcriptional responses in the living body are not affected under these conditions.

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.

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 b,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.

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

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.

Persistence of red blood cells with Pig-a mutation in p53 knockout mice exposed to X-irradiation.

The Pig-a mutation assay is becoming one of the major experimental procedures used to assess in vivo genotoxicity. The assay allows simple in vivo analysis and enables gene mutations in the hematopoietic system to be measured using high throughput flow cytometry. Previously, we demonstrated that X-irradiation increased the Pig-a mutant frequencies in red blood cells (RBCs) of mice in a radiation dose-dependent manner. In this study, to understand how RBCs with Pig-a mutation induced by X-irradiation persist, we compared Pig-a mutant frequencies between irradiated C57BL/6J (p53(+/+)) mice and irradiated p53 homozygous knockout (p53(-/-)) mice by using the RBC Pig-a assay. After the peak in radiation-induced Pig-a mutant frequencies, a gradual decrease in mutant frequencies in irradiated p53(-/-) mice was observed, while irradiated p53(+/+) mice had a rapid decrease, which suggests that RBCs with Pig-a mutation are eliminated normally in irradiated p53(+/+) mice but not in irradiated p53(-/-) mice due to lack of p53 function. In addition, we also found that the p53 function affected the regulation of Pig-a mutagenesis in aging mice. Our results suggest that p53 function, distinct types of mutation, and the life span of RBCs play key roles in the persistence of Pig-a mutation in the hematopoietic system of RBCs after irradiation.

Can intermediate-frequency magnetic fields affect memory function-related gene expressions in hippocampus of C57BL/6J mice?

Recently, a cooking appliance based on the principle of electromagnetic induction has come to be used domestically on a widespread basis; this induction heating cooking hob mainly generates intermediate-frequency magnetic fields (IF-MF). However, whether electromagnetic fields originating from household appliances represent a health risk remains uncertain. We investigated the effect of IF-MF on the expressions of memory function-related genes and related transduction molecules in the mouse hippocampus. Male and female C57BL/6J mice were allotted to a control (sham-exposed), an exposure, or a recovery (one week after exposure) group and were exposed to IF-MF (21 kHz, 3.8 mT) one hour per day for 2 weeks. Twenty-four hour after final exposure, the expression levels of memory function-related genes and the mRNA levels for signal transduction pathway molecules in the hippocampi were examined using real-time RT-PCR. The relative mRNA expression levels of the N-methyl-D aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B as well as transcription factors (calcium/calmodulin-dependent protein kinase (CaMK) -IV, cyclic AMP responsive element binding protein (CREB) -1) and neurotrophins (nerve growth factor (NGF), and brain-derived neurotrophic factors (BDNF)) were not significantly altered in the IF-MF-exposed mice. We also examined the morphology of the hippocampus using a histological analysis, but no changes in the IF-MF-exposed mice were seen. This is the first in vivo study to show that IF-MF exposure did not affect the expression levels of memory function-related genes in the hippocampus of C57BL/6J mice. The present findings suggest that IF-MF exposure may not affect cognitive function in the present animal model.

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.

Autoantibodies recognizing native MOG are closely associated with active demyelination but not with neuroinflammation in chronic EAE.

It is important to find biomarkers for autoimmune inflammation and demyelination in the CNS to monitor disease status in patients with multiple sclerosis (MS). For this purpose, we determined the titers of antibodies (Ab) reacting with native myelin oligodendrocyte glycoprotein (MOG)-expressing cells to evaluate the disease activity of chronic experimental autoimmune encephalomyelitis (EAE) in rats and the relationship between anti-MOGcme (cell membrane-expressed MOG), Ab titers and clinical and pathological parameters were evaluated. Consequently, we found that elevation of anti-MOGcme Ab titers was associated with clinical severity, except for some cases in very late stages and with severe and widespread demyelination but with dominant inflammation. In contrast, antibodies detected by standard ELISA using recombinant MOG were elevated in both symptomatic and asymptomatic rats and were not associated with parameters such as inflammation and demyelination. Longitudinal examination of anti-MOGcme Ab titers in individual rats revealed that Ab titers accurately reflect disease activity. Furthermore, anti-MOGcme Ab titer was not elevated in acute EAE without demyelination. These findings suggest that autoantibodies reacting with native and glycosylated MOG play an important role in the progression of demyelinating diseases and could be biomarkers for monitoring the status of patients with MS.

Role of pathogenic T cells and autoantibodies in relapse and progression of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis in LEW.1AV1 rats.

Accumulating evidence suggests that T cells and autoantibodies reactive with myelin oligodendrocyte glycoprotein (MOG) play a critical role in the pathogenesis of multiple sclerosis (MS). In the present study, we have tried to elucidate the pathomechanisms of development and progression of the disease by analysing T cells and autoantibodies in MOG-induced rat experimental autoimmune encephalomyelitis (EAE), which exhibits various clinical subtypes mimicking MS. Analysis using overlapping peptides revealed that encephalitogenic epitopes resided in peptide 7 (P7, residue 91-108) and P8 (residue 103-125) of MOG. Immunization with MOGP7 and MOGP8 induced relapsing-remitting or secondary progressive EAE. T cells taken from MOG-immunized and MOGP7-immunized rats responded to MOG and MOGP7 and sera from MOG-immunized rats reacted to MOG and MOGP1. Significant epitope spreading was not observed at either T-cell or antibody levels. Interestingly, sera from MOGP7-immunized rats with clinical signs did not react to MOG and MOG peptides throughout the observation period, suggesting that disease development and relapse in MOGP7-induced EAE occur without autoantibodies. However, MOGP7 immunization with adoptive transfer of anti-MOG antibodies aggravated the clinical course of EAE only slightly. Analysis of antibodies against conformational epitope (cme) suggests that anti-MOG(cme) may play a role in the pathogenicity of anti-MOG antibodies. Collectively, these findings demonstrated that relapse of a certain type of MOG-induced EAE occurs without autoantibodies but that autoantibodies may play a role in disease progression. Relapses and the progression of MS-mimicking EAE are differently immunoregulated so immunotherapy should be designed appropriately on the basis of precise information.

A deletion in the endothelin-B receptor gene is responsible for the Waardenburg syndrome-like phenotypes of WS4 mice.

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomic sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.

Reduced expression of the endothelin receptor type B gene in piebald mice caused by insertion of a retroposon-like element in intron 1.

Mice carrying the piebald mutation exhibit white coat spotting due to the regional absence of neural crest-derived melanocytes. We reported previously that the piebald locus encodes the Ednrb gene and that piebald mice express low levels of structurally intact Ednrb mRNA and EDNRB protein (Hosoda, K., Hammer, R. E., Richardson, J. A., Baynash, A. G., Cheung, J. C., Giaid, A., and Yanagisawa, M. (1994) Cell 79, 1267-1276). Here, we report that both the life span of the Ednrb mRNA and the promoter activity of the Ednrb gene are indistinguishable between wild-type and piebald mice. Introns 2-6 of the Ednrb gene in piebald mice were correctly excised with an efficiency indistinguishable from those in wild-type mice in exon trapping experiments. We found that the piebald allele of the Ednrb gene has a 5.5-kb retroposon-like element in intron 1 possessing canonical sequences of a polyadenylation signal and a splice acceptor site. Abnormal hybrid transcripts carrying exon 1 of the Ednrb gene and a portion of the 5.5-kb element are expressed in piebald mice. The insertion of the 5.5-kb element into a heterologous intron in a mammalian expression vector markedly reduced the expression of the reporter gene. Premature termination and aberrant splicing of the Ednrb transcript caused by the retroposon-like element in intron 1 lead to a reduced level of the normal Ednrb transcript, which is responsible for the partial loss-of-function phenotype of piebald mice.

Aortic valve replacement in a woman with osteogenesis imperfecta.

The mortality rate in cardiac surgery patients with heritable generalized connective tissue disorders, such as Marfan s syndrome and osteogenesis imperfecta, is high due to tissue friability. We describe a successful open heart surgery for repair of aortic regurgitation in a woman with osteogenesis imperfecta (OI). Although tissue friability caused no problems during surgery in this case, it should be kept in mind when operating on patients with OI.