In vitro and in vivo genotoxicity of radiofrequency fields.

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation.

Effects of amiloride on thermosensitivity of Chinese hamster cells under neutral and acidic pH.

The modifying effects of amiloride on the thermosensitivity of Chinese hamster V-79 cells were examined under both neutral (pH 7.3) and acidic (pH 6.6) conditions. Amiloride, a diuretic drug, is known to inhibit the Na+/H+ exchange activity. Under the extracellular pH of 7.3, amiloride (0.1-0.5 mM) enhanced the thermal cell killing powers of 42 degrees C hyperthermia with increasing concentration and exposure time of the drug. The age response of cells to 42 degrees C hyperthermia in the presence or absence of amiloride (0.5 mM) showed that amiloride sensitized cells to heat, especially those at G1-S boundary through middle S phases. On the other hand, the lowering of extracellular pH to 6.6 enhanced cell killing by 42 degrees C hyperthermia. When cells were exposed to 42 degrees C hyperthermia in the presence of amiloride at pH 6.6, cell survival decreased still more. The thermosensitizing effects of the lowered pH at 6.6 and amiloride appeared to be additive. From these results, it is suggested that the thermosensitization by amiloride is probably due, in part, to the inhibition of cellular Na+/H+ exchange activity. The present study proposes the possibility that amiloride may be useful as a hyperthermic sensitizer in a clinical treatment of cancer.

Induction of c-fos gene expression by exposure to a static magnetic field in HeLaS3 cells.

Effects of a static magnetic field on cell growth and c-fos oncogene expression were investigated. HeLaS3 cells exposed to the magnetic field of 0.18-0.2 T for 1-6 days were not affected in the cell growth. Exposure to the magnetic field did not enhance the effects of X-rays or heat treatment which caused the transient cell growth delay. c-fos mRNA in the HeLaS3 cells was undetectable in untreated cells, but the expression was induced in cells by the magnetic field exposure for 2-24 h. The amounts of the mRNA expression changed time dependently with a peak at a 6-h exposure. c-fos was expressed following the heat treatment at 45 degrees C for 10 and 15 min, and the expression was further enhanced by the treatment of cells with heating followed by 4 h of the magnetic field exposure. Exposure of the cells to a static magnetic field may affect some cellular metabolic events leading to the c-fos gene expression.

Absence of IFNA and IFNB genes from human malignant glioma cell lines and lack of correlation with cellular sensitivity to interferons.

We report that 5 of 19 human malignant glioma cell lines have neither interferon alpha (IFNA) nor interferon beta (IFNB) genes that are detectable by Southern blotting. Of 5 other of these malignant glioma lines that have a single IFNB gene copy, 3 lack the IFNA genes entirely and two have one copy. One of the lines that lacks the IFNA genes entirely but has one copy of the IFNB gene has a rearrangement near the IFNB gene that is most easily interpreted as an insertion of a large segment of DNA (at least 50 kilobases) the 3' end of which is less than 1.3 kilobases 5' to the known regulatory sequences of the IFNB gene. In spite of the rearrangement, IFNB-specific RNA is highly inducible in this line by poly(I)-poly(C). The ability of interferon alpha or interferon beta to inhibit cell growth does not depend upon the presence or absence of the respective gene. This finding adds solid tumors to those tumor cell lines (acute lymphocytic leukemia, chronic myelogeneous leukemia) previously determined to lack the IFNA and IFNB genes (Diaz et al., Proc. Natl. Acad. Sci. USA, 85:5259-5263, 1988).

Activation of c-mos oncogene by integration of an endogenous long terminal repeat element during transfection of genomic DNA from mouse skin tumor cells.

An activated c-mos oncogene was identified in a transformed clone of golden hamster embryo cells transfected with DNA extracted from cells cultured from a UV-induced mouse skin tumor. Southern blot hybridization with a v-mos oncogene probe showed that the mos oncogene was amplified in the primary and secondary transformed cells but not in the original tumor cells. Expression of the mos oncogene was very high in the primary and secondary transformants, but mos mRNA was undetectable in the original tumor cells. A genomic DNA fragment containing the activated mos oncogene was cloned and sequenced. The upstream mouse sequence of the mos oncogene, which functions as the transcription terminator, was lost and replaced by a mouse endogenous long terminal repeat (LTR) element that provides the promoter sequence, resulting in high expression of the gene. The rearrangement apparently occurred during transfection, since the polymerase chain reaction (PCR) product encompassing the junction region was present in the primary and secondary transformants but not in the original tumor cells. The LTR element is likely to have been amplified during the skin tumor development caused by UV irradiation. Southern blot hybridization showed that the copy number of LTR in the tumor cells was significantly higher than that in normal skin cells. The amplification of the LTR in the cells may have increased the chance of recombination between the LTR and c-mos gene during the DNA transfection.

Lack of expression of tumor-suppressor genes in human malignant glioma cell lines.

Human malignant gliomas (glioblastomas and anaplastic astrocytomas) are the most frequent brain tumors and are associated with a variety of genetic alterations including retinoblastoma (RB) and p53 gene mutations, loss of interferon alpha and beta (IFNA, IFNB) genes and lack of O6-methylguanine-DNA methyltransferase (MGMT) expression. Yet, in the studies performed to date, the relationship between these alterations has not been addressed. In this report, we have studied gene expression in 29 malignant glioma cell lines and have determined that, although loss of the interferon genes and loss of RB, p53 and MGMT mRNAs are frequent events, combinations of genetic alterations involving these four proven or putative tumor-suppressor genes are relatively infrequent. The exception was loss of RB mRNA, which may be associated with lack of MGMT mRNA.

Skin responses to step-up and step-down heating in C3H mice.

The effects of step-up (42 leads to 44 degrees C sequence) and step-down (44 leads to 42 degrees C sequence) heating were studied on the skin of C3H/He mouse feet. Skin damage in mouse feet exposed to 44 degrees C alone became proportionally more severe with increasing exposure time, while it was slight or not observed at 42 degrees C for up to 2 hr. Preheating to 42 degrees C for 60 or 120 min (step-up) had little effect on the damage from 30-60 min exposure at 44 degrees C, but increased the damage seen with 90 min at 44 degrees C. However, skin damage was markedly enhanced by exposure to step-down heating. On the other hand, thermal resistance was induced in the mouse skin by 44 degrees C fractionated treatments. The induced thermal resistance reached a maximum with a 1 day interval, and then disappeared after a 3 day interval. In another fractionation schedule of step-down heating, the enhanced skin damage which was observed when here was no interval, recovered rapidly and attained an additive response within a 1 day interval. No thermal resistance was observed.

Increase in radiation sensitivity of human malignant melanoma cells by expression of wild-type p16 gene.

The influence of wild-type p16 expression on the radiation sensitivity of human melanoma cell lines was investigated. MeWo cells, which alone expressed intrinsic wild-type p16 among six melanoma cell lines examined, showed higher radiosensitivity in comparison with the other five melanoma cells. The introduction of human wild-type p16 cDNA into A875 cells, which homozygously lost p16 genes, and AKI cells, which retained p16 gene but did not express p16 mRNA, led to increased sensitivity of those cells to X-ray irradiation. The radiosensitizing effect by the p16 introduction to those cells was prominent after rather higher doses of X-rays (8 and 10 Gy). In both A875 and AKI, no significant difference in sensitivities to UVC and cisplatin was observed between the parental and p16-transfectant cells. These results suggest that the loss or dysfunction of p16 gives melanoma cells the radioresistant characteristics.

Reduced UV-induced mutations in human osteosarcoma cells stably expressing transfected wild-type p53 cDNA.

We constructed the plasmid which can express human wild-type p53 cDNA and introduced it into the human osteosarcoma cell line SAOS-2 that lacks the chromosomal p53 gene. A cell clone stably expressing p53 protein was isolated and UV sensitivity and UV-induced mutation frequencies of the clone were examined. The UV sensitivity of the clone was slightly higher and UV-induced hprt mutation frequencies of the clone were markedly lower than those of parental SAOS-2 cells. The capability to repair UV-induced DNA damage assessed by the amount of unscheduled DNA synthesis or DNA single strand breaks as well as cell cycle progression after UV irradiation were not different between the clone and SAOS-2 cells. These results indicate that wild-type p53 protein would be involved in the human DNA damage-processing pathway other than the genome-overall excision repair.

Thermosensitization by methylglyoxal bis(guanylhydrazone) in Chinese hamster cells.

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.

Effects of m-aminobenzamide on the response of Chinese hamster cells to hyperthermia and/or radiation.

The modifying effects of m-aminobenzamide (m-ABA), an inhibitor of poly(ADP-ribose) synthesis, on 42 degrees C hyperthermia- and/or radiation-induced cell killing were examined in Chinese hamster V-79 cells. When cells were exposed to 42 degrees C hyperthermia in combination with m-ABA (10 mM), cell survival decreased compared with that for 42 degrees C hyperthermia alone. Thermosensitizing effects of m-ABA changed with treatments in a decreasing order of during and after heating greater than during heating greater than after heating. Treatments with m-ABA during and/or after X irradiation enhanced radiation-induced cell killing. When cells were exposed to combined treatment with X irradiation, 42 degrees C hyperthermia (60 min), and m-ABA (24 hr), cell survival decreased markedly compared with that for X irradiation alone. However, with both X----42 degrees C and X----42 degrees C----m-ABA, the enhancement ratios (ER), designated as D0 ratio, were similar. These results suggest that the mechanisms of radiosensitization by m-ABA may be similar to those of 42 degrees C hyperthermia.

Development of a cryopreservation procedure employing a freezer bag for pancreatic islets using a newly developed cryoprotectant.

One of the most important requirements for success in clinical islet transplantation is the use of a large number of viable donor islets. To achieve this, the ability to cryopreserve islets and to establish an islet bank are critical. Previously, we developed a two-step cryopreservation procedure with freezing tubes utilizing low and high concentrations of dimethyl sulfoxide (DMSO) and using a fully automated cryomachine for human pancreatic islets and porcine islet-like cell clusters (ICCs). Based on these experiments, we developed a simple and efficient cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant consisting of ethylene glycol (EG) instead of DMSO for decreasing injury of the islets by freezing. A 250 ml Cryocyte blood freezer bag and our newly developed cryoprotectant containing ethylene glycol (EG) were used in the freezing procedure. The islets were frozen by a fully automated computer-controlled cryomachine (GE 9,000) with our original program of slow cooling. Nucleation occurred at -8 degrees C, and the frozen islets were stored at -196 degrees C in a liquid nitrogen tank. The frozen-stored islets were subsequently rapidly thawed in a 37 degrees C water bath and cultured before viability testing. In vitro function, the stimulation index of insulin release during the static incubation test for rat islets cryopreserved in a freezer bag vs. nonfrozen islets as control, was 2.13 +/- 0.42 and 2.02 +/- 0.38 (94.8% compared with control), respectively (n = 5, p = NS). The islet recovery compared with the nonfrozen control group was 85% (n = 5) in insulin content. When 1000 rat islets cryopreserved in a freezer bag were transplanted into the renal capsule of diabetic athymic mice, all the mice became normoglycemic within 7 days from transplantation. Before nephrectomy, the intravenous glucose torelance test (IVGTT) was performed. The fractional decay constant of the glucose level (K value) of the frozen-thawed group was 0.42 +/- 0.06%/min. A histological study of renal subcapsular grafts demonstrated the morphological integrity of the islets. These results demonstrate the utility of our cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant for the maintenance of viability and function of frozen-stored islets both in culture and after transplantation. Cryopreservation using freezer bags with the new cryoprotectant is an effective and simple method for making an islet bank for clinical trials of islet transplantation.

Decreased host-cell reactivation of UV-irradiated adenovirus in human colon tumor cell lines that have normal post-UV survival.

An ongoing study in our laboratories is to examine the relationship of DNA repair defects to human cancer. Our underlying hypothesis has been that human tumors may arise that lack interesting DNA repair pathways if these pathways are important in preventing cancer. In this study, we found that the UV-irradiated adenoviruses showed hypersensitivity when assayed on monolayers of certain human colon tumor cell lines, including three that are reported to have defects in long patch DNA mismatch repair genes and one with no reported defect in mismatch repair. The survival curves showed two components. The first sensitive component was characteristic of 77-95% of the infections depending upon the cell line and the experiment and had an average slope indicating 4.8-fold hypersensitivity to UV. The average of the second-component slopes indicated that the remainder of the infections was accompanied by near-normal repair. Although the value of the first component indicated that the colon tumor lines supported the growth of UV-damaged adenoviruses poorly, the cell lines themselves showed the same post-UV colony-forming ability as did normal human fibroblasts, and their ability to support the growth of N-methyl-N'-nitro-N-nitrosoguanidine-damaged adenoviruses was normal, i.e. it parallelled their ability to repair O6-methylguanine in vitro. We previously observed two-component survival curves when assaying UV-irradiated adenovirus on monolayers of all of seven strains of fibroblasts from Cockayne's syndrome patients. By contrast, single-component curves have been obtained using 21 strains of normal human fibroblasts and seven other tumor lines. We interpret the two-component survival curves in terms of the defective transcription-coupled repair of UV-induced DNA damage that is characteristic both of Cockayne's and certain colon tumor cell lines. In addition, four mismatch repair-deficient colon tumor lines were resistant to killing by elevated levels of dG.

Suppression of heat-induced HSP-70 by simultaneous exposure to 50 mT magnetic field.

Effect of extremely low frequency magnetic field (ELFMF) at 50 mT and 60 Hz on heat-induced expression of heat shock protein 70 (hsp-70) was examined in HL60RG cells. No increase in hsp-70 production was observed in the cells after exposure to 50 mT ELFMF alone. Simultaneous exposure to 50 mT ELFMF in combination with mild heat at 42 and 40 degrees C suppressed heat-induced hsp-70 expression. The suppression of hsp-70 occurred when cells were simultaneously exposed to both for longer periods of more than 5 h. However, the suppression of hsp-70 was not observed at a magnetic density of 5 and 0.5 mT. This result suggests that exposure to 50 mT ELFMF may act on a protection against the concomitant mild heat stress in HL60RG cells.

Increase in hypoxanthine-guanine phosphoribosyl transferase gene mutations by exposure to electric field.

Previously, we reported that exposure to extremely low frequency magnetic field (400 mT) increased in hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. However, it is unclear these mutations were induced by magnetic field (MF), electric field (EF), or both. To explore this question, a new exposure apparatus for EF was manufactured. We observed an increase in HPRT gene mutations in Chinese hamster ovary (CHO) cells after exposure to EF (10 V/m, 60 Hz) for 10 h. The mutant frequency by EF-exposure was an approximate 2-fold of that by sham-exposure. Our data suggest that the mutations induced by exposure of cells to the variable magnetic field at 400 mT may be, in part, due to the induced EF.

Enhanced radiosensitivity by inhibition of nuclear factor kappa B activation in human malignant glioma cells.

To clarify the relationship between cellular radiosensitivity and nuclear factor kappa B (NF-kappa B) activation, an expression plasmid was constructed for I kappa B-alpha, a cellular inhibitory protein of NF-kappa B, and transfected it into two human malignant glioma cell lines. Cells overexpressing the I kappa B-alpha protein were more radiosensitive than the parental cells and one transfected clone with low expression. In the parental cell lines and one transfected clone with low expression, the sequence specific DNA-binding activity of NF-kappa B was considerably increased between 1 and 2 h after irradiation. In contrast, no increase in the DNA-binding activity was observed in the transfected clone overexpressing I kappa B-alpha protein. These results suggest that the activation of NF-kappa B may be one of the intrinsic responses determining cellular radiosensitivity.

Mutation induction by high-density, 50-Hz magnetic fields in human MeWo cells exposed in the DNA synthesis phase.

Exposure of cultured human MeWo cells to high-density (400 mT at 50 Hz) extremely low frequency magnetic fields (ELF-MF) induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene. Mutation induced by the ELF-MF increased during the DNA-synthesis phase in synchronously growing cells. DNA replication errors and/or disturbance of the mismatch repair systems caused by exposure to ELF-MF may be involved in the mutagenic effect.

Inhibition of UV-induced G1 arrest by exposure to 50 Hz magnetic fields in repair-proficient and -deficient yeast strains.

PURPOSE: To assess the possibility that extremely low frequency (ELF) magnetic fields obstruct the damage repair process, the gene conversion frequency and cell cycle kinetics in a DNA repair-proficient and nucleotide excision repair (NER)-deficient strain of diploid yeast Saccharomyces cerevisiae. MATERIALS AND METHODS: DNA repair- or NER-deficient cells were irradiated with sublethal doses of ultraviolet light (UV) radiation followed by exposure to 50 Hz magnetic fields up to 30 mT for 48 h. After exposure, colony-forming ability was scored as revertants in which gene conversion had restored the functional allele of the ARG4 gene conversion hotspot. Cell cycle analysis was performed using flow cytometry. RESULTS: Gene conversion rate was increased by the combined exposure in DNA repair-proficient cells, whereas it remained unchanged between UV alone and the combined exposure in NER-deficient cells. The UV-induced G1 arrest was inhibited by exposure to 30 mT ELF magnetic fields in both repair-proficient and -deficient cells. CONCLUSIONS: The results suggest that exposure to high-density (30 mT) ELF magnetic fields decreases the efficiency of NER by suppressing G1 arrest, which in turn led to enhancement of the UV-induced gene conversion.

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 degrees 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 degrees 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 degrees 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 degrees 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.