The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation

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TOXLET 8712 1–9

Toxicology Letters xxx (2014) xxx–xxx

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation

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Kaijun Liu a,b , Guowei Zhang b , Zhi Wang b , Yong Liu b , Jianyun Dong b , Xiaomei Dong b , Jinyi Liu b , Jia Cao b , Lin Ao b,∗ , Shaoxiang Zhang b,∗ a

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Institute of Computing Medicine, Third Military Medical University, Chongqing, China Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, China

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h i g h l i g h t s

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• 1800 MHz RF exposure (4 w/kg) could enhance autophagy flux in GC-2 cells. • Intracellular ROS and ERK phosphorylation are involved in RF induced autophagy. • Inhibition of autophagy increased the percentage of apoptotic cells with RF exposure.

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Article history: Received 12 February 2014 Received in revised form 30 April 2014 Accepted 2 May 2014 Available online xxx

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Keywords: Radiofrequency electromagnetic field ROS Autophagy

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The increasing exposure to radiofrequency (RF) radiation emitted from mobile phone use has raised public concern regarding the biological effects of RF exposure on the male reproductive system. Autophagy contributes to maintaining intracellular homeostasis under environmental stress. To clarify whether RF exposure could induce autophagy in the spermatocyte, mouse spermatocyte-derived cells (GC-2) were exposed to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rate (SAR) values of 1 w/kg, 2 w/kg or 4 w/kg for 24 h, respectively. The results indicated that the expression of LC3-II increased in a dose- and time-dependent manner with RF exposure, and showed a significant change at the SAR value of 4 w/kg. The autophagosome formation and the occurrence of autophagy were further confirmed by GFP-LC3 transient transfection assay and transmission electron microscopy (TEM) analysis. Furthermore, the conversion of LC3-I to LC3-II was enhanced by co-treatment with Chloroquine (CQ), indicating autophagic flux could be enhanced by RF exposure. Intracellular ROS levels significantly increased in a dose- and time-dependent manner after cells were exposed to RF. Pretreatment with anti-oxidative NAC obviously decreased the conversion of LC3-I to LC3-II and attenuated the degradation of p62 induced by RF exposure. Meanwhile, phosphorylated extracellularsignal-regulated kinase (ERK) significantly increased after RF exposure at the SAR value of 2 w/kg and 4 w/kg. Moreover, we observed that RF exposure did not increase the percentage of apoptotic cells, but inhibition of autophagy could increase the percentage of apoptotic cells. These findings suggested that autophagy flux could be enhanced by 1800 MHz GSM exposure (4 w/kg), which is mediated by ROS generation. Autophagy may play an important role in preventing cells from apoptotic cell death under RF exposure stress. © 2014 Published by Elsevier Ireland Ltd.

Abbreviations: RF, radiofrequency; GSM, Global System for Mobile Communication; SAR, specific absorption rates; LC3, micro-tubule-associated protein 1 light chain 3; ROS, reactive oxygen species; ERK, extracellular-signal-regulated kinase; RF-EMR, radiofrequency electromagnetic radiation; NAC, N-acetyl-cysteine; CQ, chloroquine; 3-MA, 3-methyladenine; DCFH-DA, 2,7-dichloroluorescein diacetate; DCF, fluorescent dichlorol uorescein; TEM, transmission electron microscopy. ∗ Corresponding authors. Tel.: +86 02368753708; fax: +86 02368753708. E-mail addresses: [email protected] (K. Liu), [email protected] (G. Zhang), [email protected] (Z. Wang), [email protected] (Y. Liu), [email protected] (J. Dong), [email protected] (X. Dong), [email protected] (J. Liu), [email protected] (J. Cao), [email protected] (L. Ao), [email protected] (S. Zhang). http://dx.doi.org/10.1016/j.toxlet.2014.05.004 0378-4274/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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1. Introduction

2. Methods

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The rapid growth of mobile communication has been accompanied by an increase in radiofrequency electromagnetic radiation (RF-EMR) (Merhi, 2012). Public concerns have been raised by the possible hazardous health effect of exposure to RF-EMR emitted from mobile phones. Many epidemiological studies have indicated that mobile phone use is a risk factor of brain tumors (Kundi, 2009). Although evidence from human studies and animal studies is limited, the International Agency for Research on Cancer (IARC) classifies RF-EMR as “possibly carcinogenic to humans” (Group 2B) (Baan et al., 2011). In addition to concern on the association between mobile phone use and cancer, the effects of mobile phone use on the male reproductive system have also attracted public attention, particularly when considering that mobile phones are often kept in trousers pockets and therefore close to testes for a long time (Kesari et al., 2013). Studies have indicated that recent innovations in cell phone technology have a detrimental effect on male fertility, and maybe a growing factor contributory to male infertility (Agarwal et al., 2011). Taking Bluetooth as an example, these innovations may reduce the RF-EMR exposure to brain, but in turn increase exposure to reproductive organs. Recent epidemiological studies, animal studies and in vitro laboratory studies have also indicated the possible harmful effects of RF-EMR on semen quality (Agarwal et al., 2008, 2009; De Iuliis et al., 2009; Gutschi et al., 2011; Tas et al., 2013; Veerachari and Vasan, 2012; Wdowiak et al., 2007; Yan et al., 2007). Other researchers reported that mobile phone use had no harmful effects on sperm parameters (Feijo et al., 2011), therefore, there is still no clear consensus of opinion. From the results of our meta-analysis on the association between mobile phone use and semen quality, the semen parameters had no significant difference between mobile phone users and the control group in human studies (Liu et al., 2014). Moreover, it has been reported that pre-exposure to radiofrequency radiation could induce adaptive response to reduce genotoxicity (Jiang et al., 2012, 2013; Sannino et al., 2013; Zeni et al., 2012) and hematopoietic damage (Cao et al., 2011) caused by ionizing radiation or chemical agents. DNA damage responses or oxidative responses have been suggested to be partly involved in the adaptive responses induced by RF exposure at an adaptive dose (Jiang et al., 2012, 2013; Zeni et al., 2012). However, the underlying mechanism is still unclear. Autophagy is the degradation and recycling of cellular components in lysosomes, a process essential for the maintenance of cellular homeostasis and implicated in many disease processes, cell death and stress conditions (Moore et al., 2006). Under certain circumstances, autophagy is a self-defense and adaptive response mechanism used to alleviate cellular stress/damage and clear up dysfunctional or damaged organelles (Zhang et al., 2012). ROS production is an important intracellular inducer of autophagy (Garg et al., 2013). Recent studies have reported that RF exposure could increase ROS production in sperm in vitro (Agarwal et al., 2009; De Iuliis et al., 2009), which is considered to be one of the primary mechanisms involved in the bio-effects mediated by RF-EMR (Desai et al., 2009). Based on these evidences, we hypothesized that autophagy might be induced by RF-EMR as an underlying adaptive response mechanism and ROS could play a vital role in this process. To clarify this issue, we investigated RF induced autophagy in mouse spermatocyte-derived cells and the role of ROS in this process. Our present study involved a 24 h intermittent exposure (5 min on, 10 min off) to 1800 MHz GSM-Talk signals at SAR values of 1 w/kg, 2 w/kg or 4 w/kg. These results demonstrated that RF exposure under our experimental conditions had no impact on cell apoptosis but could induce autophgy and stimulate ROS generation. Meanwhile, inhibition of autophagy could significantly increase the percentage of apoptotic cells.

2.1. Cell culture

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Mouse spermatocyte-derived GC-2 spd (ts) cells (GC-2 cells) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) at 37 ◦ C in a 5% CO2 humidified incubator.

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2.2. Reagents and antibodies

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N-acetyl-cysteine (NAC), Chloroquine (CQ), 3-methyladenine (3-MA), and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2, 7dichloroluorescein diacetate (DCFH-DA) was obtained from Beyotime Company, (Shanghai, China). The antibody against micro-tubule-associated protein 1 light chain 3B (LC3B) was purchased from Cell Signaling (Boston, MA). Antibodies against phospho-ERK, GAPDH were purchased from Santa Cruz (Texas, USA).

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2.3. RF-EMR exposure system

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The exposure system was provided and set up by the Foundation for Information Technologies in Society (IT’IS Foundation, Switzerland), as described in detail previously (Franzellitti et al., 2010; Liu et al., 2013; Xu et al., 2013; Zeng et al., 2006). The RF-EMR exposure system mainly includes a RF generator, an arbitrary function generator, a narrow band amplifier and two rectangular waveguides. Both waveguides were placed inside a CO2 incubator, one was used for RF-EMR exposure, while the other one for sham exposure. The SAR values and temperature were monitored by a computer during the exposure. The exposure system kept a constant temperature and environment for the waveguides (37 ◦ C, 5% CO2 /95% atmospheric air). Six 35 mm Petri dishes can be placed in the H-field maxima and exposed to a polarized E-field (an electric field that is perpendicular to the H-field) in the same manner. This system keeps a steady frequency of 1800 MHz. The SAR variability of this system is below 6%, with a temperature rise of 0.03 ◦ C (w/kg) of the average SAR value following exposure of monolayer cells. The temperature change is uniformly distributed in monolayer cells, and the temperature difference between the RF-exposed and shamexposed chambers does not exceed 0.1 ◦ C (Schuderer et al., 2004). Double-blind experiments can be performed, as the computer randomly defines the exposure waveguides in each experiment. All of the exposure conditions and monitor data including temperature, SAR value, etc.., were recorded and encoded in a file. Then it was e-mailed to the IT’IS Foundation and decoded by that foundation with the data analysis.

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2.4. Cell exposure procedure

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Cells were seeded into 35 mm Petri dishes (Corning, UK) at a density of 1 × 105 /ml before exposure. At 24 h after cell seeding, the culture medium was renewed, and dishes were exposed to 1800 MHz GSM-Talk signals with an intermittent cycle of 5 min on and 10 min off for the indicated time intervals. To explore the SAR-related effects of RF exposure, dishes were randomly divided into the following groups: (1) sham exposure; (2) 1 w/kg; (3) 2 w/kg and (4) 4 w/kg.

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2.5. Western blot

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The cells were harvested and lysed in cell lysis buffer (Beyotime, China) on ice. Then, the cell lysates were centrifuged at 12,000 rpm for 15 min. The supernatant was collected and heated at 100 ◦ C for 10 min, and the concentration of protein was determined by BCA Protein Assay Kit (Beyotime, China). Identical amounts of protein (60 ␮g) from each sample were loaded and run on 12% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA) by Semi-Dry Electrophoretic Transfer (Bio-rad, US). After membrane blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h, the membranes were incubated with specific primary antibodies at 4 ◦ C overnight, washed with TBST, and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST, the immunoblots were visualized by chemiluminescence using HRP substrate (Millipore, Billerica, MA, USA). GAPDH was used to ensure equal protein loading.

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2.6. Plasmid and transient transfection

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Cells were seeded on 35 mm petri dishes (Corning, UK) at a density of 5 × 104 ml−1 . After 24 h, cells were transiently transfected with GFP-LC3 plasmid using Lipofectamine® 2000 (Life Technologies Corporation, USA) according to the manufacturer’s protocol. The cells were then incubated for 12 h before exposure to RF. After RF exposure, the cells were observed with a fluorescent microscope. The cells with more than five GFP-LC3 dots were considered to be autophagic, and was calculated and quantified. The GFP-LC3 plasmid was gifted from Dr. Guobing Li (Third Military Medical University, Chongqing, China).

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Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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Fig. 1. RF exposure induces autophagy in mouse spermatocyte-derived cells. (A) GC-2 cells were exposed to RF at SAR values of 2 w/kg, 4 w/kg for 24 h, respectively. Then, the cell lysates were subjected to western blot using antibody against LC-3B. GAPDH was used as a loading control. Rapamycin (Rap) was used as a positive control. The LC3-II/LC3-I ratio was calculated based on densitometry analysis of both bands by Image J2x software. (B) After exposure to RF for the indicated time intervals at SAR value of 4 w/kg, the cells were lysed and immunoblotted with an anti-LC3B antibody. (C) GC-2 cells were transiently transfected with the GFP-LC3 plasmid for 24 h, subsequently exposed to RF at the SAR value of 4 w/kg for 24 h and observed with a fluorescence microscope. Representative images are shown to indicate the cellular localization patterns of the

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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Fig. 2. RF induced autophagy flux could be blocked by the autophagic inhibitor chloroquine. (A) GC-2 cells were exposed to RF (4 w/kg), with or without chloroquine CQ (10 ␮M) co-treatment for 24 h. Then the expressions of LC3-I, LC3-II and p62 were detected by Western blot. GAPDH was used as a loading control. (B) and (C) LC3-II/LC3-I and P62/GAPDH ratio were calculated based on densitometry analysis of both bands by Image J2x software. * Indicated the p < 0.05 vs comparison group.

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2.7. ROS generation detection

2.10. Statistical analysis

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Data are represented as the mean ± SD from three independent experiments. One-way analysis of variance (ANOVA) was used to compare means between groups. A threshold of p < 0.05 was defined as statistically significant.

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Intracellular ROS was measured by detecting the conversion of cell permeable DCFH-DA to fluorescent dichlorol uorescein (DCF). Cells were seeded in 35 mm petri dishes and exposed to RF at the SAR values of 1 w/kg, 2 w/kg or 4 w/kg for 24 h, respectively. In another experiment, cells were exposed to RF for 3 h, 6 h, 12 h and 24 h at the SAR value of 4 w/kg, respectively. Then, the cells were harvested, washed twice with PBS, incubated with DCFH-DA at 37 ◦ C for 25 min. After being washed twice with PBS, the fluorescence densities of cells were analyzed by Cytomics FC500 flow cytometer (Beckman Coulter, Inc.) within 20 min at an excitation wavelength of 488 nm and fluorescence emission was measured in channel FL-1. The results were analyzed by using CXP analysis software (Beckman Coulter, Inc.). ROS level (% of sham exposure) was expressed as the fluorescence of the exposure samples compared to that of the sham exposure ones.

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2.8. Transmission electron microscopy

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For the transmission electron microscopy studies, cells were exposed to RF (4 w/kg) for 24 h, then harvested and washed twice with PBS and fixed with 2.5% glutaraldehyde. The cells were postfixed in 2% osmium tetroxide, embedded and stained with uranyl acetate/lead citrate. Cells were observed using a Hitachi-7500 electron microscope (Japan) to detect autophagic vacuoles.

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2.9. Detection of apoptosis

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Apoptosis was evaluated by the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China) following the manufacturer’s instructions. After RF exposure, cells were harvested and incubated with Annexin V-FITC and propidium iodide (PI) at room temperature for 10 min, respectively. Subsequently, Annexin V-positive cells were analyzed by Cytomics FC500 flow cytometer (Beckman Coulter, Inc) within 20 min and the results were analyzed by using CXP analysis software (Beckman Coulter, Inc). At least 10,000 cells were counted in each sample. Annexin V-FITC conjugates were detected with the FL1 channel of the Cytomics FC500. PI was read on the FL2 channel.

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3. Results 3.1. RF exposure induces autophagy in mouse spermatocyte-derived cells To determine whether RF exposure could induce autophagy in mouse spermatocyte cells, GC-2 cells were exposed to RF according to the cell exposure procedure mentioned above and LC3-I and LC3-II expressions were analyzed by western blot assay. The conversion of LC3-I to LC3-II, a membrane bound formation of LC3, is a well-established indicator of autophagy induction. The expression of LC3-II gradually increased in a dose- and time-dependent manner with RF exposure and showed a significant change at the SAR value of 4 w/kg (Fig. 1A and B). Rapamycin, a well known inducer of autophagy was used as a positive control. The LC3-II/LC3-I ratio was calculated based on densitometry analysis of both bands (Fig. 1A and B). The translocation of LC3 from the cytosol to the autophagic vacuoles is another hallmark of autophagy and can be identified by the formation of puncta with the GFP-LC3 fusion protein in autophagosomes (Klionsky et al., 2012). To further confirm that RF induces autophagy in spermatocyte cells, GC-2 cells were transiently transfected with the GFP-LC3 plasmid and then treated with RF (4 w/kg) for 24 h. Fluorescence microscopic examination showed

GFP-LC3 fusion protein (×40 magnification). The percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells. The data are represented as mean ± SD of at least three independent experiments. At least 150 GFP-LC3-transfected cells were counted in each experiment. (D) Transmission electron microscopy pictures of autophagosomes. GC-2 cells were exposed to RF at SAR value of 4 w/kg for 24 h. Cells were harvested and fixed for observation by TEM. Representative images are shown to indicate the autophagosomes in cytoplasm. The arrows indicate autophagosomes. * Indicates the p < 0.05 vs sham exposure group.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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Fig. 3. Intracellular ROS is involved in RF induced autophagy. (A) GC-2 cells were exposed to RF for the indicated SAR values for 24 h, then incubated with 1 ␮M DCFH-DA at 37 ◦ C for 25 min, and subsequently subjected to DCF fluorescence analysis to evaluate the levels of intracellular ROS. Changes in the mean fluorescence intensity are expressed as the percentage of sham exposure. ROSup was used as a positive control. (B) Cells were pretreated with NAC (10 mM) for 2 h then exposed to RF at the SAR value of 4 w/kg for 24 h and subsequently subjected to DCF fluorescence analysis. (C) Pretreatment with NAC attenuated RF-induced autophagy in GC-2 cells. Cells were pretreated with 10 mM NAC or PBS for 2 h and then exposed to RF at the SAR value of 4 w/kg for 24 h. The expression of LC3 and p62 was assessed by Western blot. GAPDH was used as a loading control. LC3-II/LC3-I and P62/GAPDH ratios were calculated based on densitometry analysis of both bands by Image J2x software. (D) GC-2 cells were transiently transfected with the GFP-LC3 plasmid for 24 h, pretreated with 10 mM NAC or PBS for 2 h, subsequently exposed to RF at the SAR value of 4 w/kg for 24 h and observed with a fluorescence microscope. Representative images are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (×40 magnification). The percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells. The data was represented as mean ± SD of at least three independent experiments. At least 150 GFP-LC3-transfected cells were counted in each experiment. * Indicates p < 0.05 vs comparison group.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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the characteristic punctate fluorescent pattern of GFP-LC3, indicating autophagosome formation and the occurrence of autophagy (Fig. 1C). The percentage of GFP-LC3 transfected cells with punctate fluorescence in RF exposed dishes was significantly higher than that in sham exposed dishes (Fig. 1C). The use of transmission electron microscopy (TEM) is a valid and important method both for the qualitative and quantitative analysis of changes in various autophagic structures (Klionsky et al., 2012) and was employed in this case to inspect autophagic cells. As shown in Fig. 1D, compared with the sham exposed group, autophagic vacuoles were observed in larger quantities in the RF exposed GC-2 cells (Fig. 1D). Furthermore, RF inducing LC3-II expressing was enhanced by cotreatment with CQ (10 ␮M) (Fig. 2A and B), a classic lysosome inhibitor that blocks autophagic protein degradation. Meanwhile, P62, reported to interact with LC3 and is degraded through the autophagy–lysosome pathway, decreased after RF exposure, but recovered after co-treatment with CQ (10 ␮M). This result confirmed that RF increased the autophagic flux. Altogether, these data suggested that 1800 MHz RF-EMR could induce autophagy at the SAR value of 4 w/kg after 24 h exposure in spermatocyte derived cells.

(but not 1 w/kg) for 24 h, ROS levels significantly increased compared to the sham exposure group (Fig. 3A). ROS production is a rapid response, so we detected the ROS levels in different time points at the SAR value of 4 w/kg. The results showed that ROS levels did not increase after 3 h and 6 h exposure, but slightly increased after 12 h exposure, while significantly increased after 24 h exposure (supplementary figure). Meanwhile, pretreatment with the classic antioxidant NAC 1 h prior to RF exposure significantly reduced ROS levels compared to the RF exposure group alone (Fig. 3B). Next, we investigated whether RF-induced autophagy was related to the production of ROS in GC-2 cells. As shown in Fig. 3C, pretreatment with NAC obviously decreased RF-induced LC3-I conversion and increased p62 accumulation in GC-2 cells. The LC3-II/LC3-I ratio and P62/GAPDH ratio were calculated based on densitometry analysis (Fig. 3C). Meanwhile, we examined the levels of autophagy in GC-2 cells transfected with GFP-LC3 plasmid by observing the fluorescent GFP-LC3 dots. As shown in Fig. 3D, pretreatment with NAC for 1 h significantly suppressed the formation of GFP-LC3 puncta induced by RF exposure. These results indicate that intracellular ROS is involved in RF-induced autophagy in GC-2 cells and antioxidant pretreatment can inhibit autophagy induced by RF exposure.

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3.2. Intracellular ROS are involved in RF-EMR induced autophagy 3.3. ERK phosphorylation is involved in RF-induced autophagy

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Many studies have reported that some chemical or physical factors could activate the production of intracellular ROS and then induce cellular apoptosis and/or autophagy in certain types of cells (Chakraborty et al., 2012; Cheng et al., 2013; Fukui et al., 2013; Ghavami et al., 2010; Gong et al., 2012). To further investigate whether a possible increase in ROS production occurred due to exposure to RF, intracellular ROS were examined by flow cytometry using the specific ROS-detecting fluorescent dye DCFH-DA (fluoresces green in the presence of ROS). After cells were exposed either to ROSup (a positive control provided by ROS-detecting Kit, Beyotime, China) or to RF-EMR at the SAR values of 2 w/kg or 4 w/kg

The exposure to RF-EMR affects the expression of many proteins, including the activation of the ERK cascade mediated by ROS production (Friedman et al., 2007). Meanwhile, the ERK signaling pathway is involved in the activation of autophagy induced by environmental stress (Hu et al., 2012). To further explore the molecular mechanism contributing to the mediation of RF-induced autophagy, we evaluated the phosphorylation statuses of ERK. The results showed that the levels of phosphorylated ERK significantly increased after RF exposure at the SAR value of 2 w/kg and 4 w/kg (Fig. 4A). Meanwhile, NAC pretreatment could also block the

Fig. 4. ERK phosphorylation is involved in RF-induced autophagy. (A) GC-2 cells were exposed to the indicated SAR values for 24 h. The expression of phosphor-ERK was detected by western blot. Rapamycin was used as a positive control. (B) GC-2 cells were exposed to RF in the absence or presence of pretreatment of 10 mM NAC for 2 h. P-ERK/GAPDH ratio was calculated based on densitometry analysis of both bands by Image J2x software. * Indicates that p < 0.05 compared to sham exposure group or RF exposure group.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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Fig. 5. RF exposure could not induce apoptosis and inhibition of autophagy increased the percentage of apoptotic cells. (A) GC-2 cells were exposed to RF at the indicated SAR values for 24 h. Then, cells were harvested and incubated with Annexin V-FITC and propidium iodide (PI) at room temperature for 10 min, respectively. Subsequently, samples were analyzed using flow cytometry. (B) GC-2 cells were exposed to RF alone or with the autophagy inhibitor 3-MA (5 mM) or NAC (10 mM). Then apoptotic cells were analyzed by the methods previously described. (C) and (D) The percentage of apoptotic cells was represented as the mean ± SD of three independent experiments. Representative images of three independent experiments are shown. * Indicates that p < 0.05, while # indicates p > 0.05.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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phosphorylation of ERK (Fig. 4B). P-ERK/GAPDH ratio was calculated based on densitometry analysis (Fig. 4A and B). 3.4. Inhibition of autophagy increases apoptotic cells To evaluate whether RF could induce cell toxicity, we examined Annexin V and PI stained apoptotic cells by flow cytometry. RF could not increase the percentage of apoptotic cells at the SAR values of 1 w/kg, 2 w/kg, and 4 w/kg for 24 h (Fig. 5A and C). To understand the role of ROS-mediated autophagy on cell survival after RF exposure, 3-MA, a common specific inhibitor of autophagic/lysosomal degradation was used to block autophagy and then apoptosis was determined. Compared to RF exposure alone, co-treatment with 3-MA increased the percentage of apoptotic cells (Fig. 5B and D). Meanwhile pretreatment of NAC did not significantly change the percentage of apoptotic cells (Fig. 5B and D). These data suggest that RF-induced ROS production and autophagy may protect cells from apoptotic cell death, and inhibition of autophagy could increase the percentage of apoptotic cells. 4. Discussion Autophagy acts as a survival mechanism under conditions of environmental stress, maintaining cellular homeostasis by regenerating metabolic precursors and clearing subcellular debris (Doria et al., 2013). RF-EMR exposure is increasing with the development and adoption of mobile devices, which has raised public concerns on its biological effects. Many studies have reported that RF-EMR could induce adaptive response to protect against chemical mutagen and ionizing radiation (Cao et al., 2011; Jiang et al., 2012, 2013; Sannino et al., 2009, 2013, 2011; Zeni et al., 2012). Such protective phenomenon was generally described as an adaptive response and was well documented in human and animal cells (Jiang et al., 2013). However, the underlying mechanism is still unknown. The results of the present study indicated that exposure to 1800 MHz RF could induce autophagy in mouse spermatocyte cells at a SAR value of 4 w/kg and this process was mediated by ROS generation. Classic antioxidant NAC inhibited autophagy by suppressing RF-induced ROS production. Moreover, specific inhibitors of autophagy blocked autophagy protecting cells from apoptotic cell death at the same time. These data indicated that autophagy may play an important role in the adaptive response mechanism to protect against RF-EMR and ensure cell survival. The biological effects of EMR comprise both thermal effect and non-thermal effects dependent on the temperature rise at particular frequencies of EMR (Agarwal et al., 2011). To avoid thermal effects, the maximum SAR value was maintained under 4 w/kg in our experiment, with a temperature rise of approximately 0.08 ◦ C. The GSM-Talk mode of the exposure system was used in our experiment to mimic real-life exposure conditions. The intermittent exposure procedure with 5 min on and 10 min off, which closely resemble real-life exposure conditions, had stronger effects than the continuous exposure procedure (Chauhan et al., 2007; Focke et al., 2010; Ivancsits et al., 2002). Previous in vitro studies demonstrated that RF-EMR enhanced ROS production in human spermatozoa (De Iuliis et al., 2009) and spermatocyte cells (Liu et al., 2013). ROS could trigger various biological outcomes, such as autophagy, apoptosis, and DNA damage. In this study, we did not observed a significant increase in apoptotic cells in the RF exposure group, compared with sham exposure group. But a significant increase of autophagic cells was observed upon exposure to RF at SAR value of 4 w/kg. Inhibition of autophagy by 3-MA could increase the percentage of apoptotic cells. These evidences suggested that autophagy plays a protective role in maintaining cell survival under the stress of RF. The crosstalk between

apoptosis and autophagy is complex and may often appear contradictory, yet is critical to the overall fate of the cell (Booth et al., 2013). Autophagy and apoptosis may be triggered by common upstream signals, and sometimes autophagy prevents cells from apoptotic cell death (Maiuri et al., 2007). In our study, autophagy plays a critical role in preventing cells from apoptosis. If autophagy was blocked, cells would initiate the process of apoptosis. However, in our experiment, in order to exclude the thermal effects induced by RF, we did not adopt higher doses. According to the way by which cargo is delivered to the lysosomes, autophagy is divided into three different pathways; macroautophagy, microautophagy and chaperone-mediated autophagy (Mizushima and Klionsky, 2007). During macroautophagy cytosolic components are delivered to lysosomes by intermediate vesicles called ‘autophagosomes’, while in chaperone-mediated autophagy (CMA) protein substrates are translocated across the lysosomal membrane by a complex of chaperone proteins. Chaperone-mediated autophagy is a selective form of autophagy where autophagosomes do not form (Johansen and Lamark, 2011). Previous studies on GSM-900 MHz at a low SAR value (0.25 w/kg) showed that chaperone-mediated autophagy was not altered, but CMA-related protein heat shock cognate 70 (HSC70) and HPS90 changed, which indicated that the GSM signal is a stress signal. The delayed and long term consequences of these changes on cell fate should be examined (Terro et al., 2012). In our study, we adopted a higher dose (4 w/kg) to test whether GSM signal could induce autophagy, and our results showed that autophagy flux could be enhanced by a 1800 MHz GSM signal at a relatively higher dose. The ERK signaling pathway can be activated by various intracellular and extracellular stresses, such as ROS, growth factors, and cytokines (Cagnol and Chambard, 2010). Our study indicated that total ERK could be activated by RF exposure, which was consistent with the results of Friedman et al. (2007). Their results showed that the ERK cascade could be activated within 5 min. In our experiment, the ERK pathway was activated after 24 h intermittent exposure with 5 min on and 10 min off, which may induce further transcription of downstream genes. In addition, other signaling pathway should be further detected to determine the whole view on autophagy induced by RF exposure. In our study, we demonstrated that RF could induce autophagy under our experimental conditions, which may be a reasonable explanation for the adaptive response induced by RF exposure. Autophagy induced by RF exposure may trigger a particular mechanism to prevent cells from further damage by the subsequent harmful factors. Future investigations should be carried out to explore the underlying mechanism.

Conflict of interest statement Authors declare that there is no conflict of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgments This study was funded by National Basic Research Program (973 Program) (No. 2011CB503705). We thank Dr. GB Li for the gift of the GFP-LC3 plasmid and the Department of Occupational Health, Third Military Medical University for the use of the exposure devices.

Please cite this article in press as: Liu, K., et al., The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.05.004

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