The role of the NF-κB, SAPK/JNK, and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves
Glushkova OV, Khrenov MO, Novoselova TV, Lunin SM, Parfenyuk SB, Alekseev SI, Fessenko EE, Novoselova EG. The role of the NF-κB, SAPK/JNK, and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves. Int J Radiat Biol. 2014 Dec 15:1-24. [Epub ahead of print].
Abstract
Purpose: To investigate the role of the toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB ), and stress activated protein kinases/Jun N-terminal kinase (SAPK/JNK) signalling pathways in the responses of RAW 264.7 macrophages to low-intensity microwaves (MW).
Materials and methods: Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment.
Results: MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells.
Conclusions: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.
http://1.usa.gov/1w34thB
Excerpts
A sweep-type generator YA2R-76/2 (Russia) providing swinging frequencies in the range of 8.15 to 18 GHz at an output power of 2 mW was used as the source of MW. The direct and reversed sweep times were 1.0 s and 16 ms, respectively ... The average IPD across the full area of the flask was 1.4 μW/cm2. The cells were exposed for 1 h. The temperature elevation following MW exposure was within the accuracy of the temperature measurement with a thermocouple of ±0.1°C ...
The static component of magnetic field was about 42 μT. The magnitude of the industrial AC component at 50 Hz was less than 30 nT.
The studies performed at the end of the last and the beginning of the present centuries reported the positive MW effects at a cellular level (Goswami et al. 1999, Czyz 2004, Franzelliti 2008, Cleary 1997, Leszczynski 2002, Fritze 1997). However, recent publications often report the absence of any MW effect on the production of stress and signalling proteins and on the expression of their genes in mammalian cells (Lee et al. 2006, Whitehead et al. 2005, 2006, Hirose et al. 2007, Lim et al., 2005). In addition, many publications have reported that MW does not produce any genotoxic, cancerogenic, and mutational effects (Juutilainen et al. 2011, Leszczynski 2013). However, there is no doubt that modulated MW can cause neural effects. As shown in some previous studies, MW modifies electroencephalography (Hinrikus et al. 2008, Croft at al. 2010, Regel et al. 2007a), cerebral blood flow (Huber et al. 2005, Fritze 1997), and memory processes (Regel et al. 2007b) in humans and animals. Undoubtedly, such marked changes in physiological processes in human and animal organisms produced by low-intensity MW exposure are accompanied with modifications of cellular responses. However, to date, the molecular mechanisms of the effects of MW remain unclear. In the present study, we showed that MW can not only produce immunomodulatory action but also change the intracellular signalling profile. In accordance with the obtained data, participants of the NF-κB signalling pathway were found to be potential triggers of the MW effect on cells. Friedman with co-author detected the immediate effect of mobile phone frequencies irradiation on the MAPK cascades, and found that extracellular-signal-regulated kinases, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities (Friedman et al. 2007). Further investigation of the MW sensitivity of receptor systems causing an activation of the NF-κB and other cellular signalling pathways will allow us to identify the mechanisms underlying the primary acceptance of MW by living cells.
--
Joel M. Moskowitz, Ph.D., Director
Center for Family and Community Health
School of Public Health
University of California, Berkeley
Electromagnetic Radiation Safety
Website: http://www.saferemr.com
Facebook: http://www.facebook.com/SaferE
News Releases: http://pressroom.prlog.org/
Twitter: @berkeleyprc
Materials and methods: Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment.
Results: MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells.
Conclusions: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.
http://1.usa.gov/1w34thB
Excerpts
A sweep-type generator YA2R-76/2 (Russia) providing swinging frequencies in the range of 8.15 to 18 GHz at an output power of 2 mW was used as the source of MW. The direct and reversed sweep times were 1.0 s and 16 ms, respectively ... The average IPD across the full area of the flask was 1.4 μW/cm2. The cells were exposed for 1 h. The temperature elevation following MW exposure was within the accuracy of the temperature measurement with a thermocouple of ±0.1°C ...
The static component of magnetic field was about 42 μT. The magnitude of the industrial AC component at 50 Hz was less than 30 nT.
The studies performed at the end of the last and the beginning of the present centuries reported the positive MW effects at a cellular level (Goswami et al. 1999, Czyz 2004, Franzelliti 2008, Cleary 1997, Leszczynski 2002, Fritze 1997). However, recent publications often report the absence of any MW effect on the production of stress and signalling proteins and on the expression of their genes in mammalian cells (Lee et al. 2006, Whitehead et al. 2005, 2006, Hirose et al. 2007, Lim et al., 2005). In addition, many publications have reported that MW does not produce any genotoxic, cancerogenic, and mutational effects (Juutilainen et al. 2011, Leszczynski 2013). However, there is no doubt that modulated MW can cause neural effects. As shown in some previous studies, MW modifies electroencephalography (Hinrikus et al. 2008, Croft at al. 2010, Regel et al. 2007a), cerebral blood flow (Huber et al. 2005, Fritze 1997), and memory processes (Regel et al. 2007b) in humans and animals. Undoubtedly, such marked changes in physiological processes in human and animal organisms produced by low-intensity MW exposure are accompanied with modifications of cellular responses. However, to date, the molecular mechanisms of the effects of MW remain unclear. In the present study, we showed that MW can not only produce immunomodulatory action but also change the intracellular signalling profile. In accordance with the obtained data, participants of the NF-κB signalling pathway were found to be potential triggers of the MW effect on cells. Friedman with co-author detected the immediate effect of mobile phone frequencies irradiation on the MAPK cascades, and found that extracellular-signal-regulated kinases, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities (Friedman et al. 2007). Further investigation of the MW sensitivity of receptor systems causing an activation of the NF-κB and other cellular signalling pathways will allow us to identify the mechanisms underlying the primary acceptance of MW by living cells.
--
Joel M. Moskowitz, Ph.D., Director
Center for Family and Community Health
School of Public Health
University of California, Berkeley
Electromagnetic Radiation Safety
Website: http://www.saferemr.com
Facebook: http://www.facebook.com/SaferE
News Releases: http://pressroom.prlog.org/
Twitter: @berkeleyprc
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