Cell Type-Dependent Induction of DNA Damage by 1800 MHz Radiofrequency Electromagnetic Fields Does Not Result in Significant Cellular Dysfunctions

<div><h3>Background</h3><p>Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF) as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity.</p> <h3>Objectives</h3><p>To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation.</p> <h3>Methods</h3><p>Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay.</p> <h3>Results</h3><p>Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs), but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level.</p> <h3>Conclusions</h3><p>RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.</p> </div

1800 MHz RF-EMF induces cell type-dependent DSBs as evaluated by the γH2AX foci formation assay.

<p>(A) Representative images of γH2AX immunofluorescent staining of CHL, astrocytes, FL, HUVEC, HLEC, and HSF exposed to radiation at 3.0 W/kg for either 1 h or 24 h. Red dots indicate γH2AX foci; nuclei are stained blue with DAPI. Scale bar, 10 µm. (B) Histograms showing the average numbers of γH2AX foci per cell by scoring ∼200 cells per sample. Values are mean ± SEM of at least 4 independent experiments. *<i>p</i><0.05 compared with the sham-exposed sample.</p

RF-EMF-induced γH2AX foci formation does not change cell cycle distribution in HSF cells.

<p>Histograms show the percentages of HSF cells in different phases of the cell cycle at 0 (left), 6 (middle), and 12 h (right) after 24 h exposure to 1800 MHz RF-EMF at 3.0 W/kg. Values represent mean ± SEM of 5 independent experiments. *<i>p</i><0.05 compared with sham-exposed sample (Student’s t-test).</p

RF-EMF-induced γH2AX foci formation does not result in more DNA nicks in HSF cells.

<p>Histogram of DNA fragment levels in HSF cells. The background fluorescence value of the cells (NC) was determined without adding rTdT, and 1 µM 4NQO treatment for 1 h serves as positive control. Values represent mean ± SEM of 3 independent experiments. *<i>p</i><0.05 compared with sham-exposed sample (Student’s t-test).</p

RF-EMF-induced γH2AX foci formation does not affect cell proliferation and viability in HSF cells.

<p>(A) HSF cell numbers at 0, 12, 24, and 48 h after 24 h exposure to 1800 MHz RF-EMF at 3.0 W/kg. (B) HSF cell viability at 0, 1, 2, 3, and 4 days after re-seeding at 1000 cells/well (B, left) and 2000 cells/well (B, right) immediately after 24 h exposure. 1 µM 4NQO treatment for 1 h serves as positive control. Values represent mean ± SEM of 3 independent experiments. *<i>p</i><0.05 and **<i>p</i><0.01 compared with sham-exposed sample (Student’s t-test).</p

Effect of RE-EMF exposure on ROS production in HSF cells.

<p>Histogram of ROS levels in HSF cells. The intracellular ROS level of 24 h exposed cells was measured by flow cytometry using DCFH-DA. The background fluorescence value of the cells (NC) was determined without adding DCFH-DA, and 1 µM 4NQO treatment for 1 h serves as positive control. Values represent mean ± SEM of 7 independent experiments. **<i>p</i><0.01 compared with sham-exposed sample (Student’s t-test).</p

Inter-operator variability across structures assessed on the axial slices.

<p>Inter-operator variability across structures assessed on the axial slices.</p

Segmentation of the bone.

<p>Top: coronal view of a T2-weighted MRI slice (a) with the skull, vertebrae, and intervertebral disks outlined (b). The T2-weighted MRI, in which the intensity of the cancellous bone inside the diploë is enhanced compared to that of the cortical bones of the inner and outer tables made the subdivision of the skull into the main three layers, i.e., outer table (white outline), diploë (yellow outline), and inner table (white outline) possible. Bottom: 3D reconstruction of the skull, vertebrae, and intervertebral disks (c and d).</p

Segmentation of the cerebrum.

<p>(a) Original axial T1-weighted MRI, (b) intra-dural space obtained as the space surrounded by the previously segmented dura mater, (c) masked T1-weighted MRI, and (d) the image resulting from application of the <i>k</i>-means algorithm.</p

Inter-operator variability across structures assessed on the coronal slices.

<p>Inter-operator variability across structures assessed on the coronal slices.</p