**Experimental Requirements for** *in vitro* **Studies Aimed to Evaluate the Biological Effects of Radiofrequency Radiation**

Olga Zeni and Maria Rosaria Scarfì

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51421

### **1. Introduction**

In the last years human exposure to electromagnetic fields (EMF) in the radiofrequency (RF) range has increased rapidly becoming unavoidable. As a matter of fact, many sources of RF fields are present at home, at work, and in the environment. In addition, other sources of occupational RF field exposure include equipments such as medical devices, dielectric heaters, induction heaters, diathermy machines, plasma discharge equipment and radars. Moreover, the rapidly increased use of mobile telecommunication has lead to exposure of a large amount of the population to RF fields. It has been estimated that 4.9 billions of mobile phone subscriptions will be active by the end of 2012. Although these technologies have highly improved the quality of life, at the same time they have given rise to great concern about possible health effects of such non-ionizing radiation at low exposure levels, with particular attention to cancer risk. As a matter of fact, heating is the most widely accepted mechanism of RF radiation with biological systems, and the current guidelines of human exposure are based on thermal effects, but subtle effects due to chronic exposures cannot be excluded. The latter, non thermal effects, are hypothesized to occur in the absence of local or whole body increases in temperature, although there are no generally accepted biophysical mechanisms that could explain such effects.

Three main approaches, epidemiological studies, in vivo studies and in vitro studies, providing different and complementary information, can be followed in addressing the evaluation of biological effects induced by exposure to RF fields. The epidemiological studies aim to test, on a statistical basis, whether a causal nexus between exposure to an environmental agent and its putative health effects on the health status of the exposed subjects could exist. They use specially designed studies that try to determine statistical

associations between independent (level of exposure) and dependent (health status, disease occurrence, etc.) variables by collecting data from population samples. In addition, in relation to RF-based wireless communications, there are two different exposure situations: to RF in the far field, emitted by base stations, WiFi access points, etc. and to RF in the near field, emitted by handheld devices (e.g. mobile phones). According to the World Health Organization publication on Electromagnetic Fields, Environmental Health Criteria series [1, 2], to proper address human health risk assessment, epidemiological research should allow for sufficient latency, sufficient range of exposure, including high exposure, and ability to accurately classify individuals into several exposure groups.

In vivo studies are carried out on human volunteers or animals and provide information concerning the interaction of RF radiation with living systems displaying the whole body functions, such as immune response, cardiovascular changes, and behaviour. There are obvious limitations in the exposure conditions to be tested on humans due to ethical issues, therefore most of the in vivo studies are carried out on laboratory animals (mainly rodents). However, extrapolation to humans to provide an estimation of health risk is not straightforward due to the differences in physiology and metabolism between species as well as differences in life expectancy and many other variables.

In vitro studies, carried out mainly on cell cultures or isolated tissue samples, are used extensively in toxicological investigations. This is because they can provide essential information about the potential effects of chemicals and other agents such as radiation on specific cell properties, and provide a more rapid and cost-effective approach to molecular and mechanistic studies than conventional laboratory animal models.

In the last 20 years, scientific investigations on whether RF radiation used in these technologies could have short-, medium- or long-term biological effects, and whether they could represent a health hazard to human population have largely increased, since any detectable detrimental effect of RF radiation, even small, could be very important, due to its widespread use, the large numbers of people exposed on a daily basis, and the social, economical and health impact this could have.

Recently, on May 2011, the International Agency for Research on Cancer (IARC) classified RF electromagnetic fields as possibly carcinogenic to humans (Group 2.B) [3, 4]. The IARC evaluated available literature about the carcinogenicity of RF electromagnetic fields and found the evidence to be "limited for carcinogenicity of RF-EMF, based on positive associations between glioma and acoustic neurinoma and long term exposure". The conclusion of the IARC was mainly based on the INTERPHONE epidemiological study, which found an increased risk for glioma in the highest category of heavy users (30 minutes per day over a 10 year period), although no increased risk was found at lower exposure. The evidence for other types of cancer was found to be "inadequate" [5]. In vivo and in vitro studies, carried out so far, have provided only limited support for the above mentioned classification [6], mainly due to the difficulty in comparing the results of the available studies to draw general conclusion. The difficulty often arises from the quality of the studies, in terms of study design, specific methodologies and analysis of the results.

In this chapter the focus is on in vitro studies, which are the studies providing insight into the basic mechanisms by which effects might be induced in more complex animal or human organisms. As a matter of fact, in vitro studies, carried out on tissue or cell cultures of animal or human origin, transformed or not transformed, are relatively simple and represent well described models, where the control of exposure and experimental conditions is significantly greater than in live animals or human volunteers.

122 Microwave Materials Characterization

associations between independent (level of exposure) and dependent (health status, disease occurrence, etc.) variables by collecting data from population samples. In addition, in relation to RF-based wireless communications, there are two different exposure situations: to RF in the far field, emitted by base stations, WiFi access points, etc. and to RF in the near field, emitted by handheld devices (e.g. mobile phones). According to the World Health Organization publication on Electromagnetic Fields, Environmental Health Criteria series [1, 2], to proper address human health risk assessment, epidemiological research should allow for sufficient latency, sufficient range of exposure, including high exposure, and ability to

In vivo studies are carried out on human volunteers or animals and provide information concerning the interaction of RF radiation with living systems displaying the whole body functions, such as immune response, cardiovascular changes, and behaviour. There are obvious limitations in the exposure conditions to be tested on humans due to ethical issues, therefore most of the in vivo studies are carried out on laboratory animals (mainly rodents). However, extrapolation to humans to provide an estimation of health risk is not straightforward due to the differences in physiology and metabolism between species as

In vitro studies, carried out mainly on cell cultures or isolated tissue samples, are used extensively in toxicological investigations. This is because they can provide essential information about the potential effects of chemicals and other agents such as radiation on specific cell properties, and provide a more rapid and cost-effective approach to molecular

In the last 20 years, scientific investigations on whether RF radiation used in these technologies could have short-, medium- or long-term biological effects, and whether they could represent a health hazard to human population have largely increased, since any detectable detrimental effect of RF radiation, even small, could be very important, due to its widespread use, the large numbers of people exposed on a daily basis, and the social,

Recently, on May 2011, the International Agency for Research on Cancer (IARC) classified RF electromagnetic fields as possibly carcinogenic to humans (Group 2.B) [3, 4]. The IARC evaluated available literature about the carcinogenicity of RF electromagnetic fields and found the evidence to be "limited for carcinogenicity of RF-EMF, based on positive associations between glioma and acoustic neurinoma and long term exposure". The conclusion of the IARC was mainly based on the INTERPHONE epidemiological study, which found an increased risk for glioma in the highest category of heavy users (30 minutes per day over a 10 year period), although no increased risk was found at lower exposure. The evidence for other types of cancer was found to be "inadequate" [5]. In vivo and in vitro studies, carried out so far, have provided only limited support for the above mentioned classification [6], mainly due to the difficulty in comparing the results of the available studies to draw general conclusion. The difficulty often arises from the quality of the

studies, in terms of study design, specific methodologies and analysis of the results.

accurately classify individuals into several exposure groups.

well as differences in life expectancy and many other variables.

and mechanistic studies than conventional laboratory animal models.

economical and health impact this could have.

In vitro studies, the most common in the evaluation of biological effects of RF radiation, are mainly aimed to investigate cellular endpoints related to cancer occurrence. Carcinogenesis is a multi-step process in which direct (genotoxic) and indirect (non-genotoxic) DNA damage is involved, as schematically depicted in figure 1. Therefore, cancer related studies can be classified as genotoxic and non-genotoxic. Genotoxic effects include DNA strand breaks, micronucleus formation, mutation and chromosomal aberration. Non-genotoxic effects refer to changes in cellular function, and include cell proliferation, oxidative metabolism, apoptosis or programmed cell death, cellular signal transduction, and gene expression (RNA and protein).

**Figure 1.** Schematic representation for genotoxic and non-genotoxic carcinogenesis

On the basis of the conclusions reported in the above mentioned reviews [1-3, 6], also confirmed by more recent scientific literature, the following considerations can be drawn. Results on genetic effects and other cellular endpoints, like cell proliferation and differentiation, apoptosis and cell transformation, are mainly negative, and some of the few positive findings may be attributable to a thermal insult rather than to the RF-exposure as such. As a matter of fact, some studies following replication, under more controlled conditions, failed to be confirmed by independent research groups [7]. The same is for expression of cancer-related genes (e.g., proto-oncogenes and tumor suppressor genes), and studies, carried out using powerful high-throughput screening techniques capable of examining changes in the expression of very large numbers of genes and proteins. It should be pointed out that the results achieved by high-throughput techniques need to be confirmed by quantitative methods since methodologies are not sufficiently standardized.

In order to improve the quality of in vitro studies and of the future research, high methodological quality is needed to address uncertainties in technical and biological aspects, and to determine whether the achieved results reflect "true" biological response or they are related to some unknown uncontrolled variable.

The aim of this chapter is to describe the main requirements for a well conducted in vitro study to overcome methodological limitations. Due to the inter-disciplinary nature of the bioelectromagnetic research, basically, biological and electromagnetic requirements can be identified.
