Effects of 1800 MHz radiofrequency fields on signal transduction and antioxidant proteins in human A172 glioblastoma cells.

PURPOSE: To assess the effects of 1800 MHz radiofrequency electromagnetic field (RF-EMF) exposure on the expression of signal transduction and antioxidant proteins in a human-derived A172 glioblastoma cell line. MATERIALS AND METHODS: Adherent human-derived A172 glioblastoma cells (1.0 × 105 cells per 35 mm culture dish, containing 2 mL DMEM media) were exposed to 1800 MHz continuous-wave (CW) or GSM-modulated RF fields, in the presence or absence of serum for 5, 30 or 240 min at a specific absorption rate (SAR) of 0 (sham) or 2.0 W/kg. Concurrent negative (vehicle) and positive controls (1 µg/mL anisomycin) were included in each experiment. Cell lysates were collected immediately after exposure, stabilized by protease and phosphatase inhibitors in lysis buffer, then frozen and maintained at -80 °C until analysis. The relative expression levels of phosphorylated- and total-signal transduction proteins (CREB, JNK, NF-κB, ERK1/2, Akt, p70S6K, STAT3 and STAT5) and antioxidant proteins (SOD1, SOD2, CAT, TRX1, PRX2) were assessed using Milliplex magnetic bead array panels and a MagPix Multiplex imaging system. RESULTS: In cells exposed to 1800 MHz continuous-wave RF-EMF with the presence of serum in the culture medium, CAT expression was statistically significantly decreased after a 30 min exposure, total JNK was decreased at both 30 and 240 min of exposure, STAT3 was decreased after 240 min of exposure and phosphorylated-CREB expression was decreased after 30 min of exposure. In cells exposed to 1800 MHz GSM-modulated RF-EMF in serum-free cultures, the expression level of total STAT5 was decreased after 30 and 240 min of exposure. These observed changes were detected sporadically across time-points, culture conditions and RF-EMF exposure conditions indicating the likelihood of false positive events. When cells were treated with anisomycin for 15 min as a positive control, dramatic increases in the expression of phosphorylated signaling proteins were observed in both serum-starved and serum-fed A172 cells, with larger fold change increases in the serum-free cultures. No statistically significant differences in the expression levels of SOD1, SOD2 or TRX1 were observed under any tested conditions after exposure to RF-EMF. CONCLUSIONS: The current study found no consistent evidence of changes in the expression of antioxidant proteins (SOD1, SOD2, CAT or TRX2) or a variety of signal transductions proteins (CREB, JNK, NF-κB, ERK1/2, Akt, p70S6K, STAT3, STAT5) in a human-derived glioblastoma A172 cell line in response to exposure to 1800 MHz continuous-wave or GSM-modulated RF-EMF for 5, 30 or 240 min in either serum-free or serum-containing cultures.

Analysis of gene expression in mouse brain regions after exposure to 1.9 GHz radiofrequency fields.

PURPOSE: To assess 1.9 GHz radiofrequency (RF) field exposure on gene expression within a variety of discrete mouse brain regions using whole genome microarray analysis. MATERIALS AND METHODS: Adult male C57BL/6 mice were exposed to 1.9 GHz pulse-modulated or continuous-wave RF fields for 4 h/day for 5 consecutive days at whole body average (WBA) specific absorption rates of 0 (sham), ∼0.2 W/kg and ∼1.4 W/kg. Total RNA was isolated from the auditory cortex, amygdala, caudate, cerebellum, hippocampus, hypothalamus, and medial prefrontal cortex and differential gene expression was assessed using Illumina MouseWG-6 (v2) BeadChip arrays. Validation of potentially responding genes was conducted by RT-PCR. RESULTS: When analysis of gene expression was conducted within individual brain regions when controlling the false discovery rate (FDR), no differentially expressed genes were identified relative to the sham control. However, it must be noted that most fold changes among groups were observed to be less than 1.5-fold and this study had limited ability to detect such small changes. While some genes were differentially expressed without correction for multiple-comparisons testing, no consistent pattern of response was observed among different RF-exposure levels or among different RF-modulations. CONCLUSIONS: The current study provides the most comprehensive analysis of potential gene expression changes in the rodent brain in response to RF field exposure conducted to date. Within the exposure conditions and limitations of this study, no convincing evidence of consistent changes in gene expression was found in response to 1.9 GHz RF field exposure.

Cylindrical waveguide electromagnetic exposure system for biological studies with unrestrained mice at 1.9 GHz.

This paper presents the development of an in vivo exposure system for exposing small rodents. The system consists of four identical cylindrical waveguide chambers, each with a plastic cage for housing the animal. The chamber is fed by circularly polarized radiofrequency power in the 1.9 GHz cellular frequency band and is vertically mounted so that the long axis of the animal is co-planar with the rotating incident electric field. Power sensors were used along with directional or hybrid couplers and a digital voltmeter for data acquisition for real-time dose rate monitoring. The system was tested to evaluate its dose rate performance when a mouse phantom or a mouse cadaver was inside the cage. The dose rate was quantified in terms of whole-body-average (WBA) specific absorption rate (SAR) per input power using both measurement and computational methods. The exposures of the mouse phantom and cadaver were evaluated for various possible postures and positions. The measurement results showed that the highest WBA-SAR was 16.9 W kg per 1 W incident power when the cadaver was lying prone against the cage wall and the lowest WBA-SAR was 10.4 W kg per 1 W incident power when the cadaver was standing upright in the cage center. These results were found to be in good agreement with those obtained from the computational method.

Dosimetry evaluation of a cylindrical waveguide chamber for unrestrained small rodents at 1.9 GHz.

An exposure system, consisting of four identical cylindrical waveguide chambers, was developed for studying the effects of radiofrequency (RF) energy on laboratory mice at a frequency of 1.9 GHz. The chamber was characterized for RF dose rate as a function of animal body mass and dose rate variations due to animal movement in the cage. Dose rates were quantified in terms of whole-body average (WBA) specific absorption rate (SAR), brain average (BA) SAR and peak spatial-average (PSA) SAR using measurement and computational methods. Measurements were conducted on mouse cadavers in a multitude of possible postures and positions to evaluate the variations of WBA-SAR and its upper and lower bounds, while computations utilizing the finite-difference time-domain method together with a heterogeneous mouse model were performed to determine variations in BA-SAR and the ratio of PSA-SAR to WBA-SAR. Measured WBA-SAR variations were found to be within the ranges of 9-23.5 W/kg and 5.2-13.8 W/kg per 1 W incident power for 20 and 40 g mice, respectively. Computed BA-SAR variations were within the ranges of 3.2-10.1 W/kg and 3.3-9.2 W/kg per 1 W incident power for 25 and 30 g mouse models, respectively. Ratios of PSA-SAR to WBA-SAR, averaged over 0.5 mg and 5 mg tissue volumes, were observed to be within the ranges of 6-15 and 4-10, respectively.

Biological effects of alpha particle radiation exposure on human monocytic cells.

Radon ((222)Rn) gas produces decay progeny that emits high energy alpha (α)-particles. Epidemiological studies have shown that exposure to (222)Rn is linked with elevated risk of developing lung cancer, however clear mechanisms leading to such effects have not been delineated. Cytokines play a critical role in inflammation and their dysregulated production often contributes to disease pathogenesis. In this study, Bio-plex multiplex technology was employed to investigate modulations of 27 pro-inflammatory cytokines following exposure of human monocytic cells to 1.5 Gy of α-particle radiation. Concurrently, DNA damage was assessed by examining the formation of phosphorylated H2A histone family X (γ-H2AX) sites. Of the 27 cytokines assessed, 4 cytokines were shown to be statistically downregulated by ∼2 fold relative to the untreated controls and included the interleukin (IL) family of proteins (IL-2, IL-15 and IL-17) and macrophage inflammatory protein 1 beta (MIP-1b). Interferon-inducible protein-12 (IP-12), vascular endothelial growth factor and regulated on activation normal T cell expressed and secreted (RANTES) were shown to be high expressors and upregulated. Cells irradiated with α-particles ranging from 0.27 to 2.14 Gy showed statistically significant, dose-dependant increases in γ-H2AX formation. These data suggest that α-particle radiation causes dysregulation in the production of a number of pro-inflammatory cytokines and results in significant DNA damage.

The genotoxicity of mainstream and sidestream marijuana and tobacco smoke condensates.

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.

Assessment of genetic damage in peripheral blood of human volunteers exposed (whole-body) to a 200 muT, 60 Hz magnetic field.

AIM: To investigate the extent of damage in nucleated cells in peripheral blood of healthy human volunteers exposed to a whole-body 60 Hz, 200 microT magnetic field. MATERIALS AND METHODS: In this study, 10 male and 10 female healthy human volunteers received a 4 h whole-body exposure to a 200 microT, 60 Hz magnetic field. In addition, five males and five females were treated in a similar fashion, but were exposed to sham conditions. For each subject, a blood sample was obtained prior to the exposure period and aliquots were used as negative- (pre-exposure) and positive- [1.5 Gray (Gy) (60)Cobalt ((60)Co) gamma-irradiation] controls. At the end of the 4 h exposure period, a second blood sample was obtained. The extent of DNA damage was assessed in peripheral human blood leukocytes from all samples using the alkaline comet assay. To detect possible clastogenic effects, the incidence of micronuclei was assessed in phytohemagglutinin (PHA)-stimulated lymphocytes using the cytokinesis-block micronucleus assay. RESULTS: There was no evidence of either increased DNA damage, as indicated by the alkaline comet assay, or increased incidence of micronuclei (MN) in the magnetic field exposed group. However, an in vitro exposure of 1.5 Gy gamma-irradiation caused a significant increase in both DNA damage and MN induction. CONCLUSIONS: This study found no evidence that an acute, whole-body exposure to a 200 microT, 60 Hz magnetic field for 4 hours could cause DNA damage in human blood.

Analysis of gene expression in two human-derived cell lines exposed in vitro to a 1.9 GHz pulse-modulated radiofrequency field.

There is considerable controversy surrounding the biological effects of radiofrequency (RF) fields, as emitted by mobile phones. Previous work from our laboratory has shown no effect related to the exposure of 1.9 GHz pulse-modulated RF fields on the expression of 22,000 genes in a human glioblastoma-derived cell-line (U87MG) at 6 h following a 4 h RF field exposure period. As a follow-up to this study, we have now examined the effect of RF field exposure on the possible expression of late onset genes in U87MG cells after a 24 h RF exposure period. In addition, a human monocyte-derived cell-line (Mono-Mac-6, MM6) was exposed to intermittent (5 min ON, 10 min OFF) RF fields for 6 h and then gene expression was assessed immediately after exposure and at 18 h postexposure. Both cell lines were exposed to 1.9 GHz pulse-modulated RF fields for 6 or 24 h at specific absorption rates (SARs) of 0.1-10.0 W/kg. In support of our previous results, we found no evidence that nonthermal RF field exposure could alter gene expression in either cultured U87MG or MM6 cells, relative to nonirradiated control groups. However, exposure of both cell-lines to heat-shock conditions (43 degrees C for 1 h) caused an alteration in the expression of a number of well-characterized heat-shock proteins.

Fractionated X-radiation treatment can elicit an inducible-like radioprotective response that is not dependent on the intrinsic cellular X-radiation resistance/sensitivity.

Inducible responses are well documented to play a role in the radiation response of cells. However, it is not known whether clinically relevant fractionated X-radiation treatment could elicit an inducible-like radioprotective response and whether there is a direct correlation between the inducible radiation response phenomenon and the intrinsic radiation response of the cell. Therefore, the purpose of this study was to determine whether closely related human colorectal tumor (HCT116) clones treated with fractionated X rays could elicit an inducible-like radiation response to a subsequent acute (i.e. single) X-ray challenge, and whether the magnitude of the inducible-like response correlates with the intrinsic X-ray resistance of the responding clones. After fractionated X irradiation, only the radiosensitive clone showed enhanced clonogenic survival with a subsequent acute X-ray exposure. Cell cycle changes or the selection of subclones with increased intrinsic radiation resistance induced by the fractionated X rays were excluded as the basis of this enhanced tolerance, suggesting the presence of an inducible-like radioprotective response. Using the comet assay, we found similar amounts of intrinsic DNA damage among the clones after acute X irradiation. Our findings demonstrate that fractionated X-ray treatment can elicit an inducible-like radioprotective response and represent the first evidence that this response is independent of the intrinsic radiation resistance/sensitivity of the responding cells.

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood.

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.

Prompt and delayed nonsteroidal anti-inflammatory drug-photoinduced DNA damage in peripheral blood mononuclear cells measured with the comet assays.

Nonsteroidal anti-inflammatory drug (NSAID)-photoinduced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid-photoinduced DNA damage was promptly induced (i.e. observed at relatively low radiation doses), Ketoprofen-photoinduced DNA damage was delayed. This prompt and delayed effect is observed with UVA (320-400 nm), UVB (290-320 nm) and solar-simulated radiation and is attributed to the different photochemical properties of NSAID. The results from these experiments, carried out in living cells, confirm the speculations of NSAID-photoinduced DNA damage brought up by the many experiments conducted in solution.