Biophysical Evaluation of Radiofrequency Electromagnetic Field Effects on Male Reproductive Pattern

DOI 10.1007/s12013-012-9414-6 REVIEW PAPER Biophysical Evaluation of Radiofrequency Electromagnetic Field Effects on Male Reproductive Pattern Kavindra Kumar Kesari Sanjay Kumar Jayprakash Nirala Mohd. Haris Siddiqui Jitendra Behari Ó Springer Science+Business Media, LLC 2012 Abstract There are possible hazardous health effects of exposure to radiofrequency electromagnetic radiations emitted from mobile phone on the human reproductive pattern. It is more effective while keeping mobile phones in pocket or near testicular organs. Present review examines the possible concern on radio frequency radiation interaction and biological effects such as enzyme induction, and toxicological effects, including genotoxicity and carcinogenicity, testicular cancer, and reproductive outcomes. Testicular infertility or testicular cancer due to mobile phone or microwave radiations suggests an increased level of reactive oxygen species (ROS). Though generation of ROS in testis has been responsible for possible toxic effects on physiology of reproduction, the reviews of last few decades have well established that these radiations are very harmful and cause mutagenic changes in reproductive pattern and leads to infertility. The debate will be focused on bio-interaction mechanism between mobile phone and testicular cancer due to ROS formation. This causes the biological damage and leads to several changes like decreased sperm count, enzymatic and hormonal changes, DNA damage, and apoptosis formation. In the present review, physics of mobile phone including future research on various aspects has been discussed. Keywords ROS Mobile phone Microwave Infertility Testicular cancer Apoptosis K. K. Kesari (&) S. Kumar J. Nirala J. Behari (&) School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India e-mail: kavindra_biotech@yahoo.co.in; kkkesari@mail.jnu.ac.in J. Behari e-mail: masijnudelhi@gmail.com Mohd. H. Siddiqui Department of Biotechnology, Integral University, Lucknow, India Introduction Over the last decade, the exponential growth of mobile communications has been accompanied by a parallel increase in the density of electromagnetic field (EMF). The increased use of EMF in modern society has subjected the general and working population to unprecedented levels of exposure to these fields. Radiofrequency (RF) and microwave radiations emitted from mobile phones and their towers are the measurement of the macroscopical interaction between EMF and the biological system in terms of absorbed power. Exposure to RF fields from a base station is measured as power density which is a measure of power per unit area. The power density is often expressed in watts per square meter (W/m 2 ) or microwatts per square centimeter (lw/cm 2 ). The measurement of RF energy dose from a cell phone absorbed by the body terms as specific absorption rate (SAR), is expressed in units of watts per kilogram (W/kg) or milliwatts per gram (mw/g). It is well accepted that low frequency electric field is attenuated by the surface of any physical body, including biological bodies, while magnetic field of the same frequency penetrates the human bodies without any loses [1]. Among many biological targets, the DNA molecule has received the greatest attention with respect to potential EMF damage because of its relevance for cell function, proliferation, viability, mutation, and cancer. Due to these radiations, the problems of the human reproductive tract lead to a variety of undesired outcomes such as low sperm count, apoptosis, invasion, metastasis, cell proliferation, and changes in enzyme level. It is understood that such changes are complex phenomena controlled by complex series of pathways, which communicate with each other through a myriad of signaling cascades and contribute to tumor promotion. Kesari et al. [2 4] have shown tumor promotion in testicular cells by

identifying several parameters i.e., protein kinase C, (PKC) histone kinase, apoptosis, change in cell cycle, and low sperm count. Desai et al. [5] suggested that any tumor promoting effects of radiofrequency electromagnetic waves (RF-EMW) might be due to the effect it has on PKC, ODC, intracellular calcium spikes, and stimulation of stress kinase. Authors also indicated that the stimulation of plasma membrane NADH oxidase might a play central role in abovementioned effects, where commercially available cellular phones might affect cell function via non-thermal effects. Authors hypothesized that the plasma membrane might be the target of cell phone radiation and this can increase ROS formation by increasing the activity of plasma membrane NADH oxidase. In most cases of infertility, high concentration of reactive oxygen species (ROS) are reported [4, 6]. Kumar et al. [7] also reported an increased level of ROS due to microwave exposure. The generation of ROS in the testis is responsible for possible toxic effects on physiology of reproduction. However, cells have their defense mechanism i.e., antioxidants [glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD)] to fight against increased production of ROS. Kesari et al. [3, 4, 8 11] have shown antioxidant changes in these enzyme level at various radiofrequency/microwave frequencies (900 MHz, 2.45 GHz, 50 GHz) in different type of cells (brain and sperm). Several studies since decades have recommended that men should not carry mobile phones in their front trouser pocket. Fejes et al. [12], Aitken et al. [13], Erogul et al. [14], Yan et al. [15], Agarwal et al. [16, 17], and Falzone et al. [18] concluded that usage of mobile phones, exposure to mobile phone signals, or storage of a mobile phone close to the testis affect sperm counts, motility, viability, and structure. Deepinder et al. [19] has also reported that testis is most sensitive part which exposed due to keeping mobile phones in trouser pocket. In a study of males attending an infertility clinic in Poland, Wdowiak et al. [20] found an increased percentage of sperm cells of abnormal structure and a decrease in sperm motility associated with the duration of exposure to mobile phone radiations. Mailankot et al. [21] has found that rats exposed to mobile phone emissions for 1 h/day for 28 days had impaired semen quality, and the authors believed this may affect male fertility adversely. Research also suggests that microwave radiation from mobile phones may induce chromosomal instability and lead to increased cancer risk [22 24]. Exposure to various kinds of frequencies present in environment can evoke a number of alterations at the cellular level, such as increased Ca 2? efflux [25], reduced melatonin secretion [26], disturbed balance of antioxidant enzymes [8], and induction of micronuclei [27]. Microwave exposure disrupts the seminiferous tubules and reduces leydigs cells population and testosterone concentration in rats with increased luteinizing hormone (LH) level [28]. It is well established that testosterone is essential for the spermatogenesis and formation of spermatozoa [29]. In the present review, testosterone and other hormonal changes occurred due to microwave or a mobile phone frequency has been discussed. Present study has been carried out to describe the biophysics of mobile phones at several frequencies, and their associated gross effects on male fertility. Further, factors affecting male fertility will be discussed followed by a theory of field interaction mechanism and ongoing studies surrounding mobile phone research. Biophysical Mechanism of Body Interaction with Mobile Phone Radiations and SAR Measurement The risk assessment is generally based on experimental data from biological systems. A possible mechanism is nevertheless pertinent for two reasons. First, the experimental data regarding biological effects of RF fields are fragmentary and inconsistent in many respects, and understanding of the biophysical mechanisms on reported effects can help to rationalize and understand the data. Second, it is necessary to extrapolate data from one exposure condition to others, and reliable extrapolation with understanding of the underlying mechanisms is needed. The electric and magnetic fields produced in body by a nearby electromagnetic source may cause both thermal and non-thermal biological effects. The effects of magnetic fields vary with frequency, and are probably higher in biological tissue containing small amounts of magnetite. Magnetite (Fe 3 O 4 ) is a naturally occurring oxide of iron. It is a ferrimagnet but behaves similar to a ferromagnet such as iron in magnetic fields. Magnetite is found in certain bacteria and in cells of many animals and human beings. It is believed to be used by some species of birds and fish to provide magnetic sensitivity, which employed in navigation. Furthermore, no other effects associated with the interactions of EMFs with magnetite are demonstrated in animals. The bio-interaction resulting from the higher radio frequency (RF) has been calculated. A magnetic field generated by mobile phones is extremely small and any other effects of magnetic fields at these frequencies should be even smaller. Indeed, it is generally agreed that any biological effects from mobile phones are much more likely to result from electric rather than from magnetic fields. The RF energy radiated by Meter test equipment is absorbed in the body and produces heat, which might be carried away by the body s thermoregulatory processes. The rate at which RF-EMW energy is imparted to an element or mass of a biological body is called SAR. SAR is expressed as energy flow (power) per unit of mass in units of W/kg. SAR values are complex to measure as the penetration

of energy within human tissues. It depends on many factors, such as the type of the phone, the shape of the user s head, or the frequency. Even though SAR measurement is performed in laboratory using specific instrumentation defined in the standards, i.e., head phantom, liquid, and E-field probe, measurement process consists in using a scanning system to measure the internal E-field distribution in a phantom model filled with liquid. Shape of the head and the liquids simulate the human head and tissue where device under test emits at maximum power is positioned. According to the procedure of standards, E-field value is measured with a miniature E-field probe. Indeed, E-field probes/monopole antenna is the most appropriate sensor to measure the SAR, due to their sensitivity and fast time response. E-field maybe calculated as SARðW=KgÞ ¼rE 2 =q where sigma (r) is conductivity of the liquid and rho (q) is the density of liquid. The measured E-field values and SAR distribution are 1 g and 10 g mass averaged SAR values. Mechanism of Heat Transfer When a power source is applied to an absorbing medium, the rate of energy transfer to the medium can be described by a spatially distributed function Q em (x,y,z). Usually, the energy transferred to the medium will be converted to heat and exact method of energy transfer is dependent on the modality of the power source. However, with respect to power source, an infinite homogeneous medium of heat capacity (c) and density (p), an initial temperature rise due to the conversion of deposited energy to heat is dt ¼ð1=qcÞQ em dt ð1þ Convection and Radiation When the surface of a medium with uniform temperature T s is exposed to a moving fluid of temperature T F, it maybe exchanged between the surface and the fluid. Since the mechanism of heat transfer is not dependent on the interaction between adjacent particles, the rate of convective heat transfer is proportional to the temperature differential, rather than to the temperature gradient. The resulting heat flow per unit area can be expressed with surface of uniform temperature T s and a fluid of temperature T F as QC ¼ hðt s T F Þ ð3þ where, h is the mean coefficient of heat transfer, which is dependent on both the flow rate and physical properties of fluid. Thermal equation is commonly referred to as the Newton s law of cooling. Although forced convection of heat by blood flow plays a considerable role in heat transfer within perfused biological tissue, it is often useful to design simple, non-perfused models to study basic heat transfer principles within tissue. For non-perfused medium, convective heat flow occurs only at boundary interfaces that are exposed to fluids such as air or water, and can be ignored for a material of near-infinite dimension. Heat transfer at boundary skin/air shows that the first law of thermodynamics related heat transfer, work transfer, and internal energy of a body. It is important to know how heat transfers in a solid medium as per Fourier law of heat conduction proposes. Practically, it is interesting to see a heated body as defined in Fig. 1. Moreover, an active heat production or dissipation maybe defined as (i) External irradiation and (ii) Mechanisms in living tissue. Consideration of Basic Mechanisms of Microscopic Regions Inside Bodies of Animals or Humans Conduction When a thermal gradient exists within a medium, heat is transferred by physical interaction between particles with different temperatures, i.e., different kinetic energy. In a solid form, thermal conduction results from the movement of free electrons and vibrational energy in the material. The rate of heat transfer is related to the thermal gradient by the thermal conductivity (k). Although in isotropic medium, the temperature rise associated with deposition of energy, described by heat transfer equation, results in a heat flow of kd 2 T, for k independent of temperature. Increased temperature rate is described as qcðot=otþ ¼Q em þ kd 2 T ð2þ Fig. 1 Showing thermal effect of microwave radiations on human model in a solid form of body

Blood Flow, Metabolism, and Evaporation In order to derive the conduction equation, a differential control volume in the shape of a parallelepiped of dimensions dx, dy, dz, inx, y, z directions as illustrated in the Fig. 2 is constructed, where q x and q(x? dx) is the heat entering and leaving the control volume in the x-direction, q y and q(y? dy) is the heat entering and leaving the control volume in the y-direction, q z and q(z? dz) is the heat entering and leaving the control volume in the z-direction, and Q em is the heat generation in the control volume. The energy conservation principle applied to the control volume can be written as heat conduction with internal heat generation by RF-field o=oxðk ot=oxþþq em ¼ qcðot=otþ dt=dt ¼ K=qcðo 2 =ox 2 T þ o 2 =oy 2 T þ o 2 =oz 2 TÞ þð1=qcþq em ð4þ Conductivity k, can be a function of space and temperature. However, we assume that the conducting medium is homogeneous and isotropic; under this condition, the thermal conductivity depends only on temperature and because of this dependence in Eq. (4), it is left in the derivatives. Q em is the heat generation rate due to deposited electromagnetic power density P d, and DV small volume of tissue H b ¼ bðt T b Þ: ð5þ SAR can be measured by the processing of human/ animal with Eqs. (4) and (5) ðq c ÞdT=dt ¼ K=q c ðo 2 =ox 2 T þ o 2 =oy 2 T þ o 2 =oz 2 TÞ þð1=q c ÞQ em bðt T b ÞþM 0 ð6þ Total SAR of microwave or mobile phone radiations are quantified in absorbed human body using Eq. (6). The EMF interaction to biological system is a major challenge where Eq. (6) maybe useful to find the solution. However, if this may elaborate for all the three directions x, y, and z in Cartesian coordinates, then it would be ðo 2 =ox 2 T þ o 2 =oy 2 T þ o 2 =oz 2 TÞ¼0 ð7þ The bio-heat equation is often used for calculating temperature elevation in numeric phantoms. Heat transfer in vivo factors are 1. Difficult task due to complex morphology, 2. Bio-heat transfer equation by Pennes seems to be the best practical approach, 3. Effect of tissue perfusion and metabolism are introduced in the heat conduction equation, 4. Heat exchange takes place across the wall of capillaries in which blood velocity becomes very low, 5. Blood enters the tissue with a temperature T (blood) and immediately reached thermal equilibrium. Blood perfusion of metabolism ðqc p Þ t ot t =ot ¼ K t o=rorðr ot t =orþþq m p þ Qm m ð8þ where (qc p ) is the tissue effective density, specific heat at constant pressure; K t is the tissue thermal conductivity; qt t is the tissue temperature; Q m m is the tissue metabolic heat generation. Blood perfusion (Pennes) Q m p ¼ðqC pþ b V m ðt a T v Þ ð9þ where C p is the blood density and specific heat; T a is the arterial blood; T v is the venous blood. T t and T b denote the temperatures of tissue and blood, respectively, where r and t are the position vector and the time. C represents the specific heat of tissue, K is the thermal conductivity of tissue, A is the basal metabolism per unit volume, and b is the term associated with blood perfusion. Mobile Phone Exposure and Spermatogenesis Fig. 2 Showing control volume for conduction analysis in a rectangular coordinate system The testis descends from a retroperitoneal position through the inguinal canal to take its place in the scrotum during the eighth fetal month. The reason for its unusually vulnerable position is uncertain, but may well be due to the lower temperature required for spermatogenesis. One of the most important concepts in male reproduction is that the testis requires a specialized environment to produce sperm and thus developed a specialized pouch, the scrotum, for

spermatogenesis at relatively low temperature to body. In human beings, at least three causes of male infertility are related to heat i.e., varicocele, cryptorchidism, and fever. In these cases of infertility, heat might be the main reason which may cause rise in body temperature due to certain reasons, where EMF is one of them. In general males keep their mobile phones in pockets near to the testis which increases the risk of infertility. However, most of the attention regarding heat and testicular function has been focused on spermatogenesis. There is evidence that electromagnetic exposure can cause a reduction of serum testosterone level, shrinkage of seminiferous tubules, and reduction of sperm motility and count. It is also possible that function of Leydig cells are affected by EMF, a major cause in reduction of intrascrotal testosterone levels (Fig. 3). It is predicted that a change in scrotal temperature of only 1 C is enough to cause infertility. Dasdag et al. [30] observed changes in the rat s testicular function through decreased sperm count and narrower seminiferous tubules after exposing rats to 915 MHz from a cell phone having SAR of 0.141 W/Kg. Mobile Phone Exposure and Testosterone Level Testosterone is a primary male gender hormone and plays a significant role in the spermatogenesis, and is vital to general metabolism of human body. Seminiferous tubules are the place for the production of spermatocytes. It is possible that EMFs can damage these tubules. Studies conducted by Ozguner et al. [31] on male rats showed that the diameter of seminiferous tubules and the mean height of the germinal epithelium were significantly decreased after 900 MHz EMF exposure. Follicle-stimulating hormone (FSH) is responsible for the activation of the seminiferous tubules, resulting in the production of sperm as well as the conversion of testosterone to estradiol [32]. Testosterone which is formed in the Leydig cells also acts Mobile Phone Exposure Mobile phone CNS (Hypothalamus) GnRH secretion Anterior Pituitary Semniferous Tubule Reduction in diameter of seminiferous tubules Free radicals formation Overproduction of ROS Decrease FSH Secretion Increase LH Secretion Imbalance in antioxidant level Sertoli cells Leydig cells Deficiency of energy Spermatogenesis Decrease Testosterone secretion Increase in phosphorylation 1) Sperm Count 2) Apoptosis 3) Sperm viability & morphology 4) DNA (S & D) strand break Inhibit maintenance of accessory reproductive organ and secondary sex characteristics Affect functions of various tissue Alteration Restrict sex drive Protein synthesis in skeletal muscle Bone growth in adolescence Decrease of Histone kinase, Protein kinase C, Creatine kinase Affect mobility of sperm Infertility Fig. 3 Possible pathways between central nervous system (CNS) and testis for the mechanism of damage caused to spermatozoa by electromagnetic waves (EMW) emitted from cell phones. It also represents the several proposed mechanisms of damage to spermatozoa and testosterone by cell phone radiation based on the preliminary findings in the studies discussed above

on seminiferous tubules and stimulates spermatogenesis. The greater part of the testosterone is produced in the interstitial cell of Leyding in the testis. Regulation of testicular production occurs via a negative feedback loop system involving the anterior pituitary gland, hypothalamus, and testis [32, 33]. Mobile phone or microwave radiations may exert an effect on the state of polarization of the cellular membrane. An inadequate polarization of cellular membrane is responsible for the process of various abnormalities of testosterone synthesis, secretion of which may impair spermatogenesis and ultimately become a cause of infertility [34]. Sarookhani et al. [35] evaluated the influence of different powers (3 and 6 W) of 950 MHz (mobile phone radiation) magnetic field on testosterone and FSH levels of male rabbits. Authors concluded that the concentrations of testosterone and FSH after exposure in both 3 and 6 W groups are decreased compared to the control group. When the 6 W group was compared with the 3 W group, the concentration of testosterone was decreased in the 6 W but the concentration of FSH increased (P \ 0.05). Meo et al. [34] have experimented on Wistar Albino rats with different mobile phone exposure duration. They have shown that the exposure to mobile phone radiation for 60 min/day for the total period of 3 months significantly decrease the serum testosterone level (P = 0.028) as compared with control rats. They also indicated that the testosterone is the primary male gender hormone and any changes in normal level maybe devastating for reproductive and general health. Moreover, Kumar et al. [36] have also showed interesting results on testosterone level at 2.45 GHz of exposure. Authors reported that serum testosterone level decreased significantly (P \ 0.002) in 2.45 GHz exposed animals (1.36 ± 0.54), when compared to sham exposed group (5.41 ± 1.25). In addition to this, they have also reported a retrieval effect of pulsed magnetic field in response to microwave field (2.45 GHz). This is first time that our group has reported therapeutic response against microwave exposure. More importantly we revealed with their studies that oxidative stress is the major mechanism affecting health, and microwave field causes chronic stress effects through overproduction of ROS. Pulsed magnetic field therapy provides a significant protection by way of controlling ROS production. Similarly, Mailankot et al. [37] reported that EMF radiation generated from mobile phone in active mode disturbs free radical metabolism by enhancing ROS in reproductive tissue and thereby leads to change in reproductive parameters. Although male partner produces billions of sperm in a single ejaculation to achieve fertilization but functional defects in these sperms prevent conception. Despite several decades of research, the cause of infertility due to EMF remains unsolved. Fragmentation and strand break of DNA in germ line has been associated with male infertility. Human reproduction is a complex process requiring high energy. This system produces the gametes which carry the genetic information from one generation to other generation. The development of sperm is controlled by a complex interplay of genetic, physiological, hormonal, and environmental factors. Testicular hormones regulated spermatozoa differentiation, homeostasis, and reproduction. Hormones impart their effects on spermatozoa and cells through several different mechanisms. Testis is the central organ of male reproduction and controlled by central nervous system through neuroendocrine feedback loop. The primary function of testicular Leydig cells is the production of androgens i.e., testosterone. Testicular function is controlled by CNS through gonadotropin releasing hormone involving FSH and LH as the key hormonal signals. Testosterone promotes sexual differentiation in the fetus, secondary sexual maturation, and spermatogenesis in the adult. Figure 3 represents the mobile phone exposure effects on male fertility pattern by affecting spermatogenesis and hormonal level. Mobile Phone Causes Oxidative Stress and Possible Mechanism of ROS Formation Oxidative stress is a cellular or physiological condition. Elevated concentrations of reactive oxygen species cause molecular damage to vital structures and their functions. Free radicals and ROS are formed as by-products by nonionizing radiation. ROS are highly reactive molecules that cause dramatic effects on macromolecules like DNA, RNA, and proteins. Reactive oxygen species maybe defined as formation of free radicals, a group of highly reactive molecules consisting of unpaired electrons in the outer orbit. Free radicals that are derived from oxygen metabolism are known as reactive oxygen species (ROS) [38]. ROS are a necessary biochemical component for the maintenance of healthy cells and tissues. However, it can be highly cytotoxic, leading to cell and tissue damage in excess concentration [39]. Mobile phone radiation initiates an iron-mediated process (Fenton reaction) that increases hydroxy free radical formation in cells and causes severe damage. Hydroxy radicals are generated from hydrogen peroxide via the Fenton reaction in the presence of iron. A simple mixture of H 2 O 2 and Fe 2? salt forms OH, as given by the following Fenton reaction [40] Fe 2þ þ H 2 O 2! Fe 3þ þ OH þ O ~H: Traces of Fe 3? can react further with H 2 O 2 to form the following products: Fe 3þ þ H 2 O 2! Fe 2þ þ O 2 þ Hþ : Thus, a free-radical mechanism for the generation of OH maybe deduced as follows:

O 2 þ H 2O 2! OH þ OH þ O 2 : Cells with high metabolic rate generate high amount of hydrogen peroxide via the mitochondrial electron transport pathway, and thus are more vulnerable to EMF. Mitochondria are the main site of reactive oxygen species (ROS) production at a rate proportional to mobile phone exposure. De Iuliis et al. [41]havereportedthatanenhancedactivityofmitochondrial NADH oxidase under short-term exposure to electromagnetic wave field may change ROS level. ROS may originate from a number of intracellular redox systems such as mitochondrial respiratory chain enzymes, cytochrome P450 enzymes, xanthine oxidase, NAD(P)H oxidase, lipoxygenase, and cycloxygenase. Several studies on human semen suggest that an increase in ROS production due to cell phone radiation is responsible for such cause [17, 41]. Desai et al. [5] and Agarwal et al. [42] showed a possible mechanism in various cellular targets of RF-EMW that stimulates plasma membrane NADH oxidase which can increase ROS formation. Thus, RF-EMW-mediated increases in ROS production can trigger cell differentiation by its action on mitogen-activated protein kinase (MAPK), heat shock protein, protein kinase C, and ornithine decorboxylase (ODC). EMFs disturbed the balance of antioxidants, which leads to activation of cell defense mechanism. H 2 O 2 is relatively stable; higher oxidant potential due to this and since it is uncharged, it can freely cross cell membranes [43]. The cell defense systems include enzymes (SOD, Catalase, glutathione peroxidase) that catalytically remove free radicals and ROS. SOD is a family of enzymes responsible for dismutating superoxide to form hydrogen peroxide. Catalase is another antioxidant enzyme that is involved in breakdown of H 2 O 2 to ground state Oxygen and water. GPx is also a family of enzymes that catalyze the removal of H 2 O 2 by coupling its reduction to H 2 O with oxidation of reduced glutathione (GSH). Dietary antioxidants including vitamin A, E, and C are also important in defending oxidative stress. Other protecting agents include heat shock proteins and endogenous enzymes that are responsible for minimizing the availability of pro-oxidants such as iron ions, copper ions, and heme. Oxidative stress depends on a number of factors i.e., free radical productions, strength of antioxidative defense, and rate of repair. Free radicals are involved in the pathogenesis and development of many kinds of diseases, such as atherosclerosis [44], hypercholesterolemia, hyperlipidemia [45 47], Alzheimer s, Parkinson s, and Schizophrenia [48 50], and increases the implication in development of insulin resistance and diabetes. Desai et al. [5] hypothesized that the plasma membrane might be the target of cell phone radiation. RF-EMW can increase ROS formation by increasing the activity of plasma membrane NADH oxidase. A possible mechanism of interaction between the biological systems and the EMF is a process which involved free radical. Ground state oxygen maybe converted to more reactive ROS forms either by energy transfer or by electron transfer reactions after the exposure of EMFs. This leads to the formation of singlet oxygen and results in its sequential reduction to superoxide (O 2 - ), hydrogen peroxide (OH ), and hydroxyl radical (H 2 O 2 ). EMFs are responsible for the generation of free radicals which may link to sperm deformities but the mechanism of cascades is still unknown. Many of the events related to these processes include increase in camp concentration, stimulation of protein kinases and phosphorylation, and calcium influx. Sperm membrane is the first target for ROS. It is well recognized that, owing to its low reactivity, O 2 - rarely induces lipid peroxidation unless it is produced or solubilized in the interior of hydrophobic membrane, or is present in its protonated form (HO 2 )[43]. There are several environmental factors which may cause oxidative damage to cell membrane. EMF exposure to a biological system may cause a weak induced signal near the cellular boundary and lead to the penetration of energy from a coherent signal, with amplification derived from noise via stochastic phenomena [36]. Thus, the signal can circumvent the barrier height of plasma membrane and penetrate inside the nucleus to cause DNA damage (strand and double breaks). This signal may also provide sufficient energy for overproduction of ROS [7]. Kesari et al. [4] have reported a significant change (P \ 0.05) in enzymes due to cell phone exposure. Authors suggested that the changes may occur due to significant (P \ 0.05) increase in ROS level of cell phone exposed (58.25 ± 10.36 mg/l) animals as compared with sham exposed (41.78 ± 12.93 mg/l). Furthermore, mobile phones might induce vasodilatation and increase the production of NO as stated by Paredi et al. [51] and Yariktas et al. [52]. Moreover, such stress is already known to induce the functional and structural lesions observed in this study. The main damage of increased ROS production results from the alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, vital proteins, and DNA [53]. Mobile phone or microwave radiations are one of them which may generate free radical formation and enhance the ROS level in cellular metastasis. An increased level of ROS may cause cell damage and progression or tumor promotion. This study clearly demonstrates that EMF can damage sperm function via mechanisms that involve the creation of oxidative stress. The event of possible correlation between mobile phone exposure, ROS formation, and tumor promotion has been presented in Fig. 4. Measurements of Damage There are several factors which may affect the biological system. Mobile phone or microwave radiations are one of

Environmental Contamination Aging Inflammation PAF alter RF-EMF (Mobile phone) O 2 Free-radicals formation Ligand Receptor Chemical and Drugs Endogenous mediator ROS/NOS (O 2 -, H 2 O 2, OH - ) Adaptor protein Procaspase-8 Mitochondrial caspase-9 (ca +2 released from mitochondrial sac) Cytochrome c DNA / Protein/ Lipid Modified Molecules Caspase-8 Effector Caspases (3,6,7) Apoptosis Activation of repair Systems (caused by prooxidants) Stress Stress Activation of transcription factor/protein (transfer genetic inforamtion DNA-RNA) Gene activation/inactivation (free-radicals play active role in gene activation) Activation of Kinases (i.e. PKC, MAP Kinase; phenolic antioxidants) Induction of oncogenes Inhibition of anti-oncogenes Altered apoptosis (PCD) Cancer Progression Adoptation Induction of defense system Homeostatic response Normal Metabolism of Sperm Cells Damage DNA Single and Double strand break Cell Death Fig. 4 Interaction mechanism between free radicals formation and cell function due to radiofrequency/microwave radiation exposure on human system. The pathway indicates overproduction of ROS and their effects on different types of cell them which can cause biological damage. It is important to measure the damage by exploring several parameters. The most effective parameters are discussed below. The Comet Assay: Measurement of DNA Strand Breaks to Check Genotoxicity Assessment The comet assay, also known as the single-cell gel electrophoresis assay, is a rapid and sensitive method for the detection and visualization of DNA damage (single and double strand breaks) in individual cells. The comet assay can be applied to all types of nucleated eukaryotic cell exposed to a variety of physical or chemical agents [54]. It was first described by Singh et al. in 1988 [55]. The comet assay measures the damage in the form of DNA, accessed by fluorescent microscope. Any lesions which reduce the size of large duplex DNA molecule and affect the supercoiled chromatin generally take the comet shape. Comet assay can be done through two methods using alkaline and neutral ph. Recently, Kesari et al. [9] and Kesari and Behari [8] have shown DNA double strand breaks at different frequency level. Authors observed increased DNA break with tail and head length migration and intensity. The first study of DNA strand break was observed by Lai and Singh [56] in rat brain cells at continuous and pulsed RF radiation. An addition to this flowcytometer is another method to measure variability in DNA content among cells and is a recognized method. Using flow cytometry to measure clastogenic effects is based on the principle that fluorescent emissions from cells stained with DNA-binding fluorochromes can be measured and positively associated with DNA content. The DNA damage or DNA fragmentation may occur mainly due to micronuclei formation. Mobile phone or microwave radiations have ability to damage the cells at micronucleus level. The micronuclei damage caused due to these radiations maybe measured by flowcytometer. Kesari et al. [4] recently showed a significant decrease (P \ 0.002) in micronuclei (MN) (measured through flowcytometer) of mobile phone exposed group (0.67 ± 0.15) as compared with control group (1.36 ± 0.07), where a decrease was recorded by comparing the ratio of PCE (polychromatic

erythrocyte) and NCE (normochromatic erythrocyte) in blood cells. Authors found a significant decrease of MN levels in cultures irradiated at mobile phone frequency for 35 days at the SAR of 0.9 W/kg. Moreover, Kumar et al. [7, 27] have also shown the causative effect by lower percentage of PCE/NCE and suggested that such effects maybe caused by higher level of ROS at different frequencies of microwave exposure. Several reports suggested that DNA damage in cells could have an important implication on human health because they are cumulative. Normally, DNA is capable of repairing itself efficiently through a homeostatic mechanism, whereby cells maintain a delicate balance between spontaneous and induced DNA damage. DNA damage accumulates if such a balance is altered. Most cells have considerable ability to repair DNA single strand breaks; for example, some cells can repair as many as 200,000 breaks in 1 h. However, DNA Double strand breaks, if not properly repaired, are known to lead to cell death or apoptosis. Indeed, we have observed an increase in apoptosis, decrease in sperm count [57, 58], and DNA double strand break in sperm and brain cells exposed to microwave RF radiation (RFR) [56, 59 61]. The results of Ivancsits et al. [62 64] indicate that the interaction of these fields with DNA is quite complicated and apparently depends on many factors, such as the mode of exposure, the type of cells, and the intensity and duration of exposure. RF-EMF and Apoptosis Apoptosis or programmed cell death in the tissues of an organism is an important and inevitable event in the remodeling of tissues during development, spermatogenesis, and aging. It is a very crucial process for elimination of cancer cells [65]. Commonly used techniques for the detection of apoptosis are agarose gel electrophoresis, flow cytometry analysis, caspase-3 quantification, TUNEL (TdT-mediated dutp nick-end labeling) assay, microscopic estimation of apoptosis, annexin V labeling, etc. Apoptosis is generally an energy dependent process induced by mild genotoxic exposure. Apoptosis plays a fundamental role in maintaining the normal homeostasis through entire lifetime process. Disorder in any step of apoptosis pathways is likely to promote disease. EMFs could interfere with apoptosis as well apoptosis-related genes and genes related to signals transduction. There are several genes that are related to apoptosis pathways like p21 and p53. The p21 gene is thought to directly regulate the cell cycle, and the p21 protein product inhibits DNA replication by blocking cyclin dependent kinase (CDK) [66]. The p53 tumor suppressor gene is thought to play a critical role in the cellular response like or i.e., DNA damage, cell cycle arrest, and apoptosis [7]. Caspase-3 plays a direct role in apoptosis and is essential in other apoptotic situations, showing a remarkable tissue, cell type, or death stimulus specificity [4, 36]. These apoptosis-related genes should be affected by EMF. The power level of EMF (or SAR) is associated with a defect in DNA double strand breaks. Kesari and Behari [2] and Kesari et al. [3] have reported an increased apoptosis level due to the exposure of microwave at 2.45 GHz and mobile phone at 900 MHz. Authors have shown a significant increase in apoptosis level of microwave exposed at 2.45 GHz of group (14.30 ± 1.92 %) as compared with sham exposed (7.43 ± 1.30 %). Moreover at mobile phone frequency, authors observed significant increase in apoptotic cells of exposed group (13.15 ± 1.25) as compared with sham exposed (5.93 ± 1.64 %). Aitken et al. [67] have revealed that exposure of male mice to EMF imparts a significant effect on the mitochondrial as well as nuclear genomes integrity. Histone proteins play a vital role in maintaining chromatin structure; they also participate in the dynamics of chromatin remodeling during gene activation and gene silencing. In particular, phosphorylation of histones H1 has long been implicated in chromosome condensation during mitosis [68]. Future Research Directions Since decades, evidence for a detrimental, mutagenic effect of cell phones and microwave ovens on male fertility and nervous system are still equivocal. The inconclusive findings raise an intense debate on whether the DNA strand break, sperm quality, and sperm fertilizing potential are affected by the use of cell phones or not. The Vulnerability of spermatozoa to RF damage and the clinical significance of this damage in terms of infertility, apoptosis, tumor promotion, cancer, pregnancy, childhood health, and human studies. A careful study is needed to investigate the impact of RF waves from cell phones on whole body, especially on testicular tissue and CNS. Number of researches have discussed the effect of mobile phone and microwave radiation on human health. Now it is necessary to explore the safety criteria for these radiations in direction toward future research. Study needs to work on therapeutic effect of pulsed EMF (PEMF). Several researchers have shown positive indications toward the osteoporosis treatment and fracture healing [69, 70]. More recently, Kumar et al. [36] have reported the therapeutic impact of PEMF on RF microwave radiation affected site of male Wistar rats. Nanotechniques or nanoparticle treatment for microwave affected sites will be another option of study to inhibit the exposure effect. Specific nanoparticles maybe injected as a drug to target the affected site in body organs.

Several studies have shown hydroxyl apatite (Hap) nanoparticle s impact in osteoporosis [70] and cancer treatment. More importantly, the existing parameter, SAR measurement also needs to be explored from basic research to higher level. The controversies in response to mobile phone exposure effect maybe solved with suitable justifications by exploring the physics of mobile phone and microwave sources. The physical phenomena indicate that mobile phone antenna is very close to head. Much effort has gone into determining the dosimetry profile of microwaves in the head in various possible configurations. Keeping this in mind, it maybe proposed that the heavy users of mobile phone maybe exposed to a maximum at 30. It seems that the person using mobile phone during talking mode maybe moving their cells phone from 0 to 30 or maximum to 45. It is advisable that the futuristic model for exposure maybe based on these positions by continuous movement of mobile phone from 0 to 30. It might be grouped at various time scales for fixed minute with intermittent call of 5 min after every 20 min of call. The current debate on effect of RF has to be explored through its proper guidelines for exposure system and also their bio-interaction mechanism as well as measurement of SAR. It is necessary to find out the biomedical applications for protection against EMF. The study needs more justification and implication of experimental evaluation, so that we have an option to protect the human beings from such type of radiations. Conclusion In light of present debate, we have hypothesized that fertilizing potential of sperm cells are affected by EMF through high level of DNA damage which involves a common causative mechanism in the form of oxidative stress. Although there are mere debates that EMFs can act as promoters or co-promoters or stimulators of cancer [71, 72], some authors have revealed a positive association between EMF exposure and incidence of several types of cancers such as leukemia [73], brain tumors, genotoxicity, and neurodegenerative disease. Presently, an important mechanism of heat transfer due to mobile phone or microwave radiations has been explored. The possible mechanism of EMF interaction to biological system has been extrapolated by theoretical model as given in Eq. (6). The above study concludes that EMF initiates an ironmediated process (Fenton reaction) that increases hydroxy free radical formation in cells, leading to DNA strand break and cell death. Furthermore, free radicals play a role in the genetic and enzymatic effect of RFR. If free radicals are involved in the RFR-induced DNA strand break, changes occur in antioxidant enzymes and hormonal level in sperm cells. 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