Hideki Hirose

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity.

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.

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.

1950 MHz IMT-2000 field does not activate microglial cells in vitro

Given the widespread use of the cellular phone today, investigation of potential biological effects of radiofrequency (RF) fields has become increasingly important. In particular, much research has been conducted on RF effects on brain function. To examine any biological effects on the central nervous system (CNS) induced by 1950 MHz modulation signals, which are controlled by the International Mobile Telecommunication-2000 (IMT-2000) cellular system, we investigated the effect of RF fields on microglial cells in the brain. We assessed functional changes in microglial cells by examining changes in immune reaction-related molecule expression and cytokine production after exposure to a 1950 MHz Wideband Code Division Multiple Access (W-CDMA) RF field, at specific absorption rates (SARs) of 0.2, 0.8, and 2.0 W/kg. Primary microglial cell cultures prepared from neonatal rats were subjected to an RF or sham field for 2 h. Assay samples obtained 24 and 72 h after exposure were processed in a blind manner. Results showed that the percentage of cells positive for major histocompatibility complex (MHC) class II, which is the most common marker for activated microglial cells, was similar between cells exposed to W-CDMA radiation and sham-exposed controls. No statistically significant differences were observed between any of the RF field exposure groups and the sham-exposed controls in percentage of MHC class II positive cells. Further, no remarkable differences in the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) were observed between the test groups exposed to W-CDMA signal and the sham-exposed negative controls. These findings suggest that exposure to RF fields up to 2 W/kg does not activate microglial cells in vitro.

Extremely low frequency magnetic fields and the promotion of H2O2-induced cell death in HL-60 cells

Purpose: To test whether exposure to an extremely low frequency magnetic field (60 Hz, 5 mT) affects hydrogen peroxide (H2O2)‐induced cell death in human leukaemia HL‐60 cells. Materials and methods: Cells were treated with H2O2 with or without exposure to an extremely low frequency magnetic fields. Viable cells, apoptotic and necrotic cells were determined by annexin V flow cytometry assay. The levels of apoptosis‐related proteins (caspase‐3, caspase‐7, Bcl‐2 and Bax) and poly(ADP‐ribose) polymerase were detected using Western blotting. Results: Simultaneous treatment with exposure to the magnetic field and H2O2 (85 or 100 µM) for 24 h increased the number of apoptotic and necrotic cells significantly, and significantly decreased the number of viable cells compared with cells treated with H2O2 alone. The protein levels of Bax and Bcl‐2 showed no differences between H2O2‐treated cells and those treated with both H2O2 and an extremely low frequency magnetic field. Exposure to the magnetic field also had no effect on H2O2‐induced caspase‐3 activation. However, the protein levels of active caspase‐7 in cells simultaneously exposed to an extremely low frequency magnetic field and H2O2 for 2 and 8 h was higher than that of H2O2 treatment alone. In addition, simultaneous exposure to an extremely low frequency magnetic field and H2O2 caused poly(ADP‐ribose) polymerase cleavage and induced early inactivation at 2 h, while H2O2 treatment alone did not produce this effect until 4 h. Conclusions: The data suggest that although the magnetic field itself cannot induce apoptosis and necrosis, it exerts a promoting effect on H2O2‐induced cell death, and it demonstrates that caspase‐7 as well as poly(ADP‐ribose) polymerase might be involved in this process.