Health Risk of Radiofrequency Radiation Toxicological and Laboratory Studies

1. Health Risk of Radiofrequency RadiationToxicological and Laboratory Studies Riadh W. Y. Habash, PhD, P.Eng McLaughlin Centre for Population Health Risk Assessment, Institute of Population Health School of Information Technology and Engineering University of Ottawa rhabash@site.uottawa.ca

2. The electromagnetic (EM) field is a physical influence (a field) that permeates through all of space, and which arises from electrically charged objects and describes one of the four fundamental forces of nature, electromagnetism. Electromagnetism is found almost everywhere. All EM fields are force fields, carrying energy and capable of producing an action at a distance. These fields have characteristics of both waves and particles. This energy is utilized in various ways, though we still lack the full understanding of its fundamental properties. Many inventions of the late twentieth century, ranging from everyday home and office appliances to satellite systems and mobile phones, are so important and so advantageous; we wonder how we ever lived without them.

3. General • EM waves at low frequencies are referred to as EM fields and at very high frequencies are called EMradiation. The term EM field is generally used rather than EM radiation whenever wavelengths greatly exceed distances from exposure sources. • EM fields at all frequencies make one of the most common environmental issues, about which there is a growing concern and speculation. EM fields are present everywhere in our environment but are invisible to the human eye. • All populations are now exposed to varying degrees of EM fields, and the levels will continue to increase as technological inventions advance. These inventions have become an integral part of our modern life. We just need to know that they are safe.

4. Sources of RF Radiation (100 kHz-300 GHz) • Broadcast (AM and FM) and TV ( 0.1 W/m2) • Mobile Phones and cordless phones ( 0.1 W/m2) • Microwave Ovens ( 0.5 W/m2) • Civil and Military Radar Systems • Portable and Radio Transceivers ( 0.2 W/m2) • Medical and Industrial Applications • Anti-theft Devices • New Technologies like Worldwide Interoperability for Microwave Access (WiMax)

5. Bioeffects? • A biological effect occurs when a change in the environment causes some noticeable or detectable physiological change in a living system. These changes are not necessarily harmful to health. For example, listening, reading, eating or playing will produce a range of bioeffects. However, none of these activities is expected to cause health effects. • The body has sophisticated mechanisms to adjust to the various influences that encounter in the environment. • But the body does not possess adequate compensation mechanisms for all bioeffects. Changes that stress the biosystem for long time may lead to a health effect.

6. Biological and Health Effects • Cells and Membranes • Tissues • Changes in Protein Conformation • Changes in Binding Probability • Absorption of Vibrational States of Biological Components • Genetic Material • Carcinogenesis • Hypothesis of Melatonin • Cancer • Brain and Nervous System

7. Mechanisms for (RF) Radio Frequency Radiation • Biological effects due to exposure to EM radiation are often referred to as being thermal or nonthermal/athermal. • Heating is the primary interaction of EM radiation at high frequencies especially above about 1 MHz. Thermal effects of EM radiation depend on the specific absorption rate (SAR) spatial distribution. • Controversy surrounds issues regarding bioeffects of intermediate- and low-level EM radiation. First, whether the radiation at such low levels can cause harmful biological changes in the absence of demonstrable thermal effects. Second, whether effects can occur from EM radiation when thermoregulation maintains the body temperature at the normal level despite the EM energy deposition.

8. RF Exposure Guidelines: SAR limits for RFR

9. Epidemiological StudiesWhat is Already Known so Far? • The epidemiologic evidence is not strong enough to the level required to conclude that RF radiation are a likely cause of one or more types of human cancer. This is attributed to weak design of the studies, lack of detail on actual exposures, limitations of the ability of studies to deal with other likely factors, and in some cases there might be biases in the data used. • The current epidemiologic evidence justifies further research to clarify the situation. Moreover, since there are only a few epidemiological studies that examine the health risks associated with exposure to RF radiation, research at the cellular and animal level is needed to better understand this relationship.

10. Laboratory Studies: Cellular and Animal • Cellular studies play a supporting task in health risk assessment. Cellular model systems are good candidates for testing the plausibility of mechanistic hypotheses and investigating the ability of RF radiation to have synergistic effects with agents of known biological activity. They are significant to the optimal design of animal and epidemiological studies. • On the other hand, animal studies are used when it is unethical or impossible to perform studies on humans and have the advantage that experimental conditions can be thoroughly controlled.

11. Thermal and Non-thermal Effects • Close to the high-power transmitter, high-frequency fields may be harmful to human beings by producing thermal effects that may sometimes, when thermoregulation processes are insufficient, produce irreversible damage (for example, cataract). This kind of effect is well known from animal experiments and for that reason does not constitute a particular health problem, as measures can be taken to prevent excessive exposure. • Also low-level exposures leading to non-thermal effects are, at least according to certain investigators, possible. Non-thermal effects have been reported in cell cultures and animals, in response to exposure to low-level fields. They are not well established and therefore highly controversial.

12. Chain of Events Leading from RF exposure to Disease RF radiation Interaction: RF force induce currents Transduction: Modify tissues and membranes or ion currents Not perceptible by cells; No amplification triggered Cell Signal: Signal cascade or amplification Signal within normal variation; No functional consequences Biological Response: Changes in cell behavior Sensory effects; Neutral effects ; No adverse effects Cell Dysfunction: Adverse Effects; Progress Toward Disease Transient; Reversible .. No effect Repair adaptation .. No effect With reserve capacity .. No effect

13. Cancer! • There are no laboratory studies in humans with cancer as the direct end point of investigation. • Carcinogen activity could be identified by: • Evidence from human populations, including epidemiology, clinical studies, and case reports. • Animal studies. • Cellular (in vitro) studies and studies of biophysical and biochemical mechanisms. • As a useful concept for assessing risk, scientists and regulatory agencies have generally divided carcinogens into two types: genotoxic and non-genotoxic (epigenetic).

14. Genotoxicity and Non-genotoxic Carcinogens • Genotoxicity does have a clear cancer endpoint or any other adverse health outcome, however, there is the possibility that genotoxic effects on cells might lead to adverse health effects such as cancer or other diseases. Studies in this regard have been performed at a variety of levels including damage to DNA in vitro or in vivo, damage to chromosomes, induction of sister chromatid exchange (SCE), or induction of mutations. • Non-genotoxic carcinogens do not directly damage the genetic material of cells. Rather, they affect carcinogenesis indirectly by increasing the probability that other agents will cause genotoxic injury or that genotoxic injury caused by other agents will lead to cancer.

15. Genotoxicity (In Vivo) StudiesWhat is Already Known so Far? • The in vivo experiments by Lai and Singh (1995,1996) are special, in view of the attention they have received. They reported an increase in DNA strand breaks in the brain cells of rats exposed for two hours to pulsed or CW 2450 MHz RF fields at averaged whole body SARs of 0.6 and 1.2 W/kg. In 1997, they found that melatonin, and another compound known to be a free radical scavenger, blocked the RF effect of increased DNA strand breaks. They have also reported that a temporally incoherent magnetic field (noise) blocked RF-induced increases in DNA strand breaks (Lai and Singh, 2004). • Diem (2005) reported that exposure to 1800 MHz RF radiation was associated with DNA strand breaks in human and rat cells. • Malyapa (1997) could not replicate Lai and Singh's results at 2450 MHz or at a frequency of 835/847 MHz. Lagroye (2004), using methods identical to those of Lai and Singh, could find no evidence that pulsed-wave 2450 MHz RF produced DNA damage in rat brain cells. Hossmann and Hermann (2003) suggest that the experiments by Lai and Singh used peak power that was much higher than the mean power, which may have accounted for the observed DNA damage. • Vijayalaxmi (2003) found no evidence of genotoxicity in rats exposed for 2 years to near-field RF exposure.

16. Genotoxicity(In Vivo) Studies • Repacholi et al. (1997) have induced a two-fold increase in lymphoma incidence in a strain of lymphoma-prone transgenic mice following daily exposure ( 2  30 min) up to 18 months to 900 MHz radiation with a signal similar to the GSM modulation ( pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms). Utteridge et al. (2002) and Oberto et al. (2007) failed to replicate or confirm the above results. • Several other studies have evaluated the carcinogenicity of RF fields at nonthermal levels in various models. Many studies have tested whether RF radiation alone induce any kind of cancer in normal or genetically predisposed animals, and other studies investigated whether exposure to RF radiation could enhance the development of tumors induced by chemical carcinogens, X-ray, or UV radiation. No significant increase of tumor incidence has been reported in any of these studies.

17. Genotoxicity (In Vitro) Studies • In vitro studies in general have failed to show evidence of DNA damage (Malyapa 1997; Vijayalaxmi 2000, 2001, 2006; Gos, 2000; Roti Roti, 2001; Maes, 2001, Lagroye, 2004, Hook, 2004, Zeni, 2005, Sakuma, 2006, Stronati, 2006). • Tice et al. (2002) found an increased frequency of micronucleated lymphocytes exposed to different RF signals for 24 hours at an average SAR of 5.0 W/kg or 10 W/kg. Micronuclei arise from chromosomal damage. Others have found an increased frequency of micronucleated cells following exposure to RF signals (Garaj-Vrhovac, 1991; Maes, 1995; d'Ambrosio 2002; Trosic 2002). Zotti-Martelli (2005) also found an increase in micronuclei in lymphocytes exposed to 1800 MHz at different power densities and for different time periods, but also found a wide inter-individual variability in the response. However, Vijayalaxmi (2001, 2006) Bisht (2002) and McNamee (2002; 2003), Zeni (2003), Gorlitz (2005), Scarfi (2006) and Juutilainen (2007) did not find an icrease in micronucleated cells after RF exposure.

18. Genotoxicity (In Vitro) Studies • In vitro studies in general have failed to show evidence of DNA damage (Malyapa 1997; Vijayalaxmi 2000, 2001, 2006; Gos, 2000; Roti Roti, 2001; Maes, 2001, Lagroye, 2004, Hook, 2004, Zeni, 2005, Sakuma, 2006, Stronati, 2006). • It is possible that certain cellular constituents altered by exposure to RF radiation, such as free radicals, indirectly affect DNA. • Much of the in-vitro work focused on genotoxic effects. Few studies indicated genotoxic effects from RF radiation. Those studies should be followed up. • In most studies, the genetoxic effect have been investigated after short-term exposure (Moulder et al., 1999, Vijayalaxmi and Obe, 2004).

19. Reviews • In a review, Verschaeve and Maes [1998] concluded that: “According to a great majority of papers, RF fields, and mobile telephone frequencies in particular, are not genotoxic: they do not induce genetic effects in vitro and in vivo, at least under nonthermal conditions, and do not seem to be teratogenic (cause birth defects) or to induce cancer.” • The Royal Society of Canada Expert Panel Report [1999, 2001, 2007] reviewed the subject and concluded that: “A large number of laboratory studies of the potential health effects of RF fields have focused on genotoxicity, including studies of tumorigenesis, promotion, progression, altered cell proliferation, and DNA damage. The great majority of these studies have failed to demonstrate genotoxic effects due to exposure to RF fields.”

20. The UK Independent Expert Group on Mobile Phones (IEGMP) [2000] summarized the situation as follows: “The balance of evidence, from both in vitro and in vivo experiments, indicates that neither acute nor chronic exposure to RF fields increased mutation or chromosomal aberration frequencies when temperatures are maintained within physiological limits. This suggests that RF exposure is unlikely to act as a tumor initiator.” • Meltz [2003] reviewed the in vitro literature pertinent to the issue of the possible induction of toxicity, genotoxicity, and transformation of mammalian cells due to RF exposure. The author concludes: “The weight of evidence available indicates that, for a variety of frequencies and modulations with both short and long exposure times, at exposure levels that do not (or in some instances do) heat the biological sample such that there is a measurable increase in temperature, RF exposure does not induce (a) DNA strand breaks, (b) chromosome aberrations, (c) sister chromatid exchanges (SCEs), (d) DNA repair synthesis, (e) phenotypic mutation, or (f) transformation (cancerlike changes).” The author further conclude: “While there is limited experimental evidence that RF exposure induces micronuclei formation, there is abundant evidence that it does not. There is some evidence that RF exposure does not induce DNA excision repair, suggesting the absence of base damage.”

21. Cell Proliferation • The Independent Expert Group on Mobile Phones comments: "Changes in the kinetics of cell division and in the proliferation of cells play a crucial role in the generation of cancer". Several studies have examined the relationship between RF radiation, in the mobile phone frequencies, and cell proliferation. • Cleary et al. (1990a, 1990b, 1995) have reported increased cell proliferation in experiments using brain glioma cells, human lymphocytes, and hamster ovary cells. The SARs were high in these studies, but temperature levels were well controlled. • Donnellan (1997) also found changes in cell proliferation when mast cells were exposed to 835 MHz under athermal conditions.

22. Kwee and Raskmark (1998), found a decrease in cell growth of human epithelial amnion cells exposed to 960 MHz microwave fields. • Capri (2004) also found a slight decrease in cell proliferation after exposure to 900 MHz RF radiation. • On the other hand Stagg (1997) and Vijayalaxmi (1997) found no increase in cell proliferation in their studies using 836.55 MHz and 2450 MHz respectively. • Zeni (2003) found no change in cell cycle kinetics when lymphocytes were exposed to various signals at 900 MHz frequency. • Scarfi (2006), in a two-laboratory study, found no changes in cell proliferation in lymphocytes exposed to RF radiation at 900 MHz. • Byus (1984) and Paulraj (2002) have reported decreased protein kinase activity in rats following exposure to RF radiation. Byus used modulated fields, while Paulraj used continuous wave radiation. • Pacini (2002) reported that exposure of skin fibroblasts to 900 MHz RF radiation for 1 hour altered gene expression, proliferation, and morphology.

23. Ornithine Decarboxylase (ODC) • ODC is the initial enzyme involved in the production of polyamines, which in turn are involved in the growth of normal and cancer cells. • The report by the Expert Panel of the Royal Society of Canada (1999) extensively reviewed the relationship between relationship between ODC activity and cancer. It pointed out that ODC activity may be increased in pre-malignant conditions, or following exposure to chemical carcinogens, and that ODC may function as an oncogenic protein at high levels of activity. • Several studies have shown increased ODC levels after RF exposure (Byus, 1997, Paulraj, 2002). Desta (2003), however, showed a decrease in ODC levels with exposure to RF radiation at 835 MHz, but only at SAR levels above 5 W/kg. These levels were associated with temperature increase in the medium used. Höytö (2006) also showed decreased ODC levels after RF radiation, but only in primary astrocytes and not in secondary neural cell lines. The same group (Höytö, 2007) found no increase in ODC activity on murine fibroblasts exposed at 835 or 872 MHz at SARs of 2.5 and 6.0 W/kg.

24. Hormonal Secretion • An area attracting attention, as a likely potential mechanism for RF radiation intervention in living organisms is consideration of a cancer promoting effect of RF radiation by altered circadian rhythms of pineal activity and melatonin release. Several investigations examined to what extent hormonal secretion is influenced by RF radiation. • Exposure at up to 0.3 W/kg did not disturb the normal circadian profile of melatonin of the hypothalamo-pituitary-adrenal axis [de Seze et al. 1998, 1999]. • However, Stark et al. [1997] conducted a pilot study to investigate the influence of RF radiation at 3-30 MHz on salivary melatonin concentration in dairy cattle.

25. In-Vitro Studies of Non-genotoxic Effects? • Several studies investigated the effect of RF radiation on cell cycle kinetics, however, the majority of these studies found no effect (Vijayalaxmi et al., 2001, Zeni et al., 2003, Lantow et al., 2006). • Several studies investigated whether RF radiation can induce apoptosis in human peripheral blood mononuclear cells, lymphoblastoid cells, epidermis cancer cells. No difference in apoptosis induction was detected. Apoptosis is a physiological mode of cell death occurring in development and cell differentiation. It is an important protection mechanism against cancer. • Some investigators have described increased heat shock protein level after RF radiation, however, there are other negative findings (for review see Cotgreave, 2005). This research area would benefit from independent replication. • Influence of RF radiation on immune system cells were investigated in a few studies. No significant effects were observed.

26. Nervous SystemWhat is Already Known about Nervous System Effects? • Due to the proximity of mobile phones to the head, public concerns are raised regarding a potentially toxic effect of RF radiation on the nervous system. The following aspects are usually considered in toxicology regarding this effect: • Morphology • Brain Function • Electrophysiology • Behavior • Development • Several investigations have been published regarding the potential neurotoxic effects of RF from mobile phones. Minor effects were observed on the electroencephalogram (EEG), sleep structure, and cognitive processes in human objects.

27. In Animals • In animals, few previous studies did show disturbance of work memory in rats exposed to RF (Lai et al., 1994, Wang and Lai, 2000). • Surprisingly, low SAR values caused increased permeability of the blood-brain barrier (BBB) in rats (Salford et al., 1994, 2003, Fritz, 1997; Persson et al., 1997). Recent animal studies failed to detect any changes in the BBB (Tsurita, 2000; Finnie, 2002; Nylund, 2004; Kuribayashi, 2005; Cosquer, 2005; Finnie, 2006; Kumlin, 2007). • The BBB isolates the CNS from the rest of the organism, controls molecules fluxes, and protects the brain. Increased permeability of BBB can allow unwanted substances to reach the CNS, with possible pathological consequences (inflammation, neurone death)

28. Review • For more details, see the review on this subject by D’Andrea et al. [2003]. The authors concluded: • Effects of RF exposure on the BBB have been generally accepted for exposures that are thermalizing. • Low level exposures that report alterations of the BBB remain controversial. • Exposure to high levels of RF energy can damage the structure and function of the nervous system. • Much research has focused on the neurochemistry of the brain and the reported effects of RF exposure. Research with isolated brain tissue has provided new results that do not seem to rely on thermal mechanisms.”

29. Developmental Effects • Various studies have evaluated developmental effects of RF radiation on mamals, birds, and other non-mamalian species. These studies reviewed by Heynick and Merritt (2003) and Juutilainen (2005), have shown that: • RF radiation are tertogenic at exposure levels that are sufficiently high to cause significant increase of temperature and exceed reference levels from exposure guidelines. • There is no consistent effects at non-thermal exposure levels. • Only few studies have evaluated possible effects on postnatal development using sensitive endpoints, such as behavioral effects.

30. Conclusion • There is no reliable indication from the in vitro research that RF fields affects cells at non-thermal effects. However, results of few results suggest genotoxic effects need to be better understood. • Animal cancer studies have not provided evidence that RF radiation could induce cancer, enhance the effects of known carcinogens, or accelerate the development of transplanted tumors. • Technology is advancing very fast and sources of RF exposure become increasingly common. Yet, there is a lack of understanding for interaction mechanisms below the guidelines and the information on individual RF exposure and contribution of various sources to the overall exposure. • An important area of research could be the effect of modulated or pulsed RF radiation especially on brain function, when compared with CW RF radiation of the same characteristics. • Another important area of research that might need further investigation is health risk associated with children’s use of mobile phones.

31. Strength of Evidence for Biological Effects from Exposure to RF fields