Takehisa Nakahara

Strong static magnetic field and the induction of mutations through elevated production of reactive oxygen species in Escherichia coli soxR.

Purpose : Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently. Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF. Materials and methods : An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF. Various mutant strains of E. coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined. The expression of the soxS::lacZ fusion gene was assessed by measurement of β-galactosidase activity. Results : The results for survival or mutation were obtained with wild-type E. coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative. On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress. Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E. coli when exposed to 5 and 9 T, respectively. Conclusions : These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.

Exposure to 2.45 GHz electromagnetic fields induces hsp70 at a high SAR of more than 20 W/kg but not at 5W/kg in human glioma MO54 cells.

Purpose : To determine potential hazards from exposure to a high-frequency electromagnetic field (HFEMF) at 2.45 GHz by studies of the expression of heat-shock protein 70 (hsp70) in MO54 cells. Method : MO54 cells were exposed to a HFEMF at average specific absorption rates (SAR) of 5, 20, 50 and 100 W/kg, using input powers of 0.8, 3.2, 7.8 and 13 W, at a temperature of up to 39°C. An annular culture dish provided three levels of exposure for a given input power, designated inner, middle and outer rings. Two control groups were used: the first was subjected to sham exposure and the second was a temperature control, used to determine the effect of high temperature using incubation in a conventional incubator at 39°C. Cell survival was determined in intervals up to 24 h. Protein was extracted from MO54 cells in both groups after 2, 4, 8 and 16 h exposure times. Changes in the hsp70 protein levels were analysed by Western blots. Results : Little or no cell death was observed in the sham-exposed cells, nor for incubation at 39°C for up to 16 h. Cell survival decreased to about 30% after exposure to HFEMF for 24 h at an average SAR of 100 W/kg. A slight increase in hsp70 was observed in cells in both the inner and outer rings of the plate after exposure at SAR levels of 25 and 78 W/kg, respectively, for 2 h. With increasing exposure time, hsp70 expression increased except for an SAR of 5 W/kg. In the raised temperature control at 39°C, hsp70 expression also increased as the incubation time increased. However, the expression level of hsp70 for the HFEMF exposure was greater than that for the raised temperature control. Conclusion : HFEMF can produce an increased level of hsp70 expression in MO54 cells at SAR levels above 20 W/kg, even when the effect of raised temperature is taken into account.

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

SummaryWe 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.

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.

Radiosensitization by Inhibition of IκB-α Phosphorylation in Human Glioma Cells

Abstract Ding, G-R., Honda, N., Nakahara, T., Tian, F., Yoshida, M., Hirose, H. and Miyakoshi, J. Radiosensitization by Inhibition of IκB-α Phosphorylation in Human Glioma Cells. Radiat. Res. 160, 232–237 (2003). To assess the role of nuclear factor κB (NFKB) in cellular radiosensitivity, three different IκB-α (also known as NFKBIA) expression plasmids, i.e., S-IκB (mutations at 32, 36Ser), Y-IκB (a mutation at 42Tyr), and SY-IκB, 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 (15Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IκB-α 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.

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.

Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA.

The pathways leading to G:C-->C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C-->C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E. COLI: By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.COLI: The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.COLI: Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.COLI: MutM protein, could specifically bind to the C/C mismatches in DNA.