Alteration of gene expression by exposure to a magnetic field at 23 kHz is not detected in astroglia cells.

The increasing use of induction heating (IH) cooktops has roused public concern in Japan and Europe regarding potential health effects. The purpose of this study was to evaluate the effects of exposure to a magnetic field at 23 kHz (which is the maximum output power frequency of most IH cooktops) on gene expression in a human-fetus-derived astroglia cell line, SVGp12. The cells were exposed to the magnetic field at 2 mTrms [which is approximately 74 times higher than the reference level in the most recent International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines], for 2, 4 and 6 h, using a previously reported exposure system. Gene expression was evaluated using an Agilent cDNA microarray. We did not detect any significant effects of the magnetic field on the gene expression profile. On the contrary, heat treatment at 43°C for 2 h used as a positive control significantly affected gene expression, including inducing heat shock proteins, which indicated that our protocol for microarray analysis was appropriate. From these results, we conclude that exposure of human-fetus-derived astroglia cells to an intermediate-frequency magnetic field at 23 kHz and 2 mTrms for up to 6 h does not induce detectable alteration of gene expression.

Intermediate frequency magnetic field at 23 kHz does not modify gene expression in human fetus-derived astroglia cells.

The increased use of induction heating (IH) cooktops in Japan and Europe has raised public concern on potential health effects of the magnetic fields generated by IH cooktops. In this study, we evaluated the effects of intermediate frequency (IF) magnetic fields generated by IH cooktops on gene expression profiles. Human fetus-derived astroglia cells were exposed to magnetic fields at 23 kHz and 100 µT(rms) for 2, 4, and 6 h and gene expression profiles in cells were assessed using cDNA microarray. There were no detectable effects of the IF magnetic fields at 23 kHz on the gene expression profile, whereas the heat treatment at 43 °C for 2 h, as a positive control, affected gene expression including inducing heat shock proteins. Principal component analysis and hierarchical analysis showed that the gene profiles of IF-exposed groups were similar to the sham-exposed group and were different than the heat treatment group. These results demonstrated that exposure of human fetus-derived astroglia cells to an IF magnetic field at 23 kHz and 100 µT(rms) for up to 6 h did not induce detectable changes in gene expression profile.

Myotube orientation using strong static magnetic fields.

In this experiment, we evaluated the effects of strong static magnetic fields (SMF) on the orientation of myotubes formed from a mouse-derived myoblast cell line, C2C12. Myogenic differentiation of C2C12 cells was conducted under exposure to SMF at a magnetic flux density of 0-10 T and a magnetic gradient of 0-41.7 T/m. Exposure to SMF at 10 T led to significant formation of oriented myotubes. Under the high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient, myotube orientation increased as the myogenic differentiation period increased. At the 3 T exposure position, where there was a moderate magnetic flux density and moderate magnetic field gradient, myotube orientation was not observed. We demonstrated that SMF induced the formation of oriented myotubes depending on the magnetic flux density, and that a high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient induced the formation of oriented myotubes 6 days after myogenic differentiation. We did not detect any effect of the static magnetic fields on myogenic differentiation or cell number. To the best of our knowledge, this is the first report to demonstrate that myotubes orient to each other under a SMF without affecting the cell number and myogenic differentiation.

Analysis of gene expression in a human-derived glial cell line exposed to 2.45 GHz continuous radiofrequency electromagnetic fields.

The increasing use of mobile phones has aroused public concern regarding the potential health risks of radiofrequency (RF) fields. We investigated the effects of exposure to RF fields (2.45 GHz, continuous wave) at specific absorption rate (SAR) of 1, 5, and 10 W/kg for 1, 4, and 24 h on gene expression in a normal human glial cell line, SVGp12, using DNA microarray. Microarray analysis revealed 23 assigned gene spots and 5 non-assigned gene spots as prospective altered gene spots. Twenty-two genes out of the 23 assigned gene spots were further analyzed by reverse transcription-polymerase chain reaction to validate the results of microarray, and no significant alterations in gene expression were observed. Under the experimental conditions used in this study, we found no evidence that exposure to RF fields affected gene expression in SVGp12 cells.

Ionising irradiation-induced inhibition of differentiation of C3H10T1/2 cells to the osteoblastic lineage.

PURPOSE: Previous studies using mouse osteoblast derived MC3T3-E1 and mouse myoblast derived C2C12 cells have not completely explained the mechanisms responsible for osteoradionecrosis. Thus, the aim of this study was to advance the in vitro experimental approaches for investigations of osteoradionecrosis. MATERIALS AND METHODS: The pluripotent stem cell line, mouse embryo derived C3H10T1/2, was treated with all-trans-retinoic acid after irradiation (1, 3 and 6 Gy), and cell growth, cell cycle distribution, apoptosis, and alkaline phosphatase (ALP) activity were assessed. RESULTS: We demonstrated that ionising radiation inhibited the growth and decreased ALP activity in C3H10T1/2 cells. The decrease in cell growth was not due to apoptosis but was due to cell cycle delay. The decrease in ALP activity persisted in cells that were induced to an osteoblastic lineage 24 h after irradiation. CONCLUSIONS: Our results suggested that C3H10T1/2 cells are suitable for investigating the effects of ionising irradiation on osteoblast precursor cells.

Intermediate frequency magnetic fields generated by an induction heating (IH) cooktop do not affect genotoxicities and expression of heat shock proteins.

PURPOSE: The aim of this study is to evaluate the effects of intermediate frequency (IF) fields generated by induction heating (IH) cooktops from the perspective of cellular genotoxicity and stress responses. MATERIALS AND METHODS: We evaluated the effects of exposure to 23 kHz magnetic fields at 6.05 mT(rms) for 2 h on cellular genotoxicity and stress responses in vitro. The maximum output power in most IH cooktops is at this frequency. The magnetic flux density is approximately 1000 times higher than the reference level in the International Commission on Non-ionising Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cell growth, comet assay, micronucleus formation and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation. Heat shock protein (Hsp) 27, 70, 105 and phosphorylated Hsp27 were evaluated as indicators of the stress responses. RESULTS: We did not detect any effects of the IF magnetic fields on cell growth, comet assay, micronucleus formation, HPRT gene mutation, expression of phosphorylated Hsp27, or nuclear translocation of Hsp27, 70 or 105. CONCLUSIONS: Our results indicate that exposure to an IF magnetic field at 6.05 mT(rms) for 2 h does not cause detectable cellular genotoxicity, and does not induce detectable cellular stress.

Protective effects of insulin-like growth factor-I on the decrease in myogenic differentiation by ionizing radiation.

PURPOSE: The aim of the work is to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the decrease in myotube formation induced by ionizing radiation. MATERIALS AND METHODS: We induced C2C12 cells to a myogenic linage following X-ray irradiation at 2 and 4 Gy. Myogenic differentiation was estimated using immnocytochemical staining with anti-myosin antibody, and the anti-myosin antibody positive areas, the total number of nuclei, the number of nuclei included in multinucleated myotubes per field, and the myotube formation ratio were analyzed. RESULTS: In the myogenic differentiation in the presence of IGF-1, the decrease in anti-myosin antibody positive areas, the nuclei included in myotubes, and the myotube formation ratio induced by X-ray irradiation at 2 Gy was restored to control levels. CONCLUSIONS: The addition of IGF-1 protected against the decrease myotube formation induced by X-ray irradiation at 2 Gy. Since X-ray irradiation at 2 Gy is usually used for multi-fractionated irradiation in radiotherapy, our findings suggest that IGF-1 could be useful to protect against impairment of muscle repair induced by therapeutic dose radiation.

Exposure to a MRI-type high-strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood.

The biological response after exposure to a high-strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34+ cells from human placental and umbilical cord blood were exposed under conditions of high-strength SMF in vitro. The high-strength SMF exposure system was comprised of a magnetic field generator with a helium-free superconducting magnet with built-in CO2 incubator. Freshly prepared CD34 cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst-forming unit erythroid- and megakaryocytic progenitor cells-derived colony formation was observed, thus producing 1.72- and 1.77-fold higher than the control, respectively. Furthermore, early hematopoiesis-related and cell cycle-related genes were found to be significantly up-regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP.

Effects of strong static magnetic fields used in magnetic resonance imaging on insulin-secreting cells.

The magnetic flux density of MRI for clinical diagnosis has been steadily increasing. However, there remains very little biological data regarding the effect of strong static magnetic fields (SMFs) on human health. To evaluate the effects of strong SMFs on biological systems, we cultured insulin-secreting cells under exposure to sham and SMF conditions (3-10 T of magnetic flux density, and 0-41.7 T/m of magnetic field gradient) for 0.5 or 1 h, and analyzed insulin secretion, mRNA expression, glucose-stimulated insulin secretion, insulin content, cell proliferation and cell number. Exposure to SMF with a high magnetic field gradient for 1 h significantly increased insulin secretion and insulin 1 mRNA expression. Exposure to SMF with a high magnetic flux density for 0.5 h significantly enhanced responsiveness to glucose stimulation. Exposure to SMF did not affect the insulin content, cell proliferation or cell number. Our results suggested that MRI systems with a higher magnetic flux density might not cause cell proliferative or functional damages on insulin-secreting cells, and that SMF with a high magnetic field gradient might be used clinically after thorough in vivo investigations are conducted.

Enhanced secretion of prostaglandin E2 from osteoblasts by exposure to a strong static magnetic field.

Exposure to static magnetic fields (SMFs) has been reported to promote osteoblast differentiation in vitro, and increase bone formation in vivo and in clinical studies. Prostaglandins respond early to exogenous mechanical loading, and play an important role in bone formation. In this study, we investigated whether exposure to a strong SMF affects prostaglandin E(2) (PGE(2)) secretion from a mouse osteoblastic cell line, MC3T3-E1. We also investigated the PGE(2)-synthesizing enzyme, cyclooxygenase 2 (Cox-2), and translocation of the transcription factor nuclear factor kappa B (NF-kappaB), which is involved in the induction of Cox-2 expression. In the SMF exposures, experiments were performed at the 10 T-exposure position, at which the magnetic flux density was highest, and at the 6 T-exposure position, at which the magnetic field gradient was highest (41.7 T/m). PGE(2) secretion was not affected by exposure at the 10 T-exposure position compared to sham-exposure, but was enhanced at the 6 T-exposure position (about 1.5-fold). Similarly, Cox-2 expression and NF-kappaB translocation were not enhanced at the 10 T-exposure position, but increased at the 6 T-exposure position (about twofold, two- to threefold, respectively). These findings suggested that exposure to a high magnetic field gradient induced secretion of PGE(2) and expression of the Cox-2 protein, which was mediated through increased translocation of NF-kappaB.

Exposure to extremely low frequency magnetic fields affects insulin-secreting cells.

To evaluate the effects of extremely low frequency magnetic field (ELFMF) on beta-cell survival and function, we cultured a hamster-derived insulin-secreting cell line (HIT-T15), which exhibits responsiveness to glucose in a semi-physiological range, under exposure to sham and ELFMF conditions, and assessed cell survival and function. We used our previously developed ELFMF exposure unit (a sinusoidal magnetic field at a frequency of 60 Hz, 5 mT) to culture cells under exposure to ELFMF conditions. We found that exposure to ELFMF for 5 days in the absence of glucose increased cell number, exposure for 2 days in the absence of glucose and for 5 days with 100 mg/dl glucose increased the insulin secretion to the culture medium, and exposure for 2 and 5 days with 40 and 100 mg/dl glucose increased intracellular insulin concentration in HIT-T15 cells. The increase in cell number under apoptotic culture conditions by exposure to ELFMF could lead to new therapeutic concepts in the treatment of diabetes. The ELFMF-induced increase in intracellular insulin concentration could be utilized to develop culture conditions to enhance intracellular insulin concentration in insulin-secreting cells that would be useful for cell transplantation to cure diabetes mellitus.

Extremely low frequency (ELF) magnetic fields enhance chemically induced formation of apurinic/apyrimidinic (AP) sites in A172 cells.

PURPOSE: To detect the effects of extremely low frequency (ELF) magnetic fields, the number of apurinic/apyrimidinic (AP) sites in human glioma A172 cells was measured following exposure to ELF magnetic fields. MATERIALS AND METHODS: The cells were exposed to an ELF magnetic field alone, to genotoxic agents (methyl methane sulfonate (MMS) and hydrogen peroxide (H2O2)) alone, or to an ELF magnetic field with the genotoxic agents. After exposure, DNA was extracted, and the number of AP sites was measured. RESULTS: There was no difference in the number of AP sites between cells exposed to an ELF magnetic field and sham controls. With MMS or H2O2 alone, the number of AP sites increased with longer treatment times. Exposure to an ELF magnetic field in combination with the genotoxic agents increased AP-site levels compared with the genotoxic agents alone. CONCLUSIONS: Our results suggest that the number of AP sites induced by MMS or H2O2 is enhanced by exposure to ELF magnetic fields at 5 millitesla (mT). This may occur because such exposure can enhance the activity or lengthen the lifetime of radical pairs.

Radiation-induced reduction of osteoblast differentiation in C2C12 cells.

Therapeutic radiation causes bone damage and may increase fracture risks in treatment for head-and-neck cancer and in pelvic irradiation. These properties can also be used for prevention of heterotopic ossification in hip arthroplasty. To evaluate the effects of ionizing radiation on osteoblast differentiation, C2C12 cells were directed into an osteogenic lineage by treatment with a combination of bone morphogenic protein 2 (BMP-2) (100 ng/ml) and heparin (30 mug/ml) 6 h after irradiation (2 and 4 Gy). Osteoblast differentiation was evaluated based on alkali phosphatase (ALP) activity and expression of mRNA encoding ALP and collagen type I. Ionizing radiation suppressed the growth of C2C12 cells and decreased expression of ALP and collagen type I mRNAs with concomitant reduction of the ALP activity. Although further studies are needed to elucidate the molecular mechanism, our findings suggest that ionizing radiation at therapeutic doses interferes with bone formation by reducing ALP activity and expression of mRNA encoding ALP and collagen type I.

Magnetic fields generated by an induction heating (IH) cook top do not cause genotoxicity in vitro.

The use of induction heater (IH) cook tops in homes has become widespread, especially in Japan, but there are concerns about the safety of intermediate frequency (IF) electromagnetic fields associated with these cooking appliances. Since the cellular genotoxicity of IF magnetic fields has not been examined in cultured cells, we examined the effects of these fields at a magnetic flux density of 532 +/- 20 microT at 23 kHz, using an exposure unit with a built-in CO2 incubator. Exposure to the IF magnetic field at 532 microT for 2 h did not affect the growth of CHO-K1 cells and caused no mutagenic effects in bacterial mutation assays. Exposure to the IF magnetic field for 2 h induced neither single nor double DNA strand breaks in comet assays, and caused no significant change in the mutation frequency at the HPRT locus compared to sham exposure. The magnetic field used in this study is more than 80 times higher than the level recommended as safe in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines. From these results, we suggest that exposure to an IF magnetic field for 2 h does not cause cellular genotoxicity in bacteria and in Chinese hamster cells. However, the possibility of effects on other cellular functions remains, and further studies on the cellular effects of IF magnetic fields are required.

Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations.

Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simultaneous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the combination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect.

Cold preservation of islets in UW solution--with special reference to apoptosis.

BACKGROUND: Apoptosis progresses in cultured islets. Little is known with regard to apoptosis under cold preservation. We examined viability and function of islets in University of Wisconsin (UW) solution. MATERIALS AND METHODS: Isolated rat islets were cultured overnight (overnight group) and further treated with 7-day culture in RPMI 1640 medium at 37 degrees C (culture group) or 7-day preservation in UW solution at 4 degrees C (preservation group). They were evaluated by glucose-stimulated insulin secretion test. Apoptosis was examined by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Expression of caspase mRNA and the ratio of Bax to Bcl-2 were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Islet recovery after 7 days was significantly lower in culture group than in preservation group (44.0 +/- 3.7% versus 75.0 +/- 4.9%, P < 0.05). The stimulation index in the culture group was significantly lower than in the overnight group (2.1 +/- 0.2 versus 4.1 +/- 0.4, P < 0.05). The apoptotic index in the culture group was significantly higher than both in the overnight group and in the preservation group (38.0 +/- 3.0% versus 10.8 +/- 2.0 and 27.0 +/- 4.0%, P < 0.05). Caspase 3, 8, and 9 mRNA in the culture group expressed more than in the other groups. Bax/Bcl-2 in the culture group was significantly lower than in the overnight group (3.2 +/- 0.66 versus 8.1 +/- 0.95, P < 0.05), suggesting that apoptosis had been already destined early after isolation. CONCLUSIONS: The preservation group showed better recovery and function than the culture group. Apoptosis contributed to islet loss under culture and it was significantly suppressed under cold preservation.

Effects of 2450 MHz electromagnetic fields with a wide range of SARs on methylcholanthrene-induced transformation in C3H10T1/2 cells.

This study examined whether 2450 MHz continuous wave high frequency electromagnetic fields (HFEMF) could induce cancer-like changes in mouse C3H10T1/2 cells, and whether HFEMF could initiate malignant or synergistic transformation. Transformed foci, Type II and Type III, were independently counted as the experiment endpoint. The cells were exposed to HFEMF alone at a wide range of specific absorption rates (SARs) of 5 to 200 W/kg for 2 h and/or were treated with a known initiating chemical, methylcholanthrene (MC) (2.5 microg/ml). No significant differences were observed in the malignant transformation (Type II + Type III) frequency between the controls and HFEMF with or without 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.5 ng/ml), a tumor promoter that could enhance transformation frequency initiated by MC in multistage carcinogenesis. However, the transformation frequency for HFEMF at SAR of more than 100 W/kg with MC or MC plus TPA was increased compared with MC alone or MC plus TPA. On the other hand, the corresponding heat groups (heat alone, heat + MC, and heat + MC + TPA) did not increase transformation compared with each control level in C3H10T1/2 cells. This result suggests that 2450 MHz HFEMF could not contribute to the initiation stage of tumor formation, but it may contribute to the promotion stage at the extremely high SAR (100 W/kg).

Effect of high-frequency electromagnetic fields with a wide range of SARs on chromosomal aberrations in murine m5S cells.

To investigate the induction of chromosomal aberrations in mouse m5S cells after exposure to high-frequency electromagnetic fields (HFEMFs) at 2.45 GHz, cells were exposed for 2 h at average specific absorption rates (SARs) of 5, 10, 20, 50 and 100 W/kg with continuous wave-form (CW), or at a mean SAR of 100 W/kg (with a maximum of 900 W/kg) with pulse wave-form (PW). The effects of HFEMF exposure were compared with those in sham-exposed controls and with mitomycin C (MMC) or X-ray treatment as positive controls. We examined all structural, chromatid-type and chromosome-type changes after HFEMF exposures and treatments with MMC and X-rays. No significant differences were observed following exposure to HFEMFs at SARs from 5 to 100 W/kg CW and at a mean SAR of 100 W/kg PW (a maximum SAR of 900 W/kg) compared with sham-exposed controls, whereas treatments with MMC and X-rays increased the frequency of chromatid-type and chromosome-type aberrations. In summary, HFEMF exposures at 2.45 GHz for 2 h with up to 100 W/kg SAR CW and an average 100 W/kg PW (a maximum SAR of 900 W/kg) do not induce chromosomal aberrations in m5S cells. Furthermore, there was no difference between exposures to CW and PW HFEMFs.

Combined exposure of ELF magnetic fields and x-rays increased mutant yields compared with x-rays alone in pTN89 plasmids.

We have examined mutations in the supF gene carried by pTN89 plasmids in Escherichia coli (E. coli) to examine the effects of extremely low frequency magnetic fields (ELFMFs) and/or X-rays to the plasmids. The plasmids were subjected to sham exposure or exposed to an ELFMF (5 mT), with or without X-ray irradiation (10 Gy). For the combined treatments, exposure to the ELFMF was immediately before or after X-ray irradiation. The mutant fractions were 0.94x10(-5 )for X-rays alone, 1.58x10(-5) for an ELFMF followed by X-rays, and 3.64x10(-5) for X-rays followed by an ELFMF. Increased mutant fraction was not detected following exposure to a magnetic field alone, or after sham exposure. The mutant fraction for X-rays followed by an ELFMF was significantly higher than those of other treatments. Sequence analysis of the supF mutant plasmids revealed that base substitutions were dominant on exposure to X-rays alone and X-rays plus an ELFMF. Several types of deletions were detected in only the combined treatments, but not with X-rays alone. We could not find any mutant colonies in sham irradiated and an ELFMF alone treatment, but exposure to ELFMFs immediately before or after X-ray irradiation may enhance the mutations. Our results indicate that an ELFMF increases mutation and alters the spectrum of mutations.

Decrease in glucose-stimulated insulin secretion following exposure to magnetic fields.

We evaluated the effects of extremely low frequency magnetic field (ELFMF) on glucose-stimulated insulin secretion from HIT-T15 cells and investigated the mechanisms of these effects. We demonstrated that exposure to ELFMF at 5mT decreased glucose-stimulated insulin secretion by preventing the increases in cellular adenosine 5'-triphosphate/adenosine 5'-diphosphate, membrane depolarization, and cytosolic free calcium ion concentration. The glucose-induced upregulation of insulin mRNA expression was also attenuated by exposure to ELFMF, although cell viability was not affected. These findings demonstrate the potential of exposure to ELFMF for clinical use as a novel inhibitory method of insulin secretion.