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.

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.

Nuclear accumulation and activation of nuclear factor kappaB after split-dose irradiation in LS174T cells.

Although radiation-induced gene expression has been extensively studied, most of the studies to date have focused on that after single-dose irradiation. As split-dose irradiation, rather than single-dose irradiation, is usual in clinical situations, we investigated the effects of split-dose irradiation on nuclear factor kappaB (NF-kappaB) in the human rectum carcinoma cell line, LS174T. After either single- or split-dose irradiation with a different interval, nuclear localization of NF-kappaB was examined by Western blot and immunofluorescence and its DNA-binding activity was measured by ELISA-based assay. Irradiation-induced NF-kappaB nuclear accumulation and DNA binding activity increased in a dose-dependent manner. The peak of NF-kappaB nuclear accumulation and DNA binding activity was seen 2 to 6 hours after a single dose of 4 Gy irradiation and returned to control levels after 12 hours. In split-dose irradiation, NF-kappaB activity was similar after the first and second doses of 4 Gy irradiation separated by 12 hours. In addition, NF-kappaB activity was decreased by lengthening the interval between irradiation. The cell survival, which was assessed by colony formation assay, showed inverse correlation to this: the surviving fraction was higher after split-dose irradiation than after single-dose irradiation of the same total dose and it increased as the interval between irradiation was lengthened. Thus the present results showed a correlation between NF-kappaB activation and the repair of sublethal damage in split-dose irradiation.

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.

ELF electromagnetic fields increase hydrogen peroxide (H2O2)-induced mutations in pTN89 plasmids.

We have examined the mutational effects of hydrogen peroxide (H(2)O(2)) in the presence and absence of an extremely low-frequency magnetic field (ELFMF), using pTN89 plasmids. Mutations were detected in the supF gene carried by these plasmids in Escherichia coli. The plasmids were either treated with H(2)O(2) (1microM) alone at 37 degrees C for 4h, or were exposed to an ELFMF (60Hz, 5millitesla (mT)) simultaneously with H(2)O(2) treatment. The mutation frequency was 2.28 x 10(-4) for H(2)O(2) treatment alone, and 5.81 x 10(-4) for ELFMF exposure with H(2)O(2) treatment. We did not observe any mutations using treatment with ELFMF exposure alone. This indicates that the ELFMF may potentiate H(2)O(2)-induced mutation. Sequence analysis of the supF mutant plasmids revealed that base substitutions, G: C-->A :T transitions and G:C-->T:A transversions were dominant in both treatment groups, and there was no difference in the mutation spectrum or the hotspots between the groups. Therefore, ELFMFs may interact and potentiate the damage induced by H(2)O(2), resulting in an increase in the number of mutations.

Effects of high frequency electromagnetic fields on micronucleus formation in CHO-K1 cells.

To investigate the effects of high frequency electromagnetic fields (HFEMFs), we assessed the frequency of micronucleus (MN) formation induced by chromosomal breakage or inhibition of spindles during cell division in Chinese hamster ovary (CHO)-K1 cells, using the cytokinesis block micronucleus method. The MN frequency in cells in the inner, middle and outer wells of an annular culture plate was determined for the following four conditions: (1) CHO-K1 cells were exposed to a HFEMF for 18 h at average specific absorption rates (SARs) of 13, 39 and 50 W/kg with input power 7.8 W, and were compared with a sham-exposed control; (2) the cells were also exposed to a HFEMF at SARs of 78 and 100 W/kg with input power 13 W, and were compared with a sham-exposed control; (3) the cells were treated with bleomycin alone or with bleomycin followed by exposure to a HFEMF for 18 h at SARs of 25, 78 and 100 W/kg, and were compared with a bleomycin-treated positive control. The cells treated with bleomycin alone were compared with sham-exposed controls; and (4) As a high temperature control, CHO-K1 cells were incubated at 39 degrees C for 18 h. In study (1), the MN frequency of cells exposed to a HFEMF at a SAR of up to 50 W/kg was not different to that in sham-exposed cells. In study (2), there were statistically significant increases in the MN frequencies of cells in the middle and outer wells of the annular culture plate caused by exposure to a HFEMF at 100 and 78 W/kg, respectively. In study (3), the MN frequencies of cells in the middle (100 W/kg) and outer wells (78 W/kg) of the annular culture plate were statistically higher than that caused by bleomycin-treatment alone. In study (4), there was a statistically significant increase of MN frequency in the cells treated by heat at 39 degrees C. These results indicate that cells exposed to a HFEMF at a SAR of 78 W/kg and higher form MN more frequently than sham-exposed cells, while exposure to a HFEMF at up to 50 W/kg does not induce MN formation. In addition, a HFEMF at a SAR of 78 W/kg and higher may potentiate MN formation induced by bleomycin-treatment.

Induction of kinetochore-positive and kinetochore-negative micronuclei in CHO cells by ELF magnetic fields and/or X-rays.

To test the genotoxic effects of extremely low frequency (ELF) magnetic fields, the induction of micronuclei by exposure to ELF magnetic fields and/or X-rays was investigated in cultured Chinese hamster ovary (CHO) cells, using the cytokinesis block method. Micronuclei derived from acentric fragments or from whole chromosomes were evaluated by immunofluorescent staining using anti-kinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. A 60 Hz ELF magnetic field at 5 mT field strength was applied, either before or after 1 Gy X-ray irradiation or without additional X-ray irradiation. No statistically significant difference in the frequency of micronuclei in CHO cells was observed between a sham exposure (no exposure to an ELF magnetic field) and a 24 h ELF magnetic field exposure. Exposure to an ELF magnetic field for 24 h before X-ray irradiation or for 18 h after X-ray irradiation did not affect the frequency of X-ray-induced micronuclei. However, the number of kinetochore-positive micronuclei was significantly increased in the cells subjected to X-ray irradiation followed by ELF magnetic field exposure, but not in the cells treated with ELF magnetic field exposure before X-ray irradiation, compared with exposure to X-rays alone. The number of spontaneous kinetochore-positive and kinetochore-negative micronuclei was not affected by exposure to an ELF magnetic field alone. Our data suggest that exposure to an ELF magnetic field has no effect on the number of spontaneous and X-ray-induced micronuclei. However, ELF magnetic field exposure after but not before X-ray irradiation may somehow accelerate X-ray-induced lagging of whole chromosomes (or centric fragments) in CHO cells.

Radiosensitization by inhibition of IkappaB-alpha phosphorylation in human glioma cells.

To assess the role of nuclear factor kappaB (NFKB) in cellular radiosensitivity, three different IkappaB-alpha (also known as NFKBIA) expression plasmids, i.e., S-IkappaB (mutations at (32, 36)Ser), Y-IkappaB (a mutation at (42)Tyr), and SY-IkappaB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 ((15)Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IkappaB-alpha phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.

Orientation of human glioblastoma cells embedded in type I collagen, caused by exposure to a 10 T static magnetic field.

We investigated the preferred orientation of human glioblastoma cells (A172) following exposure to static magnetic fields (SMF) at 10 Tesla in the presence or absence of collagen. A172 cells embedded in collagen gel were oriented perpendicular to the direction of the SMF. A172 cells cultured in the absence of collagen did not exhibit any specific orientation pattern after 7 days of exposure to the SMF. Thus we succeeded in evoking the magnetic orientation of human glioblastoma cells by exposure to the SMF. Our results suggest that the orientation of glioblastoma cell processes may be due to the arrangement of microtubules under the influence of magnetically oriented collagen fiber.

Static magnetic field with a strong magnetic field gradient (41.7 T/m) induces c-Jun expression in HL-60 cells.

We investigated the effects of 6- and 10-T static magnetic fields (SMFs) on the expression of protooncogenes using Western blot immunohybridization methods. We used a SMF exposure system, which can expose cells to a spatially inhomogeneous 6 T with a strong magnetic field (MF) gradient (41.7 T/m) and a spatially homogeneous 10 T of the highest magnetic flux density in this experiment. HL-60 cells exposed to either 6- or 10-T SMF for periods of 1 to 48 h did not exhibit remarkable differences in levels of c-Myc and c-Fos protein expression, as compared with sham-exposed cells. In contrast, c-Jun protein expression increased in HL-60 cells after exposure to 6-T SMF for 24, 36, 48, and 72 h. These results suggest that a homogeneous 10-T SMF does not alter the expression of the c-jun, c-fos, and c-myc protooncogenes. However, our observation that exposure to a strong MF gradient induced c-Jun expression suggests that a strong MF gradient may have significant biological effects, particularly regarding processes related to an elevation of c-jun gene expression.

Exposure to power frequency magnetic fields and X-rays induces GAP-43 gene expression in human glioma MO54 cells.

We investigated the distribution and expression of growth associated protein-43 (GAP-43) in human glioma cells (MO54) after exposure to a magnetic field (60 Hz, 5 mT), with or without initial X-ionizing radiation (2 Gy), by using immunocytochemistry and the reverse transcription polymerase chain reaction (RT-PCR). GAP-43 was present in the cytoplasm, accumulating in the perinuclear area. An increase in GAP-43 expression was observed with a peak at 10 h at the mRNA level and at 12 h at the protein level, after exposure to the magnetic field. The increased level of GAP-43 protein returned to a normal level within 24 h of exposure to a 5 mT magnetic field. The kinetic pattern of GAP-43 expression induced by X-ionizing radiation was very similar to that induced by the magnetic field. These results suggest that the stimulation of GAP-43 expression could occur by a similar mechanism following exposure to X-rays or magnetic fields. We have provided the first evidence that exposure to a 5 mT magnetic field can induce GAP-43 gene expression in human glioma MO54 cells.

Effects of exposure of CHO-K1 cells to a 10-T static magnetic field.

PURPOSE: To evaluate whether exposure to strong static magnetic fields (SMFs), of up to 10 T, affects the growth and cycle distribution of and the micronucleus formation in monolayered Chinese hamster ovary CHO-K1 cells. MATERIALS AND METHODS: The authors developed a system to expose cultured cells to strong SMFs immediately after the cells are seeded. Cell growth rate was evaluated according to cell number count. Cell cycle distribution experiments were performed by using flow cytometric analysis. In these experiments, the cells were exposed to SMFs for up to 4 days. The frequency of micronucleus formation with only SMF exposure at x-ray irradiation was analyzed at microscopic observation. RESULTS: Long-term exposure to a 10-T SMF for up to 4 days did not affect cell growth rate or cell cycle distribution. Exposure to SMFs alone did not affect micronucleus frequency. In x-ray-irradiated cells, exposure to a 1-T SMF did not affect micronucleus frequency, but exposure to a 10-T SMF resulted in a significant (P <.05) increase in micronucleus frequency. CONCLUSION: Strong (10-T) SMFs have no effect on cell growth, cell cycle distribution, or micronucleus frequency, but they may cause an increase in the micronucleus formation induced by 4-Gy x rays.

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells.

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.