Real-time detection of stimulus response in cultured neurons by high-intensity intermediate-frequency magnetic field exposure.

Threshold values of neuronal stimulation and modulation associated with exposure to time-varying electromagnetic fields contribute to establishing human protection guidelines and standards. However, biological evidence of threshold values in the intermediate-frequency range is limited. Additionally, although it is known that dendrites, a type of unmyelinated neuronal fibre, play an important role in information processing in the central nervous system, the stimulus threshold in dendrites has not been sufficiently investigated. We evaluated the excitation site-specific stimulus response of rat brain-derived cultured neurons by using a 20 kHz high-intensity intermediate-frequency magnetic field (hIF-MF) exposure system, a non-conductive fibre-optic imaging (NCFI) system, combined with a micro-patterning technique. Our hIF-MF exposure and NCFI system permitted real-time detection of the intracellular calcium ([Ca2+]i) spikes in neuronal cell bodies or unmyelinated neuronal fibres during exposure to a 20 kHz, 70 mT (peak), burst-type sinusoidal wave hIF-MF. Dosimetry of the induced electric fields intensities in the extracellular solution indicated that about 50% of unmyelinated neuronal fibres respond at about 147 V m-1. In contrast, the threshold of the [Ca2+]i spikes in neuronal cell bodies were lower than that in unmyelinated neuronal fibres. Our results provide a basis for understanding site-specific differences in the responses of cultured neurons to hIF-MFs.

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2, and an early developmental gene, Hba-x, in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc. in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

Evaluation of mutagenicity and co-mutagenicity of strong static magnetic fields up to 13 Tesla in Escherichia coli deficient in superoxide dismutase.

PURPOSE: To evaluate the biological effects of static magnetic fields (SMFs) up to 13 Tesla (T), with respect to superoxide behavior, by determining the effect on mutagenicity in superoxide dismutase (SOD)-deficient Escherichia coli strain QC774, and its parental strain GC4468. MATERIALS AND METHODS: Experimental strains were exposed to a 5, 10, or 13T SMF for 24 h at 37°C in Luria-Bertani medium. To evaluate mutagenicity after SMF exposure, the mutation frequency in thymine synthesis genes was determined. The effect of exposure to a 5 or 13T SMF on mutagenicity induced by plumbagin was also investigated. RESULTS: No statistically significant differences in the mutation frequency in thymine synthesis genes were observed between SMF-exposed cells and unexposed cells at any of the applied magnetic flux densities. Furthermore, exposure to SMFs up to 13T did not affect mutagenicity induced by plumbagin. CONCLUSION: Exposure to SMFs up to 13T caused neither mutagenicity nor co-mutagenicity in the SOD-deficient E. coli strain QC774 or in its parental strain GC4468, suggesting that exposure to strong SMFs does not affect the behavior of superoxides in these microorganisms.

Indium chloride-induced micronuclei in in vivo and in vitro experimental systems.

OBJECTIVES: The aim of this study was to investigate the genotoxic effects of indium trichloride (InCl(3)·4H(2)O; InCl(3)) using the in vivo bone marrow micronucleus test and the in vitro CHL/IU cell micronucleus test. METHOD: BALB/c mice were administered a single intraperitoneal (i.p.) injection of InCl (3) at a dose 0.625, 1.25, 2.5, 5, or 10 mg/kg b.w. The frequency of micronuclei, the ratio of polychromatic erythrocytes to normochromatic erythrocytes (P/N ratio) and body weight gain were determined 24 h after administration of the InCl(3). In the in vitro micronucleus test, CHL/IU cells were treated continuously for 24, 48, or 72 h in the absence of S9mix (the continuous treatment method) and/or for 6 h with or without S9 mix followed by an 18, 42 or 66 h recovery time (the short time treatment method). The frequency of micronuclei was determined at the end of each culture period. RESULTS: The frequency of micronuclei induced by InCl(3) increased in the in vivo erythroblast-erythrocyte micronucleus test using BALB/c mice at doses of 2.5 and 5 mg/kg b.w. The P/N ratio, a marker of bone marrow toxicity, decreased significantly following the injection of InCl(3). Body weight gain was also inhibited by InCl(3). InCl(3) induced micronuclei in the CHL/IU cell micronucleus test in both the continuous treatment method and the short time treatment method, both with and without S9mix. CONCLUSIONS: These results suggest that InCl(3) has a genotoxic effect on mammalian cells both in vivo and in vitro.

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests.

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.

Effect of static magnetic field on the induction of micronuclei by some mutagens.

OBJECTIVES: It is important to assess the risk of static magnetic fields (SMFs) on human health, because epidemiological studies have indicated that SMFs play a role in the development of diseases such as leukemia and brain tumor. In our environment, we have numerous chances to be exposed to not only SMFs but also many chemicals containing mutagens. The aim of this study is to investigate the effect of SMFs on the induction of micronuclei induced by some mutagens. METHODS: BALB/c mice were exposed to 4.7 tesla (T) SMF for 24 hr immediately after the injection of carboquone (alkylating agent), colcemid (spindle poison), mitomycin C (cross-linking agent), vincristine (spindle poison), sodium fluoride (a byproduct of aluminum plants under strong SMF) or 1-ethyl-1-nitrosourea (brain tumor-, gliomas- and thymic lymphoma-inducing chemical). RESULTS: The frequency of micronuclei induced by six mutagens increased after co-exposure to SMF. CONCLUSIONS: An additive/synergistic effect of SMF and chemicals was observed from the results of increased frequency of micronuclei induced by mutagens in mouse bone marrow erythrocytes.

Genotoxic effects of strong static magnetic fields in DNA-repair defective mutants of Drosophila melanogaster.

To assess the possibility that strong static magnetic fields cause DNA damage and mutation, we examined the genotoxic effects of magnetic field exposure by using the somatic mutation and recombination test system in DNA repair-proficient and -deficient strains of Drosophila melanogaster. A postreplication repair-defective mutation mei-41D5 and/or a nucleotide excision repair-defective mutation mei-9(a) was introduced into the conventional loss of the heterozygosity assay system by the use of mwh +/ + flr transheterozygotes, and were exposed to static magnetic fields of up to 14 Tesla (T). We found that exposure to 2, 5, or 14 T fields for 24 h caused a statistically significant enhancement in somatic recombination frequency in the postreplication repair-deficient flies, whereas the frequency remained unchanged in the nucleotide excision repair-deficient flies and in the DNA repair-proficient flies after exposure. An increase linearly dependent on the flux density was observed between 0.5 T and 2 T, but it was saturated at exposure levels over 2 T. These findings suggest that exposure to high-density magnetic fields induce somatic recombination in Drosophila and that the dose-response relationship is not linear.

Strong static magnetic field effects on yeast proliferation and distribution.

The present study focuses on the effects of gradient magnetic fields on the behavior of yeast, such as its proliferation and mass distribution, and evaluates the effects of magnetism on materials in the yeast culture system. Yeast, Saccharomyces cerevisiae, was incubated in a liquid medium under magnetic fields (flux density B = 14 T). When yeast in a tube was exposed to 9-14 T magnetic fields with a maximum flux density gradient of dB/dx = 94 T/m, where x is the space coordinate, the rate of yeast proliferation under the magnetic fields decreased after 16 h of incubation compared to that of the control group. The physical properties of the yeast culture system were investigated to discover the mechanism responsible for the observed deceleration in yeast proliferation under magnetic fields. Gas pressure inside the yeast culture flask was compared with and without exposure to a magnetic field. The results suggested that the gas pressure inside a flask with 6 T, 60 T/m slowly increased in comparison to the pressure inside a control tube. Due to the diamagnetism of water (medium solution) and yeast, the liquid surface distinctly inclined under gradient magnetic fields, and the hydrostatic force in suspension was strengthened by the diamagnetic forces. In addition, magnetophoresis of the yeast cells in the medium solution exhibited localization of the yeast sedimentation pattern. The roles of magnetically changed gas-transport processes, hydrostatic pressures acting on the yeast, and changes in the distribution of the yeast sedimentation, as well as the possible effects of magnetic fields on yeast respiratory systems in the observed disturbance of the proliferation are discussed.

Apoptosis or growth arrest: Modulation of tumor suppressor p53's specificity by bacterial redox protein azurin.

The tumor suppressor protein p53 is known to induce either apoptosis or growth arrest depending on cellular background. We have previously reported that a bacterial redox protein azurin induces apoptosis in J774 cell line-derived macrophages whereas a site-directed mutant M44KM64E azurin shows very little cytotoxicity and fails to induce apoptosis in J774 cells. We now report that purified M44KM64E mutant azurin protein can enter both J774 cells as well as the human breast cancer MCF-7 cells. Entry of M44KM64E mutant azurin in J774 cells causes strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene. Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells. Interestingly, M44KM64E mutant azurin protein failed to elicit inhibition of cell-cycle progression in MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level. Thus, the WT azurin induces apoptosis but little inhibition of cell-cycle progression whereas the M44KM64E mutant azurin is deficient in the induction of apoptosis but mediates strong inhibition of cell-cycle progression, demonstrating the role of a single bacterial protein and its hydrophobic patch in modulating two important functions of p53.

A threshold exists in the dose-response relationship for somatic mutation frequency induced by X irradiation of Drosophila.

The dose-response relationship of ionizing radiation and its stochastic effects has been thought to be linear without any thresholds. The basic data for this model were obtained from mutational assays in the male germ cells of the fruit fly Drosophila melanogaster. However, it is more appropriate to examine carcinogenic activity in somatic cells than in germ cells. Here the dose-response relationship of X irradiation and somatic mutation was examined in Drosophila. A threshold at approximately 1 Gy was observed in DNA repair-proficient flies. In the repair-deficient siblings, the threshold was smaller and the inclination of the dose-response curve was much steeper. These results suggest that the dose-response relationship between X irradiation and somatic mutation has a threshold and that the DNA repair function contributes to its formation.