1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis <i>In Vitro</i>

<div><p>Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome <i>c</i>-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m². Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome <i>c</i> release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome <i>c</i> from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis.</p></div

The microwave exposure system.

<p>(A) 1800MHz microwave generator. (B) Antenna in an incubator. The antenna was connected to a microwave generator via a flexible coaxial cable and enclosed in a humidified incubator. Petri dishes were positioned on the top of the antenna and cells in petri dishes were exposed to continuous microwave radiation at 1800MHz 1209 mW/m².</p

Schematic representation of p53 activation of caspase-3 signaling pathway in 1800MHz microwave induced cell apoptosis.

<p>1800MHz microwave activates p53 signaling pathway through enhancing ROS production and DNA damage, p53 further upregulates cytochrome <i>c</i>-mediated caspase-3 activation that leads to cell apoptosis. MW, microwave; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species.</p

Exposure to 1800MHz microwave induces ROS production in NIH/3T3 and U-87 MG cells.

<p>(A, B) Intracellular ROS production was measured by using DCFH-DA after 1800MHz microwave radiation for the indicated time in NIH/3T3 and U-87 MG cells. (C, D) detection of mitochondrial ROS production by using MitoSOX Red after 1800MHz microwave exposure for 6 hours in NIH/3T3 and U-87 MG cells. The imaging experiments were implemented on a two-photon laser scanning microscope system. DCF, 2’,7’-Dichlorodihydrofluorescin diacetate; mROS, mitochondrial reactive oxygen species; MW, microwave; NAC, N-acetyl-L-cysteine.Scale bars: 10 μm. Data represent mean ± S.D. (n = 3; *P < 0.05 vs. sham group.)</p

Intensity test of microwave radiation for various electronic devices (peak reading XYZ mode).

<p>Intensity test of microwave radiation for various electronic devices (peak reading XYZ mode).</p

ROS and p53 involve in cytochrome <i>c</i>-mediated caspase-3 activation during 1800MHz microwave-induced cell apoptosis.

<p>(A, B) Effects of NAC, PIF-α, Ac-DEVD-CHO and Z-VAD-FMK on cell viability and caspase-3 activity after 1800MHz microwave radiation for 24 hours in NIH/3T3 and U-87 MG cells. Cells were pretreated with treatments (NAC, PIF-α, Ac-DEVD-CHO and Z-VAD-FMK) for 20 min and then exposed to 1800MHz microwave or sham treatment for 24 hours. After microwave radiation, cells were harvested and levels of cell viability and caspase-3 activity were measured in NIH/3T3 and U-87 MG cells. Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was used to evaluate cell viability after various treatments. Caspase-3 activity was measured by the caspase-3 activity kit. (C, D) Western blotting analysis of caspase-3 activation and cytochrome <i>c</i> release from mitochondria to cytoplasm. Cells were pretreated with or without NAC and PIF-α for 20 min before exposure to 1800MHz microwave. After microwave radiation for 24 hours, cells were harvested and measured protein levels of caspase-3. The cytoplasm proteins were extracted using a mitochondria/cytoplasm fractionation kit followed by western blotting analysis of cytochrome <i>c</i> in cytoplasm. MW, microwave; NAC, N-acetyl-L-cysteine. PIF-α, pifithrin-α. All the data in these figures are presented as mean ± S.D. (n = 3; *P < 0.05 vs. control group; #P < 0.05 indicated group.)</p

ROS upregulates p53 expression during 1800MHz microwave-induced cell apoptosis.

<p>(A, B) ROS induces p53 expression upon 1800MHz microwave radiation for 24 hours in NIH/3T3 and U-87 MG cells. Cells were pretreated with or without NAC and exposed to 1800MHz. The representative western blot assay of p53 exposed to 1800MHz microwave in the presence of NAC in mouse NIH/3T3 and human U-87 MG cells, respectively. (C, D) Detection of the expression of Bax and PUMA, respectively, in mouse NIH/3T3 cells. (E,F) Detection of the expression of GLS2 and Bax, respectively, in human U-87 MG cells. The level of loading controls was used to normalize the expression of p53 target genes: Bax, PUMA, and GLS2. MW, microwave; NAC, N-acetyl-L-cysteine; PIF-α, pifithrin-α. All the data in these figures are presented as mean ± S.D. (n = 3; *P < 0.05 vs. indicated group.)</p

Exposure to 1800MHz microwave induces oxidative DNA damage in NIH/3T3 and U-87 MG cells.

<p>(A) In situ TUNEL detection of DNA fragmentation. (B) Hoechst 33258 staining assessment of nuclear condensation in NIH/3T3 and U-87 MG cells. Fluorescent images of DNA damaged cells were visualized under an Olympus fluorescent microscope (20×). (C, D) Quantitative analysis of DNA damages in NIH/3T3 and U87-MG cells stained by Hoechst 33258 shown in (B). MW, microwave; NAC, N-acetyl-L-cysteine. Data represent mean ± S.D. (n = 3; *P < 0.05 vs. sham group.)</p

1800 MHz microwave induces caspase-3 activation and decrease in cell viability.

<p>(A, B) Levels of cell viability after 1800MHz microwave radiation for the indicated time in NIH/3T3 and U-87 MG cells. Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was used to evaluate cell viability after various treatments. (C, D) Levels of caspase-3 activity after 1800MHz microwave radiation for the indicated time in NIH/3T3and U-87 MG cells. After microwave radiation, cells were harvested, and caspase-3 activity was measured by the caspase-3 activity kit. MW, microwave. Data are the mean ± S.D. (n = 3; *P < 0.05 vs. sham group.)</p