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Effects of microwave at 2.45 GHzradiations on reproductive system ofmale ratsKavindra Kumar Kesari a & Jitendra Behari aa Bioelectromagnetic Laboratory , School of EnvironmentalSciences, Jawaharlal Nehru University , New Delhi-110067, IndiaPublished online: 27 May 2010.
To cite this article: Kavindra Kumar Kesari & Jitendra Behari (2010) Effects of microwave at 2.45GHz radiations on reproductive system of male rats, Toxicological & Environmental Chemistry, 92:6,1135-1147
To link to this article: http://dx.doi.org/10.1080/02772240903233637
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Toxicological & Environmental ChemistryVol. 92, No. 6, July 2010, 1135–1147
Effects of microwave at 2.45GHz radiations on reproductive system
of male rats
Kavindra Kumar Kesari and Jitendra Behari*
Bioelectromagnetic Laboratory, School of Environmental Sciences, Jawaharlal NehruUniversity, New Delhi-110067, India
(Received 22 July 2009; final version received 28 August 2009)
Radio frequency and microwave radiations are a part of non-ionizingelectromagnetic radiations present in the environment and are now beingperceived as health risks. The present study was performed to investigatethe effect of 2.45GHz microwave radiation on reproductive pattern ofmale Wistar rats. Animals were exposed at 2.45GHz frequency with0.34mWcm�2 power density continuously for 2 h day�1 for 35 days, wherethe whole body specific absorption rate was 0.11Wkg�1. Exposureoccurred in a ventilated plexiglass cage and kept in anechoic chamber ata distance of 2m from the horn antenna. In the present study, apoptosiswas tested using terminal deoxynucleotide transferase dUTP nick endlabeling assay. Normal and apoptotic sperm cells were also quantified byflow cytometry. Antioxidant enzymes (superoxide dismutase (SOD),glutathione peroxidase (GPx), and catalase (CAT)) were also determinedto confirm any mutagenic effects, such as DNA fragmentation. Chronicexposure to these radiations produced formation of apoptotic cells in testis.In addition, a significant decrease in the levels of GPx, and SOD activitiesas well as an increase in CAT activity was observed in the exposed group.These results indicate that a low level exposure of microwave radiationsexerts a negative impact on male reproductive system function.
Keywords: DNA fragmentation; apoptosis; antioxidant enzyme; micro-wave radiation
Introduction
The effects of microwave radiation on biological systems is primarily due to anincrease in temperature i.e. thermal (Stuchley 1988), although non-thermal effectshave also been noted and adverse effects documented (Agarwal et al. 2008a; Akdaget al. 1999; Khillare and Behari 1998; Kunjilwar and Behari 1993; Lokhmatova1994; Paulraj and Behari 2004, 2006; Weyandt et al. 1996). Lai and Singh (1996)first reported DNA strand breaks due to microwave exposure at low intensitylevels following exposure to 2.45GHz continuous and pulsed radio frequency (RF)radiation for 2 h day�1. Animal studies indicated that electromagnetic waves(EMWs) may exert a wide range of damaging effects on the testicular function
*Corresponding author. Email: [email protected]
ISSN 0277–2248 print/ISSN 1029–0486 online
� 2010 Taylor & Francis
DOI: 10.1080/02772240903233637
http://www.informaworld.com
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and male germ lines (Dasdag, Akdag, and Aksen 2003; Davoudi, Brossner, andKuber 2002).
Deepinder, Kartikeya, and Agarwal (2007) examined 361 men attending aninfertility clinic who indicated that use of cell phones adversely affects the qualityof semen as evidenced by decreased sperm counts, motility, viability, andmorphology, which might contribute to male infertility. In another epidemiologicalstudy (Fejes et al. 2005) conducted on 371 men, it was concluded that prolongeduse of cell phones might exert negative effects on sperm motility. In a similar pilotstudy, Agarwal et al. (2008b) reported that the direct exposure of human semensamples to cellular phone radiation (in talking mode) decreased sperm motility andviability. In a small prospective study involving 13 normal men, GSM phone usagefor 5 days for 6 h day�1 decreased the rapid progressive motility of sperm(Davoudi, Brossner, and Kuber 2002). Investigators also found a decrease in spermmotility in semen samples of men exposed to 900MHz cell phone for 5min (Erogulet al. 2006).
Adverse effects due to microwave radiations in animals showed a decrease indiameter of seminiferous tubule (Dasdag, Akdag, and Aksen 2003; Dasdag, Ketani,and Akdag 1999), weight of testicular organs (i.e. caput, cauda, corpus), sperm count(Behari and Kesari 2006), and destruction in Leydig cells. Saunders and Kowalczuk(1981) also showed that microwave radiation of 50mWcm�2 and frequency2.45GHz for 30–40min resulted in significant degeneration of seminiferousepithelium in mice. Nakamura et al. (2003) found that exposure to 2.45GHzcontinuous-wave (CW) microwave on females at 2mWcm�2 power density for90min decreased uteroplacental blood flow and increased progesterone and PGF2�levels in pregnant rats.
The aim of the present study was to determine the influence of chronicelectromagnetic field (EMF) exposure (2.45GHz) on reactive oxygen species (ROS)generation and to examine the negative effects on male fertility. Agarwal et al.(2008b) reported that microwaves may produce ROS emitted from mobile phonesinducing infertility. It was suggested that sperm damage (DNA fragmentation) byROS may occur due to microwave field exposure (Erenpreiss et al. 2002; Marchettiet al. 2002; Saleh et al. 2002). Previous reports from our laboratory showed that theseradiations affect growth related enzymes in rats (Paulraj and Behari 2002), proteinkinase C activity in brain (Paulraj and Behari 2004), brain Naþ–Kþ ATPase activity(Behari, Kunjilwar, and Pyne 1998), single strand DNA breaks in rat brain cells(Paulraj and Behari 2004) antioxidative changes at 50GHz exposed rat brain (Kesariand Behari 2009), and effect on fertility of male rats (Behari and Kesari 2006, Kesariand Behari 2010).
Materials
Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kitwas purchased from R & D system, Inc, USA. The glutathione peroxidase (GPx,catalog no. 703102), catalase (CAT, catalog no. 707002), and superoxide dismutase(SOD, catalog no. 706002) antioxidant enzyme kit was purchased from the CaymanChemical Company, Ann Arbor, MI, USA. Propidium iodide (PI) and RNAse werepurchased from Sigma Aldrich Chemicals, USA. The rest of the chemicals werepurchased from Thomas Baker Chemicals Limited, Mumbai.
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Methods
Animal exposure
Wistar rats (70 days old and 200� 20 g body weight) were obtained from animalfacility of Jawaharlal Nehru University, New Delhi. Rats were divided into twogroups, control (n¼ 6) and exposure (n¼ 6). All experiments were repeated thriceand carried out in a blind pattern. All animals were housed in an air conditionedroom, where the temperature was maintained at 25–27�C and humidity (40–50%).Animals were provided with standard food pallets (prepared by Brook Bond IndiaLimited) and water ad libitum. The protocols for animal experimentation describedin this study were approved previously by the Institutional Animal EthicalCommittee (IAEC) and Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA). All subsequent animal experiments adhered tothe ‘‘Guidelines for Animal Experimentation’’ of the University.
Exposure chamber
Six rats at a time were placed in a plexiglass cage. The cage was ventilated with holesof 1 cm diameter. The dimension of each house in exposure cage was identical andmade in such a manner that the animal moved freely. Six rats were kept in aplexiglass cage (43 cm� 27 cm� 15 cm) exposed to 2.45GHz irradiation in ananechoic chamber at a far field distance from horn antenna (Figure 1). The anechoicchamber was lined with radar absorbing material (attenuation, 40 db) to minimizethe reflection of scattered beam. The temperature in the chamber was maintained at25–27�C throughout the experiment by circulating air. In the position of animalplacement, a horn antenna (13 cm� 9.8 cm) was placed and the field was measuredwhich is all through homogeneous in vertical plane of midline of beam. The exposurecage was placed in such a manner that all animals were irradiated homogeneouslywith same power level. Rats were exposed with 2.45GHz source (duty cycle 3/4)through the horn antenna, 2 h day�1 for 35 days. The power density at the receiving
Figure 1. Diagram of anechoic chamber using 2.45GHz.
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end was 0.34mWcm�2. Every day the cage was placed in the same position facingthe horn antenna and the same number of rat position was filled in reshuffledpositions. This was done to ensure that each animal received the same dosage. Asimilar experiment was performed with control animals (sham exposed) withoutpower input in microwave generator.
Specific absorption rate
For a plane wave exposure having random polarization and power density of0.34mWcm�2, the specific absorption rate (SAR) value at 2.45GHz turns out to be0.11Wkg�1. A similar setup was previously used by Paulraj and Behari (2004, 2006).
TUNEL assays
Testes of exposed and control rats were removed after sacrificing the rats.Paraffin blocks of testes were made and transverse sections (TS) (5 mm) weretaken. The TS of testes were deparaffinized by heating the slide at 57�C for 5minon a slide warmer. Thereafter, slides were transferred to xylene in a glass jar andkept at 18–24�C for 5min. Another change of xylene was conducted to obtaincomplete deparaffination. Slides were washed sequentially in 100, 95, and 70%ethanol at 18–24�C for 5min and twice in DNAase free water for 2min each. Theslides were transferred to 10mM phosphate buffered saline (PBS) and then 50 mLof proteinase K solution (50mL d H2O containing 1 mL proteinase K). Aftertreatment, slides were kept at 18–24�C for 15min and washed immediately inDNAase-free water twice for 2min. Washed slides were placed in quenchingsolution (3% H2O2 in methanol) at 18–24�C for 5min to terminate permeabiliza-tion reaction, washed with distilled water, and transferred to 10mM PBS. Theslides were kept in terminal deoxynucleotidyl transferase labeling buffer for 5minand dried. About 50 mL of labeling reaction mixture was added on each sampleby covering with a cover slip and the slides were kept at 37�C for 1 h in a humidchamber. Finally, the slides were transferred to buffer in coplin jar and kept for5min at 18–24�C. The slides were removed from the jar and dipped into PBS andviewed under bright field light microscope (10� and 20�).
Flow cytometry estimation of apoptosis and total sperm count
One hundred microliter sperm cells (collected from caput and cauda regions) of2� 106 cells mL�1 of concentration was taken in a Falcon 12� 75mMpolypropylene tube with snap cap and incubated in an ice bath for 15min. Onemilliliter ice-cold 70% ethanol was added at a time interval of 3–5 s per tube. Thesample was incubated overnight at 4�C. After the completion of an incubationperiod, the sample was centrifuged at 1000� g for 15min at 4�C and finally thesupernatant was decanted. One hundred microliters of RNAase (100 unit) was addedto the pellet and pipetted. Samples were incubated at room temperature for 20min.Finally, these were stained with 500 mL (20 mgmL�1) PI and incubated overnightat 4�C in the dark.
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Antioxidant enzyme estimation
In the present investigations, enzyme assay was used to determine the antioxidantenzyme activity (SOD, GPx, and CAT) in exposed and control rats. Immediatelyafter the exposure period, rats were anesthetized, sacrificed, and sperms werecollected (from caput and cauda regions) in cold buffer. The assay was performedwith positive control for enzyme estimation.
Sperm sample preparation
Sperm was added to 5–10mL of cold buffer (50mM TRIS HCl, pH 7.5, 5mMethylenediaminetetraacetic acid (EDTA) and 1mM dithiothreitol) for GPx, 20mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (1mM ethylene glycoltetraacetic acid, 210mM mannitol, and 70mM sucrose) for SOD and 5–10mL ofcold buffer (50mM potassium phosphate, pH 7, containing 1mM EDTA) for CAT.All the samples were centrifuged at 10,000� g for 15min at 4�C, supernatant wascollected and enzyme assay was performed. SOD, GPx, and CAT were determinedusing kits supplied by Cayman Chemical Company, Ann Arbor, MI, USA.
Analysis of data
Statistical analysis was performed with SX package. All data are presented asmean� standard deviation (SD). The difference between exposed and controls weretested for significance by using one-way ANOVA with Bonferroni correction forcomparison of means. A difference at p50.05 was considered statistically significant.
Results
TUNEL assay
TUNEL assay of apoptotic cells stained in brown is shown in Figure 2. It is apparentthat formation of a few apoptotic cells occurred in testes of control (Figures 2aand 2b). In exposed samples, there is an increase in the number as well as the size ofapoptotic cells (Figures 2c and 2d). In both segments, apoptotic cells were generallysurrounded by an open space, possibly resulting from shrinkage of nuclei andcytoplasm, which is an indication of DNA fragmentation (apoptosis) in exposed rats.
Flow cytometry measurement of apoptosis and sperm count
To confirm the occurrence of apoptosis in the epididymis after microwave exposure,fluorescence-activated cell sorting analysis was used and the sperm count with PIstaining. Figure 3 shows the peak of apoptosis (M2) increased significantly inexposed (Figure 3a) as compared to control samples (Figure 3b). Moreover, anotherpeak (M1) shows a significant decrease in sperm count of exposed compared tocontrol group. The comparative study between sperm count and apoptosis is shownin Figure 4. Statistical results on sperm (exposed) shows a significant decrease in themean value in the total count. The number of apoptotic cells in exposed group wassignificantly increased. Data presented in Table 1 is suggestive of possible infertilityin microwave exposed animals.
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Figure 2. Detection of apoptotic cells in transverse section of testis after TUNEL staining.
PI.004
Apo
ptos
is
Apo
ptos
is
Spe
rm c
ount
Spe
rm c
ount
M1 M1M2 M2
PI.002(a) (b)200
Cou
nts
Cou
nts
160
120
80
40
0
200
160
120
80
40
00 200 400
FL2-A600 800 1000 0 200 400
FL2-A600 800 1000
Figure 3. Flow cytometer analysis in: (a) exposed and (b) control sperm samples for apoptoticand total sperm cell counts. Histogram of fluorosphere blue-fluorescence (FL2-A) and gate Care used to count the total apoptotic cells (M2) and total sperm cell count (M1).
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Antioxidant enzyme activity
Microwave exposure significantly decreased GPx and SOD activities. However, inexposed animals, there was a significant increase in CAT activity (Figures 5–7 andTable 2).
70
60
50
40
30
20
10
0Sperm count Apoptosis
Control
Tot
al s
perm
cou
nt (
1µ10
6) Exposed
*
*
Figure 4. Comparative analysis of control and exposed sperm samples. Data are expressed asmean� SD.*Denotes significance (p50.05).
6
GP
x ac
tiviry
n m
ol/m
in/m
l
5
4
3
2
1
0Control
*
Exposed
Figure 5. Comparative analysis of antioxidant enzyme activities in rat sperm cells in exposedand control groups of rat sperms.*Denotes significance (p50.05).
Table 1. Comparative statistical analysis between apoptosis and spermcount measured by flow cytometer.
Apoptosis(gated cell %)
Sperm count(gated cell %)
Control (Mean� SD) 7.43� 1.30 56.83� 2.69Exposed (Mean� SD) 14.30� 1.92 29.64� 1.59p value p50.005 p50.001
Notes: A significance result was considered for p50.05. The analysis wasdone by one way ANOVA.
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Discussion
Concerns are growing regarding the hazardous effect of EMF, especially household
microwave sources (such as mobile phones and microwave ovens) on humans.
Studies on adverse effects were reported by Kowalczuk, Saunders, and Stapleton
(1983) and Saunders and Kowalczuk (1981), showing 1.7GHz (50mWcm�2),2.45GHz (44 and 30Wkg�1) altered seminiferous epithelia, sperm counts, sperm
morphology, and primary spermatocytes in mice. However, the level of exposure in
8
*
CA
T a
ctiv
ity n
mol
/min
/ml 7
6
5
4
3
2
1
0Control Exposed
Figure 7. Comparative analysis of antioxidant enzyme activities in rat sperm cells inexposed rats.*Denotes significance (p50.05).
200
*
SO
D a
ctiv
ity U
/ml
180160140120100806040200
Control Exposed
Figure 6. Comparative analysis of antioxidant enzyme (SOD) activities in rat sperm cells.*Denotes significance (p50.05).
Table 2. Comparative statistical analysis of GPx, SOD, and CAT.
GPx (nmolmin�1mL�1) SOD (UmL�1) CAT (nmolmin�1mL�1)
Control (Mean� SD) 5.38� 0.69 178.18� 11.26 2.63� 0.58Exposed (Mean� SD) 4.01� 0.49 159.10� 7.63 6.37� 0.99p value p50.003 p50.001 p50.003
Notes: Significance was considered as p50.05. The analysis was done by one way ANOVA.
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these experiments was high. The study of Kesari and Behari (2010) at 50GHzexposure shows alteration in sperm cell cycle and an increase in apoptosis. Moreoverin vivo studies (mice and rats) have also indicated (Lee et al. 2004) that EMF mayinduce cell death (apoptosis). Recently Agarwal et al. (2008a, 2008b) suggested thatcell phones adversely affected the quality of semen by decreasing sperm count,motility, viability, and morphology, which might contribute to male infertility. Fejeset al. (2005) observed negative effects on sperm motility due to prolonged use of cellphones. Decrease in sperm concentration was found due to keeping cell phones closeto waist (Kilgallon and Simmons 2005). Erogul et al. (2006) reported in vitro adecrease in sperm motility, when semen samples of men were exposed for 5min tothe same source (900MHz). EMF devices (telecommunication and cooking) mayincrease the incidence of cancer and DNA damage in sperm and brain cells (Kumar2000; Paulraj and Behari 2006).
The precise mechanisms underlying non-thermal interaction of microwaveradiation with reproductive system is yet to be elucidated. Microwaves may affectreproductive function by a unique non-thermal action, a thermal action, orcombination of these two (Blackwell 1979). Various investigators postulated thatthe mechanisms underlying non-thermal microwave effects are attributed to physicalalterations of non-equilibrium thermodynamics (Binhi 2002; Bischof 2003; Frohlich1968; Scott 1999). It was demonstrated that chromosomal DNA is a target forinteraction with non-thermal microwaves (Belyaev et al. 1993; Belyaev, Alipov, andShcheglov 1992; Ushakov et al. 1999). The quantum mechanical model for primaryinteraction of microwaves with DNA was proposed by Arinichev et al. (1993). DNAcontains two different codes (Belyaev 1993), the first one coding the genes and thesecond determining the spectrum of natural oscillations (electromagnetic, mechan-ical, and acoustic) in chromosomal DNA. These two codes are hypotheticallyresponsible for the gene expression at different stages and for genomic rearrange-ment in evolution.
Apoptosis was observed by TUNEL assay method induced by microwaveexposure. The number of apoptotic cells as well as their size was increased in exposedsamples. The possible mechanism of apoptosis were explored by several investiga-tors. It is speculated that the presence of DNAse after activation resulted inapoptotic degradation (Gorczyca et al. 1993; Maione et al. 1997; McCarthy andWard 1999; Weil, Jacobson, and Raff 1998). However, the DNA fragmentation is aconsequence of apoptosis in affected cells. Most studies on apoptosis in male germline focused on cell illumination during spermatogenesis (Allan, Harmon, and Kerr1987; Blanco-Rodriguez and Martinez-Garcia 1998; Blanco-Rodriguez 2002a,2002b; Brash 1996; Ricote et al. 2002; Woolveridge and Morris 2000). Our resultsalso show an increase in apoptotic cells and decrease in sperm cells due to microwaveexposure.
In the present investigation, the possible mechanism of microwave radiationappears to generate free radicals leading to the overproduction of hydrogenperoxide. This may produce a change in antioxidative levels due to free radicalsoxidative stress. In the present study of rat sperm samples, significant changes in theantioxidative system were also observed, such as a decrease in GPx and SODactivities (Ilhan, Gurel, and Armutcu 2002), and significant increase in CAT level(Irmak, Fadillioglu, and Gulec 2002). GPx is a relatively stable antioxidant enzyme;and inactivated under conditions of severe oxidation stress (Condell and Tappel1993). Alvarez et al. (1987) reported a decrease in the levels of SOD activity, which
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may lead to increase in the generation of reactive superoxide ions in biologicalsamples. A decrease in GPx activity might be due to the excessive production of freeradicals. Moreover CAT activity is enhanced when H2O2 levels are particularly high(Jones et al. 1981). Under normal conditions adequate levels of cellular antioxidantsmainly SOD, CAT, and GPx maintain free radicals scavenging potential in the testes.However, with radiation exposure the activities of these enzymes were altered,suggestive of oxidative stress.
Conclusions
In the present investigation, antioxidative changes and apoptosis were noted inreproductive pattern of male rats and our present findings indicate: (1) increase inapoptosis, (2) decrease in total sperm count, (3) decrease in the level of GPx andSOD activities, and (4) increase in the level of CAT activity. The data show thatprolonged exposure of microwave radiation may induce genetic damage inreproductive system of male rats due to overproduction of ROS. These findingsmay be helpful in initiating remedial measures to prevent hazardous effects ofmicrowave radiation.
Acknowledgment
The authors are grateful to Indian Council of Medical Research (ICMR), New Delhi, for thefinancial assistance.
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