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Induction of adaptive response in mice exposed to 900 MHz radiofrequency fields: Application of micronucleus assay
Mutation Research 751 (2013) 127–129
Contents lists available at SciVerse ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Induction of adaptive response in mice exposed to 900 MHz radiofrequency fields: Application of micronucleus assay Bingcheng Jiang, Chunyan Zong, Hua Zhao, Yongxin Ji, Jian Tong, Yi Cao ∗ School of Public Health, Soochow University, Suzhou, Jiangsu Province, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 29 October 2012 Received in revised form 5 December 2012 Accepted 19 December 2012 Available online 4 January 2013 Keywords: Mice Radiofrequency fields Gamma radiation Adaptive response Micronuclei
a b s t r a c t Adult male ICR mice were pre-exposed to non-ionizing radiofrequency fields (RF), 900 MHz at 120 W/cm2 power density for 4 h/day for 7 days (adaptation dose, AD) and then subjected to an acute whole body dose of 3 Gy ␥-radiation (challenge dose, CD). The classical micronucleus (MN) assay was used to determine the extent of genotoxicity in immature erythrocytes in peripheral blood and bone marrow. The data obtained in mice exposed to AD + CD were compared with those exposed to CD alone. The results indicated that in both tissues, the MN indices were similar in un-exposed controls and those exposed to AD alone while a significantly increased MN frequency was observed in mice exposed to CD alone. Exposure of mice to AD + CD resulted in a significant decrease in MN indices compared to those exposed to CD alone. Thus, the data suggested that pre-exposure of mice to non-ionizing RF is capable of ‘protecting’ the erythrocytes in the blood and bone marrow from genotoxic effects of subsequent ␥radiation. Such protective phenomenon is generally described as ‘adaptive response’ (AR) and is well documented in human and animal cells which were pre-exposed to very low doses of ionizing radiation. It is interesting to observe AR being induced by non-ionizing RF. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pre-exposure of animal and human cells, in vitro and/or in vivo, to a very low non-genotoxic dose of ionizing radiation (adaptation dose, AD) were found to be resistant to the damage induced by subsequent exposure to a higher dose (challenge dose, CD) of the same or similar genotoxic agents. This phenomenon is well documented in the scientific literature and is called as adaptive response (AR). Non-ionizing radiofrequency fields (RF) in the frequency range from 800 to 2000 MHz are increasingly used in wireless communication systems in recent years. The wide spread use of such devices has led to increased concern in the general public regarding potential adverse effects on human health from exposure to RF. Extensive research efforts were devoted to determine whether acute and long-term in vivo and in vitro RF exposures result in excessive genetic damage in mammalian cells since such damage is positively correlated with carcinogenesis. Several scientific reviews suggested that RF exposure is non-genotoxic [1–7]. In our earlier investigations, we have examined whether non-genotoxic RF preexposure can induce AR and thus offer protection from subsequent
Abbreviations: RF, radiofrequency fields; AD, adaptation dose; CD, challenge dose; PCE, polychromatic erythrocytes; MN, micronuclei. ∗ Corresponding author. Tel.: +86 512 65881552; fax: +86 512 65880069. E-mail address: [email protected] (Y. Cao). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2012.12.003
damage induced by genotoxic agents. Mice were pre-exposed to 900 MHz RF at different power densities for 1–14 days (AD) and then subjected to a high dose of ␥-radiation (CD). The data indicated that compared with the animals which received CD alone, mice which were exposed to AD + CD exhibited significantly increased survival, decreased hematopoietic tissue damage in the spleen and bone marrow as well as decreased primary DNA damage in peripheral blood and bone marrow cells [8–10]. Furthermore, cultured human HL-60 cells which were pre-exposed to RF (AD) and subsequently treated with doxorubicin, a chemotherapeutic drug (CD) showed decreased apoptosis and intra-cellular Ca2+ and, increased proliferation, mitochondrial membrane potential and Ca2+ –Mg2+ ATPase activity, as compared with the cells treated with CD alone [11]. In view of the above observations, the current investigation was conducted using the classical micronucleus (MN) assay to determine whether pre-exposure of mice to 900 MHz RF (AD) can induce AR and offer protection to blood and bone marrow cells from the genetic damage induced by subsequent ␥-radiation (CD). 2. Materials and methods All animal handling procedures were reviewed and approved by the animal care/user ethical committee of Soochow University, Suzhou City, PR China (approval number A68-2011). Adult male ICR mice weighing approximately 25 gm were obtained from the animal center in Suzhou University and were housed in a facility maintaining 22 ± 1 ◦ C temperature, 50 ± 5% relative humidity and 12-hour light–dark cycles. They were fed on a commercial diet (Suzhou Shuangshi Laboratory Animal Feed Science Co., Ltd., Suzhou City, Jiangsu Province, PR China) and
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B. Jiang et al. / Mutation Research 751 (2013) 127–129
Table 1 The incidence of micronuclei (MN/2000 PCE) and % polychromatic erythrocytes (PCE) in the peripheral blood and bone marrow of mice exposed to radiofrequency fields (RF) and gamma radiation (GR). Group
MN/2000 PCE Blood
1. Un-exposed controls 5.2 ± 1.83 2. GR, 3 Gy 62.6 ± 19.46 3. RF, 900 MHz 7.5 ± 2.77 14.8 ± 7.25 4. RF + GR 6.8 ± 2.09 5. Sham 57.4 ± 21.00 6. Sham + GR a Expected Values 1.11 53.2 % Changes Statistical difference between groups (p values) 0.000 1 and 2 0.054 1 and 3 0.916 1 and 5 0.000 2 and 4 0.674 2 and 6 0.001 4 and 6
% PCE Bone marrow
Blood
Bone marrow
5.9 ± 1.37 50.4 ± 14.99 7.7 ± 2.37 23.5 ± 8.26 8.0 ± 2.37 53.9 ± 18.28 16.1 109
2.3 ± 0.55 0.6 ± 0.20 2.9 ± 0.71 1.7 ± 0.86 2.4 ± 0.72 0.7 ± 0.23 58.9 74.9
46.8 ± 5.91 18.3 ± 4.03 44.6 ± 4.78 33.3 ± 4.23 46.8 ± 4.58 22.0 ± 6.51 52.2 55.0
0.000 0.363 1.000 0.000 0.134 0.000
0.000 0.690 0.781 0.000 0.368 0.000
0.000 0.713 0.668 0.000 0.475 0.000
Numbers of animals in each group: 10. Data are mean ± standard deviation. a Expected values are the sum of two individual treatment (GR alone + RF alone) minus un-exposed controls.
provided water ad libitum. After 7 days quarantine period, the animals were randomly divided into six groups of 10 mice each. Group 1: un-exposed controls, Group 2: ␥-radiation alone; Group 3 and 4: 900 MHz RF exposure at 120 W/cm2 power density for 4 h/day for 7 days; Group 5 and 6: sham exposure for 4 h/day for 7 days. On day 7, at 4 h after the last RF/sham exposure, animals in groups 2, 4 and 6 were subjected to acute whole body exposure to ␥-radiation from a 60 Co source (Nordion, Ottawa, ON, Canada, total dose 3 Gy and dose rate 0.5 Gy/min). All mice were sacrificed 72 h later to collect peripheral blood and bone marrow (see below). 2.1. RF exposure The RF exposure system was described in detail earlier [8,9]. Briefly, it consisted of GTEM chamber (Giaga-hertz transverse electromagnetic chamber which was built in-house and installed in a room which maintained 37 ± 0.5 ◦ C temperature at Soochow University, Suzhou, Jiangsu, PR China, a signal generator (PMM, Cisano sul Neva, Italy) and a power amplifier (HD Communication Corp. Ronkonkoma, NY, USA). The continuous wave 900 MHz RF signal was generated, amplified and fed into the GTEM chamber through an antenna (Southeast University, Nanjing, Jiangsu, China). A field strength meter (PMM, Cisano sul Neva, Italy) was used to determine the precise position, inside GTEM, which provided the required power intensity of 120 W/cm. The power was continuously monitored and recorded in a computer controlled data logging system. Small plastic boxes were designed in-house to hold mice (a single restrained mouse in each box). The mice inside the boxes were placed on a non-conductive table/platform for RF exposure. The whole body average specific absorption rate (SAR) was calculated using the finite-difference-time-domain model and it was 548 mW/kg. Sham exposures were conducted in the same GTEM chamber, without the transmission of RF. The environmental conditions during and after RF/sham exposures were kept identical through out the study period. 2.2. Peripheral blood and bone marrow smears All mice were anesthetized first using carbon-di-oxide. From each animal, peripheral blood was collected by heart puncture and, one or two small drops were placed on pre-cleaned microscope slides to prepare thin smears. Then, the animals were killed by cervical dislocation to collect the femurs. The bone marrow was flushed out using 1 ml fetal bovine serum, gently mixed to obtain a fine cell suspension and, one or two drops were placed on pre-cleaned microscope slides to prepare thin smears. All smears were air dried, fixed in absolute methanol, air-dried and stained using acridine orange (Sigma; 0.01 mg/ml of 0.2 M phosphate buffer, pH 7.4). Coded slides were examined under 1000x magnification using a fluorescence microscope fitted with 450–460 nm excitation and 620–670 nm emission filters. Erythrocytes which were immature (PCE) and mature (NCE) were identified by their orange–red and green colors, respectively, and MN by their bright yellowish color. For each mouse, in both blood and bone marrow tissues, 2000 consecutive PCE were examined to record the incidence of MN. In addition, the % PCE was obtained from the examination of 1000 and 200 erythrocytes in blood and bone marrow, respectively. The data were decoded after completing the microscopic evaluation [12]. 2.3. Statistical analysis The results were subjected to statistical analyses using software SPSS Statistics version 20.0. One-way analysis of variance (ANOVA) and Student’s t-test were used
to compare the differences, if any, between groups with pair wise comparisons. A p value <0.05 was considered as significant difference.
3. Results The extent of genotoxicity determined by the incidence of MN/2000 PCE and cytotoxicity measured by % PCE in blood and bone marrow cells was similar among the 10 mice in each of the 6 groups. Hence, the results were pooled and the group mean indices (±standard deviation) are presented in Table 1. The results were: (i) the incidence of MN/2000 PCE in both blood and bone marrow was not significantly different between un-exposed control mice and those that were pre-exposed to RF/sham alone (AD) and, the values ranged between 5.2 and 8.0 MN/2000 PCE; (ii) a significant increase in MN was observed in mice exposed to ␥-radiation alone (CD): 62.6 and 50.4 MN/2000 PCE in blood and bone marrow, respectively; (iii) combined exposures of RF + ␥-radiation (AD + CD) resulted in a significant decrease in MN in both blood and bone marrow, as compared with the mice exposed to ␥-radiation alone (CD): 14.8 and 23.5 MN/2000 PCE in blood and bone marrow, respectively; (iv) such a decrease was not observed in mice exposed to sham + ␥radiation (AD + CD): 57.4 and 53.9 MN/2000 PCE in blood and bone marrow, respectively. The % PCE recorded in the blood and bone marrow tissues showed no significant differences between un-exposed controls and mice exposed to RF/sham alone (AD). There was a significant decrease in % PCE in blood and bone marrow in animals exposed to ␥-radiation alone (CD). In combined AD + CD exposures however, there was an increase in % PCE in mice exposed to RF + ␥-radiation (AD + CD) while no such increase was observed in animals which received sham + ␥-radiation (AD + CD). 4. Discussion Ionizing radiation-induced AR is well documented in the scientific literature. Animal and human cells that were pre-exposed in vivo or in vitro to an extremely small adaptation dose of ionizing radiation were reported to be resistant to subsequent damage induced by a higher challenge dose of the same or similar genotoxic agent(s). A search for similar induction of AR by non-ionizing RF exposure is under way and evidence is accumulating in recent years. Sannino et al. [13] were the first to report that human blood lymphocytes which were pre-exposed in vitro for 20 h–900 MHz
B. Jiang et al. / Mutation Research 751 (2013) 127–129
at an average specific absorption rate of 1.25 W/kg (AD) and then treated with a genotoxic dose of mitomycin C, a chemotherapeutic drug (CD) exhibited significantly decreased incidence of MN when compared with the cells treated with CD alone. Such decrease in MN was confirmed in two subsequent investigations in different experimental exposure conditions [14,15]. In animal studies, our research group was the first to report RF-induced AR. Adult male Kunming mice were pre-exposed to 900 MHz RF at 12, 120 and 1200 W/cm2 power density for 1 h/day for 14 days (AD) and then subjected to ␥-radiation (CD). Compared with the animals exposed to CD alone: (i) mice exposed to RF + 8 Gy ␥-radiation showed increased survival and (ii) mice exposed to RF + 5 Gy ␥-radiation exhibited significantly decreased hematopoietic tissue damage determined from increased colony forming units in bone marrow and spleen. These changes were accompanied by elevated hematopoietic growth factors, inhibition of ␥-ray-induced suppression of hematopoietic progenitor stem cells as well as increased expression of cell cycle-related genes, viz., cyclin-D1, cyclin-E, cyclin-DK4 and cyclin-DK2 which might have helped in by-passing the G1/S-block check point (due to ␥-radiation exposure) resulting in restoration of cell cycle and cell proliferation [8,9]. In a subsequent study, ICR mice were pre-exposed to 900 MHz RF at 120 W/cm2 power density for 4 h/day for 1, 3, 5, 7 and 14 days (AD) and then subjected to 3 Gy ␥-radiation (CD). The extent of primary DNA damage was assessed in peripheral blood leukocytes using the alkaline comet assay. A significant and exposure time-dependent decrease in damage was observed in mice exposed to AD + CD compared to those exposed to CD alone. The extent of decrease was maximal in the animals pre-exposed to RF for 7 days [10]. In vitro experiments, human HL-60 cells were pre-exposed to 900 MHz RF at 12 W/cm2 power density for 1 h/day for 3 days (AD) and then treated with doxorubicin, a chemotherapeutic drug (CD). Such cells exhibited decreased apoptosis and intra-cellular Ca2+ and, increased proliferation, mitochondrial membrane potential and Ca2+ –Mg2+ -ATPase activity, as compared with the cells treated with CD alone [11]. Our earlier observations of RF-induced AR in mice gained support from similar significantly enhanced survival data reported by a research group in Iran. In the first study, adult male Sprague Dawley rats were pre-exposed to 900 MHz RF (GSM) at 2 W power density with or without flaxseed oil. The intermittent RF exposure (AD) was for 4 days, i.e. 3 h ON and 9 h OFF, twice a day (total 6 h/day). On day 5, the animals were subjected to whole body LD50/30 dose of 8 Gy ␥-radiation (CD). After 30 days, a significant increase in survival was observed in animals exposed to AD + CD compared with those subjected to CD alone [16]. In the second investigation, adult Balb/c mice and Wistar rats were pre-exposed to amplitude modulated 900 MHz RF (GSM) at 2 W power density for 5 days and then exposed to lethal dose of 8.8 Gy ␥-radiation (CD). A significantly increased survival rate was observed in mice and rats exposed to AD + CD as compared to the animals which received CD alone [17]. The results obtained in the current investigation indicated that compared with un-exposed control animals: (i) mice which were pre-exposed to RF alone (AD) exhibited no significant increase in genotoxicity (MN/2000 PCE) and thus confirmed that RF exposure is non-genotoxic, a conclusion made in several earlier reviews (also, there was no evidence of increased cytotoxicity which was assessed from % PCE), (ii) mice which were exposed to an acute whole body dose of ␥-radiation alone (CD) exhibited significant increases in genotoxicity and cytotoxicity and, (iii) mice which were pre-exposed to RF and subsequently subjected to ␥irradiation (AD + CD) exhibited significant decreases in genotoxicity and cytotoxicity. Thus, the data provided further evidence for RFinduced AR. Also, most of the research on the biological effects of RF exposure, thus far, has been focused on ‘adverse’ effects. However,
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the beneficial effect of RF pre-exposure that is observed in the study and the other studies mentioned above require special attention. Conflict of interest statement All authors declare no conflict of interest. Acknowledgments This research is supported by funding from the National Natural Science Foundation of China (Nos. 81020108028), 973 project grant from China Ministry of Science and Technology (2011CB503705) and The Priority Academic Program Development of Jiangsu Higher Education Institutions. The funding agencies had no role in designing the experiments, data collection and analysis, preparation of the manuscript, and decision to publish the results. We also thank the technicians in the laboratory for help during the experimentation. References [1] M.L. Meltz, Radiofrequency, exposure and mammalian cell toxicity, genotoxicity, and transformation, Bioelectromagnetics 6 (2003) S196–S213. [2] G. Vijayalaxmi, Obe, Controversial cytogenetic observations in mammalian somatic cells exposed to radiofrequency radiation, Radiat. Res. 162 (2004) 481–496. [3] L. Verschaeve, Genetic effects of radiofrequency radiation (RFR), Toxicol. Appl. Pharmacol. 207 (2005) S336–S341. [4] Vijayalaxmi, T.J. Prihoda, Genetic damage in mammalian somatic cells exposed to radiofrequency radiation: a meta-analysis of data from 63 publications (1990–2005), Radiat. Res. 169 (2008) 561–574. [5] L. Verschaeve, Genetic damage in subjects exposed to radiofrequency radiation, Mutat. Res. 681 (2009) 259–270. [6] L. Verschaeve, J. Juutilainen, I. Lagroye, J. Miyakoshi, R. Saunders, R. de Seze, T. Tenforde, E. van Rongen, B. Veyret, Z. Xu, In vitro and in vivo genotoxicity of radiofrequency fields, Mutat. Res. 705 (2010) 252–268. [7] Vijayalaxmi and T.J. Prihoda, Genetic damage in human cells exposed to non-ionizing radiofrequency fields: a meta-analysis of the data from 88 publications (1990–2011), Mutat. Res. Genet. Toxicol. Environ. Mutagen. (2012) http://dx.doi.org/10.1016/j.mrgentox.2012.09.007 [8] Y. Cao, Q. Xu, Z.-D. Jin, J. Zhang, M.-X. Lu, J.H. Nie, J. Tong, Effects of 900-MHz microwave radiation on ␥-ray-induced damage to mouse hematopoietic system, J. Toxicol. Environ. Health Part A 73 (2010) 507–513. [9] Y. Cao, Q. Xu, Z.-D. Jin, Z. Zhou, J.-H. Nie, J. Tong, Induction of adaptive response: pre-exposure of mice to 900 MHz. radiofrequency fields reduces hematopoietic damage caused by subsequent exposure to ionising radiation, Int. J. Radiat. Biol. 87 (2011) 720–728. [10] B. Jiang, J. Nie, Z. Zhou, J. Zhang, J. Tong, Y. Cao, Adaptive response in mice exposed to 900 MHz radiofrequency fields: primary DNA damage, PLoS ONE 7 (2012) e32040. [11] Z. Jin, C. Zong, B. Jiang, Z. Zhou, J. Tong, Y. Cao, The effect of combined exposure of 900 MHz radiofrequency fields and doxorubicin in HL-60 cells, PLoS ONE 7 (2012) e46102. [12] Vijayalaxmi, M.R. Frei, S.J. Dusch, V. Guel, M.L. Meltz, J.R. Jauchem, Frequency of micronuclei in the peripheral blood and bone marrow of cancer-prone mice chronically exposed to 2450 MHz radiofrequency radiation, Radiat. Res. 147 (1997) 495–500. [13] A. Sannino, M. Sarti, S.B. Reddy, T.J. Prihoda Vijayalaxmi, M.R. Scarfi, Induction of adaptive response in human blood lymphocytes exposed to radiofrequency radiation, Radiat. Res. 171 (2009) 735–742. [14] A. Sannino, O. Zeni, M. Sarti, S. Romeo, S.B. Reddy, A. Belisario, T.J. Prihoda Vijayalaxmi, M.R. Scarfi, Induction of adaptive response in human blood lymphocytes exposed to 900 MHz radiofrequency fields: influence of cell cycle, Int. J. Radiat. Biol. 87 (2011) 993–999. [15] O. Zeni, A. Sannino, S. Romeo, R. Massa, M. Sarti, A.B. Reddy, T.J. Prihoda, Vijayalaxmi, M.R. Scarfi, Induction of an adaptive response in human blood lymphocytes exposed to radiofrequency fields: influence of the universal mobile telecommunication system (UMTS) signal and the specific absorption rate, Mutat. Res. 747 (2012) 29–35. [16] S.M.J. Mortazavi, M.A. Mosleh-Shirazi, A.R. Tavassoli, M. Taheri, Z. Bagheri, R. Ghalandari, S. Bonyadi, M. Shafie, M. Haghani, A comparative study on the increased radioresistance to lethal doses of gamma rays after exposure to microwave radiation and oral intake of flaxseed oil, Iran J. Radiat. Res. 9 (2011) 9–14. [17] S.M.J. Mortazavi, M.A. Mosleh-Shirazi, A.R. Tavassoli, M. Taheri, A.R. Mehdizadeh, S.A.S. Namazi, A. Jamali, R. Ghalandari, S. Bonyadi, M. Haghani, M. Shafie, Increased radioresistance to lethal doses of gamma rays in mice and rats after exposure to microwave radiation emitted by a GSM mobile phone simulator, Dose Resp. (2012) Doi: 10.2203/dose-response.12-010.
Contents lists available at SciVerse ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Induction of adaptive response in mice exposed to 900 MHz radiofrequency fields: Application of micronucleus assay Bingcheng Jiang, Chunyan Zong, Hua Zhao, Yongxin Ji, Jian Tong, Yi Cao ∗ School of Public Health, Soochow University, Suzhou, Jiangsu Province, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 29 October 2012 Received in revised form 5 December 2012 Accepted 19 December 2012 Available online 4 January 2013 Keywords: Mice Radiofrequency fields Gamma radiation Adaptive response Micronuclei
a b s t r a c t Adult male ICR mice were pre-exposed to non-ionizing radiofrequency fields (RF), 900 MHz at 120 W/cm2 power density for 4 h/day for 7 days (adaptation dose, AD) and then subjected to an acute whole body dose of 3 Gy ␥-radiation (challenge dose, CD). The classical micronucleus (MN) assay was used to determine the extent of genotoxicity in immature erythrocytes in peripheral blood and bone marrow. The data obtained in mice exposed to AD + CD were compared with those exposed to CD alone. The results indicated that in both tissues, the MN indices were similar in un-exposed controls and those exposed to AD alone while a significantly increased MN frequency was observed in mice exposed to CD alone. Exposure of mice to AD + CD resulted in a significant decrease in MN indices compared to those exposed to CD alone. Thus, the data suggested that pre-exposure of mice to non-ionizing RF is capable of ‘protecting’ the erythrocytes in the blood and bone marrow from genotoxic effects of subsequent ␥radiation. Such protective phenomenon is generally described as ‘adaptive response’ (AR) and is well documented in human and animal cells which were pre-exposed to very low doses of ionizing radiation. It is interesting to observe AR being induced by non-ionizing RF. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pre-exposure of animal and human cells, in vitro and/or in vivo, to a very low non-genotoxic dose of ionizing radiation (adaptation dose, AD) were found to be resistant to the damage induced by subsequent exposure to a higher dose (challenge dose, CD) of the same or similar genotoxic agents. This phenomenon is well documented in the scientific literature and is called as adaptive response (AR). Non-ionizing radiofrequency fields (RF) in the frequency range from 800 to 2000 MHz are increasingly used in wireless communication systems in recent years. The wide spread use of such devices has led to increased concern in the general public regarding potential adverse effects on human health from exposure to RF. Extensive research efforts were devoted to determine whether acute and long-term in vivo and in vitro RF exposures result in excessive genetic damage in mammalian cells since such damage is positively correlated with carcinogenesis. Several scientific reviews suggested that RF exposure is non-genotoxic [1–7]. In our earlier investigations, we have examined whether non-genotoxic RF preexposure can induce AR and thus offer protection from subsequent
Abbreviations: RF, radiofrequency fields; AD, adaptation dose; CD, challenge dose; PCE, polychromatic erythrocytes; MN, micronuclei. ∗ Corresponding author. Tel.: +86 512 65881552; fax: +86 512 65880069. E-mail address: [email protected] (Y. Cao). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2012.12.003
damage induced by genotoxic agents. Mice were pre-exposed to 900 MHz RF at different power densities for 1–14 days (AD) and then subjected to a high dose of ␥-radiation (CD). The data indicated that compared with the animals which received CD alone, mice which were exposed to AD + CD exhibited significantly increased survival, decreased hematopoietic tissue damage in the spleen and bone marrow as well as decreased primary DNA damage in peripheral blood and bone marrow cells [8–10]. Furthermore, cultured human HL-60 cells which were pre-exposed to RF (AD) and subsequently treated with doxorubicin, a chemotherapeutic drug (CD) showed decreased apoptosis and intra-cellular Ca2+ and, increased proliferation, mitochondrial membrane potential and Ca2+ –Mg2+ ATPase activity, as compared with the cells treated with CD alone [11]. In view of the above observations, the current investigation was conducted using the classical micronucleus (MN) assay to determine whether pre-exposure of mice to 900 MHz RF (AD) can induce AR and offer protection to blood and bone marrow cells from the genetic damage induced by subsequent ␥-radiation (CD). 2. Materials and methods All animal handling procedures were reviewed and approved by the animal care/user ethical committee of Soochow University, Suzhou City, PR China (approval number A68-2011). Adult male ICR mice weighing approximately 25 gm were obtained from the animal center in Suzhou University and were housed in a facility maintaining 22 ± 1 ◦ C temperature, 50 ± 5% relative humidity and 12-hour light–dark cycles. They were fed on a commercial diet (Suzhou Shuangshi Laboratory Animal Feed Science Co., Ltd., Suzhou City, Jiangsu Province, PR China) and
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B. Jiang et al. / Mutation Research 751 (2013) 127–129
Table 1 The incidence of micronuclei (MN/2000 PCE) and % polychromatic erythrocytes (PCE) in the peripheral blood and bone marrow of mice exposed to radiofrequency fields (RF) and gamma radiation (GR). Group
MN/2000 PCE Blood
1. Un-exposed controls 5.2 ± 1.83 2. GR, 3 Gy 62.6 ± 19.46 3. RF, 900 MHz 7.5 ± 2.77 14.8 ± 7.25 4. RF + GR 6.8 ± 2.09 5. Sham 57.4 ± 21.00 6. Sham + GR a Expected Values 1.11 53.2 % Changes Statistical difference between groups (p values) 0.000 1 and 2 0.054 1 and 3 0.916 1 and 5 0.000 2 and 4 0.674 2 and 6 0.001 4 and 6
% PCE Bone marrow
Blood
Bone marrow
5.9 ± 1.37 50.4 ± 14.99 7.7 ± 2.37 23.5 ± 8.26 8.0 ± 2.37 53.9 ± 18.28 16.1 109
2.3 ± 0.55 0.6 ± 0.20 2.9 ± 0.71 1.7 ± 0.86 2.4 ± 0.72 0.7 ± 0.23 58.9 74.9
46.8 ± 5.91 18.3 ± 4.03 44.6 ± 4.78 33.3 ± 4.23 46.8 ± 4.58 22.0 ± 6.51 52.2 55.0
0.000 0.363 1.000 0.000 0.134 0.000
0.000 0.690 0.781 0.000 0.368 0.000
0.000 0.713 0.668 0.000 0.475 0.000
Numbers of animals in each group: 10. Data are mean ± standard deviation. a Expected values are the sum of two individual treatment (GR alone + RF alone) minus un-exposed controls.
provided water ad libitum. After 7 days quarantine period, the animals were randomly divided into six groups of 10 mice each. Group 1: un-exposed controls, Group 2: ␥-radiation alone; Group 3 and 4: 900 MHz RF exposure at 120 W/cm2 power density for 4 h/day for 7 days; Group 5 and 6: sham exposure for 4 h/day for 7 days. On day 7, at 4 h after the last RF/sham exposure, animals in groups 2, 4 and 6 were subjected to acute whole body exposure to ␥-radiation from a 60 Co source (Nordion, Ottawa, ON, Canada, total dose 3 Gy and dose rate 0.5 Gy/min). All mice were sacrificed 72 h later to collect peripheral blood and bone marrow (see below). 2.1. RF exposure The RF exposure system was described in detail earlier [8,9]. Briefly, it consisted of GTEM chamber (Giaga-hertz transverse electromagnetic chamber which was built in-house and installed in a room which maintained 37 ± 0.5 ◦ C temperature at Soochow University, Suzhou, Jiangsu, PR China, a signal generator (PMM, Cisano sul Neva, Italy) and a power amplifier (HD Communication Corp. Ronkonkoma, NY, USA). The continuous wave 900 MHz RF signal was generated, amplified and fed into the GTEM chamber through an antenna (Southeast University, Nanjing, Jiangsu, China). A field strength meter (PMM, Cisano sul Neva, Italy) was used to determine the precise position, inside GTEM, which provided the required power intensity of 120 W/cm. The power was continuously monitored and recorded in a computer controlled data logging system. Small plastic boxes were designed in-house to hold mice (a single restrained mouse in each box). The mice inside the boxes were placed on a non-conductive table/platform for RF exposure. The whole body average specific absorption rate (SAR) was calculated using the finite-difference-time-domain model and it was 548 mW/kg. Sham exposures were conducted in the same GTEM chamber, without the transmission of RF. The environmental conditions during and after RF/sham exposures were kept identical through out the study period. 2.2. Peripheral blood and bone marrow smears All mice were anesthetized first using carbon-di-oxide. From each animal, peripheral blood was collected by heart puncture and, one or two small drops were placed on pre-cleaned microscope slides to prepare thin smears. Then, the animals were killed by cervical dislocation to collect the femurs. The bone marrow was flushed out using 1 ml fetal bovine serum, gently mixed to obtain a fine cell suspension and, one or two drops were placed on pre-cleaned microscope slides to prepare thin smears. All smears were air dried, fixed in absolute methanol, air-dried and stained using acridine orange (Sigma; 0.01 mg/ml of 0.2 M phosphate buffer, pH 7.4). Coded slides were examined under 1000x magnification using a fluorescence microscope fitted with 450–460 nm excitation and 620–670 nm emission filters. Erythrocytes which were immature (PCE) and mature (NCE) were identified by their orange–red and green colors, respectively, and MN by their bright yellowish color. For each mouse, in both blood and bone marrow tissues, 2000 consecutive PCE were examined to record the incidence of MN. In addition, the % PCE was obtained from the examination of 1000 and 200 erythrocytes in blood and bone marrow, respectively. The data were decoded after completing the microscopic evaluation [12]. 2.3. Statistical analysis The results were subjected to statistical analyses using software SPSS Statistics version 20.0. One-way analysis of variance (ANOVA) and Student’s t-test were used
to compare the differences, if any, between groups with pair wise comparisons. A p value <0.05 was considered as significant difference.
3. Results The extent of genotoxicity determined by the incidence of MN/2000 PCE and cytotoxicity measured by % PCE in blood and bone marrow cells was similar among the 10 mice in each of the 6 groups. Hence, the results were pooled and the group mean indices (±standard deviation) are presented in Table 1. The results were: (i) the incidence of MN/2000 PCE in both blood and bone marrow was not significantly different between un-exposed control mice and those that were pre-exposed to RF/sham alone (AD) and, the values ranged between 5.2 and 8.0 MN/2000 PCE; (ii) a significant increase in MN was observed in mice exposed to ␥-radiation alone (CD): 62.6 and 50.4 MN/2000 PCE in blood and bone marrow, respectively; (iii) combined exposures of RF + ␥-radiation (AD + CD) resulted in a significant decrease in MN in both blood and bone marrow, as compared with the mice exposed to ␥-radiation alone (CD): 14.8 and 23.5 MN/2000 PCE in blood and bone marrow, respectively; (iv) such a decrease was not observed in mice exposed to sham + ␥radiation (AD + CD): 57.4 and 53.9 MN/2000 PCE in blood and bone marrow, respectively. The % PCE recorded in the blood and bone marrow tissues showed no significant differences between un-exposed controls and mice exposed to RF/sham alone (AD). There was a significant decrease in % PCE in blood and bone marrow in animals exposed to ␥-radiation alone (CD). In combined AD + CD exposures however, there was an increase in % PCE in mice exposed to RF + ␥-radiation (AD + CD) while no such increase was observed in animals which received sham + ␥-radiation (AD + CD). 4. Discussion Ionizing radiation-induced AR is well documented in the scientific literature. Animal and human cells that were pre-exposed in vivo or in vitro to an extremely small adaptation dose of ionizing radiation were reported to be resistant to subsequent damage induced by a higher challenge dose of the same or similar genotoxic agent(s). A search for similar induction of AR by non-ionizing RF exposure is under way and evidence is accumulating in recent years. Sannino et al. [13] were the first to report that human blood lymphocytes which were pre-exposed in vitro for 20 h–900 MHz
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at an average specific absorption rate of 1.25 W/kg (AD) and then treated with a genotoxic dose of mitomycin C, a chemotherapeutic drug (CD) exhibited significantly decreased incidence of MN when compared with the cells treated with CD alone. Such decrease in MN was confirmed in two subsequent investigations in different experimental exposure conditions [14,15]. In animal studies, our research group was the first to report RF-induced AR. Adult male Kunming mice were pre-exposed to 900 MHz RF at 12, 120 and 1200 W/cm2 power density for 1 h/day for 14 days (AD) and then subjected to ␥-radiation (CD). Compared with the animals exposed to CD alone: (i) mice exposed to RF + 8 Gy ␥-radiation showed increased survival and (ii) mice exposed to RF + 5 Gy ␥-radiation exhibited significantly decreased hematopoietic tissue damage determined from increased colony forming units in bone marrow and spleen. These changes were accompanied by elevated hematopoietic growth factors, inhibition of ␥-ray-induced suppression of hematopoietic progenitor stem cells as well as increased expression of cell cycle-related genes, viz., cyclin-D1, cyclin-E, cyclin-DK4 and cyclin-DK2 which might have helped in by-passing the G1/S-block check point (due to ␥-radiation exposure) resulting in restoration of cell cycle and cell proliferation [8,9]. In a subsequent study, ICR mice were pre-exposed to 900 MHz RF at 120 W/cm2 power density for 4 h/day for 1, 3, 5, 7 and 14 days (AD) and then subjected to 3 Gy ␥-radiation (CD). The extent of primary DNA damage was assessed in peripheral blood leukocytes using the alkaline comet assay. A significant and exposure time-dependent decrease in damage was observed in mice exposed to AD + CD compared to those exposed to CD alone. The extent of decrease was maximal in the animals pre-exposed to RF for 7 days [10]. In vitro experiments, human HL-60 cells were pre-exposed to 900 MHz RF at 12 W/cm2 power density for 1 h/day for 3 days (AD) and then treated with doxorubicin, a chemotherapeutic drug (CD). Such cells exhibited decreased apoptosis and intra-cellular Ca2+ and, increased proliferation, mitochondrial membrane potential and Ca2+ –Mg2+ -ATPase activity, as compared with the cells treated with CD alone [11]. Our earlier observations of RF-induced AR in mice gained support from similar significantly enhanced survival data reported by a research group in Iran. In the first study, adult male Sprague Dawley rats were pre-exposed to 900 MHz RF (GSM) at 2 W power density with or without flaxseed oil. The intermittent RF exposure (AD) was for 4 days, i.e. 3 h ON and 9 h OFF, twice a day (total 6 h/day). On day 5, the animals were subjected to whole body LD50/30 dose of 8 Gy ␥-radiation (CD). After 30 days, a significant increase in survival was observed in animals exposed to AD + CD compared with those subjected to CD alone [16]. In the second investigation, adult Balb/c mice and Wistar rats were pre-exposed to amplitude modulated 900 MHz RF (GSM) at 2 W power density for 5 days and then exposed to lethal dose of 8.8 Gy ␥-radiation (CD). A significantly increased survival rate was observed in mice and rats exposed to AD + CD as compared to the animals which received CD alone [17]. The results obtained in the current investigation indicated that compared with un-exposed control animals: (i) mice which were pre-exposed to RF alone (AD) exhibited no significant increase in genotoxicity (MN/2000 PCE) and thus confirmed that RF exposure is non-genotoxic, a conclusion made in several earlier reviews (also, there was no evidence of increased cytotoxicity which was assessed from % PCE), (ii) mice which were exposed to an acute whole body dose of ␥-radiation alone (CD) exhibited significant increases in genotoxicity and cytotoxicity and, (iii) mice which were pre-exposed to RF and subsequently subjected to ␥irradiation (AD + CD) exhibited significant decreases in genotoxicity and cytotoxicity. Thus, the data provided further evidence for RFinduced AR. Also, most of the research on the biological effects of RF exposure, thus far, has been focused on ‘adverse’ effects. However,
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the beneficial effect of RF pre-exposure that is observed in the study and the other studies mentioned above require special attention. Conflict of interest statement All authors declare no conflict of interest. Acknowledgments This research is supported by funding from the National Natural Science Foundation of China (Nos. 81020108028), 973 project grant from China Ministry of Science and Technology (2011CB503705) and The Priority Academic Program Development of Jiangsu Higher Education Institutions. The funding agencies had no role in designing the experiments, data collection and analysis, preparation of the manuscript, and decision to publish the results. We also thank the technicians in the laboratory for help during the experimentation. References [1] M.L. Meltz, Radiofrequency, exposure and mammalian cell toxicity, genotoxicity, and transformation, Bioelectromagnetics 6 (2003) S196–S213. [2] G. Vijayalaxmi, Obe, Controversial cytogenetic observations in mammalian somatic cells exposed to radiofrequency radiation, Radiat. Res. 162 (2004) 481–496. [3] L. Verschaeve, Genetic effects of radiofrequency radiation (RFR), Toxicol. Appl. Pharmacol. 207 (2005) S336–S341. [4] Vijayalaxmi, T.J. Prihoda, Genetic damage in mammalian somatic cells exposed to radiofrequency radiation: a meta-analysis of data from 63 publications (1990–2005), Radiat. Res. 169 (2008) 561–574. [5] L. Verschaeve, Genetic damage in subjects exposed to radiofrequency radiation, Mutat. Res. 681 (2009) 259–270. [6] L. Verschaeve, J. Juutilainen, I. Lagroye, J. Miyakoshi, R. Saunders, R. de Seze, T. Tenforde, E. van Rongen, B. Veyret, Z. Xu, In vitro and in vivo genotoxicity of radiofrequency fields, Mutat. Res. 705 (2010) 252–268. [7] Vijayalaxmi and T.J. Prihoda, Genetic damage in human cells exposed to non-ionizing radiofrequency fields: a meta-analysis of the data from 88 publications (1990–2011), Mutat. Res. Genet. Toxicol. Environ. Mutagen. (2012) http://dx.doi.org/10.1016/j.mrgentox.2012.09.007 [8] Y. Cao, Q. Xu, Z.-D. Jin, J. Zhang, M.-X. Lu, J.H. Nie, J. Tong, Effects of 900-MHz microwave radiation on ␥-ray-induced damage to mouse hematopoietic system, J. Toxicol. Environ. Health Part A 73 (2010) 507–513. [9] Y. Cao, Q. Xu, Z.-D. Jin, Z. Zhou, J.-H. Nie, J. Tong, Induction of adaptive response: pre-exposure of mice to 900 MHz. radiofrequency fields reduces hematopoietic damage caused by subsequent exposure to ionising radiation, Int. J. Radiat. Biol. 87 (2011) 720–728. [10] B. Jiang, J. Nie, Z. Zhou, J. Zhang, J. Tong, Y. Cao, Adaptive response in mice exposed to 900 MHz radiofrequency fields: primary DNA damage, PLoS ONE 7 (2012) e32040. [11] Z. Jin, C. Zong, B. Jiang, Z. Zhou, J. Tong, Y. Cao, The effect of combined exposure of 900 MHz radiofrequency fields and doxorubicin in HL-60 cells, PLoS ONE 7 (2012) e46102. [12] Vijayalaxmi, M.R. Frei, S.J. Dusch, V. Guel, M.L. Meltz, J.R. Jauchem, Frequency of micronuclei in the peripheral blood and bone marrow of cancer-prone mice chronically exposed to 2450 MHz radiofrequency radiation, Radiat. Res. 147 (1997) 495–500. [13] A. Sannino, M. Sarti, S.B. Reddy, T.J. Prihoda Vijayalaxmi, M.R. Scarfi, Induction of adaptive response in human blood lymphocytes exposed to radiofrequency radiation, Radiat. Res. 171 (2009) 735–742. [14] A. Sannino, O. Zeni, M. Sarti, S. Romeo, S.B. Reddy, A. Belisario, T.J. Prihoda Vijayalaxmi, M.R. Scarfi, Induction of adaptive response in human blood lymphocytes exposed to 900 MHz radiofrequency fields: influence of cell cycle, Int. J. Radiat. Biol. 87 (2011) 993–999. [15] O. Zeni, A. Sannino, S. Romeo, R. Massa, M. Sarti, A.B. Reddy, T.J. Prihoda, Vijayalaxmi, M.R. Scarfi, Induction of an adaptive response in human blood lymphocytes exposed to radiofrequency fields: influence of the universal mobile telecommunication system (UMTS) signal and the specific absorption rate, Mutat. Res. 747 (2012) 29–35. [16] S.M.J. Mortazavi, M.A. Mosleh-Shirazi, A.R. Tavassoli, M. Taheri, Z. Bagheri, R. Ghalandari, S. Bonyadi, M. Shafie, M. Haghani, A comparative study on the increased radioresistance to lethal doses of gamma rays after exposure to microwave radiation and oral intake of flaxseed oil, Iran J. Radiat. Res. 9 (2011) 9–14. [17] S.M.J. Mortazavi, M.A. Mosleh-Shirazi, A.R. Tavassoli, M. Taheri, A.R. Mehdizadeh, S.A.S. Namazi, A. Jamali, R. Ghalandari, S. Bonyadi, M. Haghani, M. Shafie, Increased radioresistance to lethal doses of gamma rays in mice and rats after exposure to microwave radiation emitted by a GSM mobile phone simulator, Dose Resp. (2012) Doi: 10.2203/dose-response.12-010.