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. 2017 Mar;21(2):179-188.
doi: 10.4196/kjpp.2017.21.2.179. Epub 2017 Feb 21.

Activation of Autophagy at Cerebral Cortex and Apoptosis at Brainstem Are Differential Responses to 835 MHz RF-EMF Exposure

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Free PMC article

Activation of Autophagy at Cerebral Cortex and Apoptosis at Brainstem Are Differential Responses to 835 MHz RF-EMF Exposure

Ju Hwan Kim et al. Korean J Physiol Pharmacol. .
Free PMC article

Abstract

With the explosive increase in exposure to radiofrequency electromagnetic fields (RF-EMF) emitted by mobile phones, public concerns have grown over the last few decades with regard to the potential effects of EMF exposure on the nervous system in the brain. Many researchers have suggested that RF-EMFs can effect diverse neuronal alterations in the brain, thereby affecting neuronal functions as well as behavior. Previously, we showed that long-term exposure to 835 MHz RF-EMF induces autophagy in the mice brain. In this study, we explore whether short-term exposure to RF-EMF leads to the autophagy pathway in the cerebral cortex and brainstem at 835 MHz with a specific absorption rate (SAR) of 4.0 W/kg for 4 weeks. Increased levels of autophagy genes and proteins such as LC3B-II and Beclin1 were demonstrated and the accumulation of autophagosomes and autolysosomes was observed in cortical neurons whereas apoptosis pathways were up-regulated in the brainstem but not in the cortex following 4 weeks of RF exposure. Taken together, the present study indicates that monthly exposure to RF-EMF induces autophagy in the cerebral cortex and suggests that autophagic degradation in cortical neurons against a stress of 835 MHz RF during 4 weeks could correspond to adaptation to the RF stress environment. However, activation of apoptosis rather than autophagy in the brainstem is suggesting the differential responses to the RF-EMF stresses in the brain system.

Keywords: Apoptosis; Autophagy; Brainstem; Cerebral cortex; RF-EMF.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1. The transcription levels of autophagic-related genes in the cerebral cortex of mice in response to RF-EMF exposure for 4 weeks.
Total RNA was extracted from the cerebral cortex of sham-exposed and RF-EMF exposed mice and were analysed by quantitative real-time PCR to determine the expression level of autophagy genes. (a~h) Quantification of Atg4A/B, Beclin1/2, Atg5, Atg9A, LC3A/B mRNA transcripts by qRT-PCR. (i) 1.5% Agarose gel electrophoresis indicating the differential expression of autophagy genes by sqRT-PCR. The expressional values of the cerebral cortex of the RF-EMF exposed mice were normalized to those of the sham-exposed mice. The relative transcriptional levels of each gene were calculated by normalizing to the expression of GAPDH using the 2−ΔΔCt method (n=5). Each bar represents the mean±SEM of three independent experiments. Statistical significance was evaluated using a t-test: *p<0.05, **p<0.01, ***p<0.001.
Fig. 2
Fig. 2. The transcription levels of autophagic-related genes in the brainstem of mice following 4 weeks of exposure to RF-EMF signals.
Total RNA was extracted from the brainstem of sham-exposed and RF-EMF exposed mice and were analysed for the expressional levels of autophagy genes by quantitative real-time PCR. (a~h) Quantification of Atg4A/B, Beclin1/2, Atg5, Atg9A, LC3A/B mRNA transcripts by qRT-PCR. (i) 1.5% Agarose gel electrophoresis showing differential expression of autophagy genes by sqRT-PCR. The expressional values of the cerebral cortex of RF-exposed mice were normalized to those of the sham-exposed mice. The relative transcriptional levels of each gene were calculated by normalizing to the expression of GAPDH using the 2−ΔΔCt method (n=5). Each bar shows the mean of three independent experiments with SEM. Statistical significance was evaluated using a t-test: *p<0.05, **p<0.01.
Fig. 3
Fig. 3. Expression level for LC3B-II and Beclin1 proteins in the cerebral cortex and brainstem of mice after 4 weeks of exposure to RF-EMF radiation.
(a) Total lysates extracted from the cerebral cortex and brainstem of mice was subjected to 15% SDS–PAGE and western-blotted with antibody against LC3B-II and Beclin1 (Cell Signaling Technology, Beverly, MA, USA). α-tubulin was used as the internal loading control. (b) The band intensity of western blot was quantified by densitometry. The protein level was normalized relative to α-tubulin. Each bar shows the mean of three independent experiments with SEM. Statistical significance was evaluated using two tailed t-test: *p<0.05, **p<0.01.
Fig. 4
Fig. 4. The expression levels of apoptosis related genes in the cerebral cortex or brainstem of mice following 835 MHz RF-EMF exposure for 4 weeks.
The cerebral cortical (A) or brainstem (B) RNA and proteins extracted from sham-exposed and RF-exposed mice were analysed to determine the expression level of apoptotic genes or proteins. (a~b) Quantification of Bcl2 and Bax mRNA transcripts by qRT-PCR. (c) 1.5% Agarose gel electrophoresis showing differential expression of Bcl2 and Bax by sqRT-PCR. The expression values of the cerebral cortex of RF-exposed mice were normalized to those of the sham-exposed mice. The relative mRNA levels of each gene were calculated by normalizing to the expression of GAPDH using the 2−ΔΔCt method (n=5). (d) Total proteins were subjected to 15% SDS–PAGE and immunoblotted with antibodies against Bcl2 and Bax. α-tubulin was used as the loading control. (e) The intensity of western blot bands was quantified by densitometry. The protein level was normalized relative to α-tubulin. Each bar represents the mean±SEM of three independent experiments. Statistical significance was evaluated using a t-test: *p<0.05, **p<0.01.
Fig. 5
Fig. 5. Representative ultrastructure images showing the autophagic structure in the neuronal cell body of the cerebral cortex or brainstem following 4 weeks of exposure to RF-EMF.
Ultrastructural comparison of autophagy between sham control vs RF-EMF exposed group. Representative TEM micrographs were acquired from sham control (a and c) and RF-EMF exposed mice (b and d). Autophagosome (Ap) and autolysosome (Aly) were clearly observed in RF-EMF exposed cortex. Abbreviations are: Ap, autophagosome; Aly, autolysosome; G, Golgi apparatus; M, mitochondria; N, nucleus; Ph, phagophore; RER, rough endoplasmic reticulum. Size bars: 500 nm.

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