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Toxicology 301 (2012) 1– 12

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Toxicology

jou rn al hom epage: www.elsev ier .com/ locate / tox ico l

Review

Is there a relation between extremely low frequency magnetic field exposure,inflammation and neurodegenerative diseases? A review of in vivo and in vitroexperimental evidence

Mats-Olof Mattssona,∗, Myrtill Simkób,1

a AIT Austrian Institute of Technology, Health and Environment Department, Environmental Resources and Technologies, Konrad-Lorenz-Strasse 24, AT-3430 Tulln, Austriab Austrian Academy of Sciences, Institute of Technology Assessment, Strohgasse 45/5, AT-1030 Vienna, Austria

a r t i c l e i n f o

Article history:Received 11 April 2012Received in revised form 25 May 2012Accepted 17 June 2012Available online 29 June 2012

Keywords:Electromagnetic fieldAlzheimer’s disease50 HzNeuroinflammation

a b s t r a c t

Possible health consequences of exposure to extremely low frequency magnetic fields (ELF-MF) havereceived considerable interest during the last decades. One area of concern is neurodegenerative diseases(NDD), where epidemiological evidence suggests a correlation between MF exposure and Alzheimer’sdisease (AD). This review is focussing on animal and in vitro studies employing ELF-MF exposures to seeif there is mechanistic support for any causal connection between NDD and MF-exposure. The hypothesisis that ELF-MF exposure can promote inflammation processes and thus influence the progression of NDD.

A firm conclusion regarding this hypothesis is difficult to draw based on available studies, since there is alack of experimental studies that have addressed the question of ELF-MF exposure and NDD. Furthermore,the heterogeneity of the performed studies regarding, e.g., the exposure duration, the flux density, the bio-logical endpoint and the cell type and the time point of investigation is substantial and makes conclusionsdifficult to draw. Nevertheless, the investigated evidence from in vivo and in vitro studies suggest thatshort-term MF-exposure causes mild oxidative stress (modest ROS increases and changes in antioxidantlevels) and possibly activates anti-inflammatory processes (decrease in pro-inflammatory and increasein anti-inflammatory cytokines). The few studies that specifically have investigated NDDs or NDD rele-vant end-points show that effects of exposure are either lacking or indicating positive effects on neuronalviability and differentiation. In both immune and NDD relevant studies, experiments with realistic long-term exposures are lacking. Importantly, consequences of a possible long-lasting mild oxidative stressare thus not investigated. In summary, the existing experimental studies are not adequate in answeringif there is a causal relationship between MF-exposure and AD, as suggested in epidemiological studies.

© 2012 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1. Immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. ROS – oxidative stress and/or cell activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.3. Chronic inflammation and neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.3.1. Neurodegenerative diseases (NDD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.3.2. Inflammation and Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1. Extremely low frequency magnetic fields and inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.1.1. In vivo studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.1.2. In vitro studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.2. EMF and NDD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

∗ Corresponding author. Tel.: +43 50550 3425; fax: +43 50550 3452.E-mail addresses: Mats-Olof.Mattsson@ait.ac.at (M.-O. Mattsson), msimko@oeaw.ac.at (M. Simkó).

1 Tel.: +43 151581 6579.

0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tox.2012.06.011

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3.2.1. In vivo studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.2. In vitro studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction

Neurodegeneration is a progressive loss of structure and func-tion of neurons (e.g., Przedborski et al., 2003). If these structuraland functional neuronal changes cannot be compensated, anovert neurodegenerative disease (NDD), of which there are hun-dreds, emerges. The most known of these are dementias such asAlzheimer’s disease (AD; the most common of the dementias),Parkinson disease (PD), Huntington disease, and Amyotrophic lat-eral sclerosis (ALS). A small fraction of the NDD has a pure geneticbackground (e.g., Huntington disease). However, most cases ofmost NDD are sporadic, implying that environmental factors, aloneor in combination with genetic susceptibility factors, are involvedin the disease process (Cannon and Greenamyre, 2011). Some exter-nal factors that are neurotoxic and causing NDD are known andwell described. However, for many of the conditions the probablecause of the disease is a subject of speculation. Many mecha-nisms are involved in neurotoxicant-induced neurodegeneration.These include blood–brain barrier (BBB) disruption, protein aggre-gation, oxidative stress, and mitochondrial dysfunction (Cannonand Greenamyre, 2011).

Exposures to anthropogenic magnetic and electromagneticfields (MF and EMF respectively) at various frequency bands havebeen discussed in terms of environmental health during the lastcouple of decades. NDD is one of the areas where exposuresto extremely low frequency (ELF) MF are considered interesting,although there is at present only suggestive evidence from a lim-ited number of epidemiological studies that there is a correlation(see SCENIHR, 2007, 2009 for comprehensive overviews). Thereare practically no epidemiological studies relating to exposures toother frequency bands of the electromagnetic spectrum and con-nections to NDD. This review is therefore focussing on in vivo(animal) and in vitro studies including ELF-MF exposures to seeif there is mechanistic support for any causal connection betweenNDD and MF-exposure. Epidemiological studies are discussed onlybecause of their background relevance. Although there are reportedeffects of ELF-MF on a number of experimental end-points, themost consistent effects are seen on radical homeostasis and oninflammation-relevant parameters. Furthermore, there are fewexperimental studies that specifically and directly address ELF-MFeffects on NDD. Accordingly, we have chosen to assess the relevantliterature from a mechanistic perspective, where our hypothesisis that ELF-MF exposure can promote inflammation processes andthus influence the progression of NDD.

1.1. Immune response

Inflammation is an adaptive immune response to infection, irri-tation, or injury. This response involves the delivery of leukocytesto the initiating site, which is mediated by chemical factors derivedfrom plasma proteins or cells, which are triggered by Toll-likereceptors (TLRs) and nucleotide-binding oligomerization-domainprotein (NOD)-like receptors (NLRs) resulting in the secretion ofinflammatory mediators, such as cytokines and chemokines. Thereare two types of inflammation: acute and chronic. Acute inflamma-tion is characterized by a fast onset combined with short duration.It manifests with the release of fluid and plasma proteins, and

the migration of leukocytes, mostly neutrophils. After a successfulacute inflammatory response repair and recovery are starting(Ivashkiv, 2011). Chronic inflammation occurs over a longerduration. If the removal of the initiating factor is not complete,inflammation manifests itself by the presence of lymphocytes (Tcells) and macrophages, resulting in fibrosis and tissue necrosisand the development of chronic diseases and even cancer.

In response to acute damage or invading pathogens, monocytessynthesise increased amounts of enzymes to eliminate the cause.Monocytes respond to chemotactic and other mobilising factorsreleased by lymphocytes and microbes and migrate into the tissuesof the body where they can differentiate into active macrophages.Monocytes and their macrophage or dendritic progeny as well asgranulocytes are capable of phagocyting micro-organisms and canafter activation release free radicals such as reactive oxygen species(ROS) or nitric oxides (NO) in large amounts (an “oxidative burst”)to destroy them. Macrophages are antigen-presenting phagocytesreleasing both antimicrobial mediators and pro-inflammatory fac-tors to stimulate other cells of the immune system. Macrophagescan be activated by the classical or the alternative pathway (Gordon,2003). The former requires a priming signal in form of IFN-�.Pathogens are subsequently taken up by phagocytosis and deliv-ered to lysosomes where they are exposed to a variety of enzymesand proteases. A battery of chemokines can be released to attractneutrophils, immature dendritic cells, natural killer cells, and acti-vated T cells. Additionally, several pro-inflammatory cytokinescan be released (IL-1�/IL-1F2, IL-6, and TNF-�/TNFSF1A) wherebyTNF-� also contributes to the pro-apoptotic activity. The alterna-tive pathway does not require any priming, although IL-4 and/orIL-13 can act as sufficient stimuli. The secreted molecules act anti-inflammatory and promote wound healing, which is consistentwith a different role than the classical pathway in humoral immu-nity and repair (Gordon, 2003).

One of the first cytokines released by macrophages is IL-1�,which can enter the blood stream and subsequently reach the brain.Cytokines are macromolecules unable to pass the blood–brainbarrier. However cytokine binding receptors (e.g., IL-1�R) onbrain vascular cells are supposed to be the link between periph-eral immune-signalling and the brain, inducing the release ofprostaglandin E2 (PGE2). PGE2 activates receptors on neurons andmicroglia (immune cells in the brain), which can then initiateother components of the acute phase response. The activation ofmicroglia to release IL-1� leads to a chain reaction (Turrin andRivest, 2004) which includes both intra and intercellular activationprocesses.

The nuclear factor-kappa B (NF-�B) transcription factor plays acrucial role in various cellular processes such as cell proliferation,cell death, development, and the activity of a number of tissuesincluding the central nervous system (Memet, 2006), but also inthe innate and adaptive immune responses (Wan and Lenardo,2010). NF-�B is involved in cellular responses to different stress-like stimuli such as cytokines, free radicals, ultraviolet irradiation,oxidized LDL, and bacterial or viral antigens. It regulates the expres-sion of several intermediates such as cytokines, inducible nitricoxide synthase (iNOS) but also several growth factors and inhibitorsof apoptosis (Gilmore, 2006; Perkins, 2007). It is involved alsoin the regulation of effector enzymes in response to ligation of

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many receptors on immune cells such as T-cell und B-cell recep-tors and also the members of the Toll-like receptor/IL-1 receptorsuper family (Damgaard and Gyrd-Hansen, 2011). Activated NF-�Bin the nucleus activates gene transcription encoding chemokinessuch as IL-8, cytokines (e.g., TNF�, IL-1, IL-2, IL-6, and GM-CSF),adhesion molecules, acute phase proteins and antimicrobial pep-tides, enzymes (e.g., COX-2, iNOS and PLA2) producing secondaryinflammatory messengers, and anti-apoptotic factors (Bonizzi andKarin, 2004; Takeuchi and Akira, 2010). These processes mediatethe recruitment of inflammatory and phagocytic cells (Charo andRansohoff, 2006) and amplify the inflammatory response.

1.2. ROS – oxidative stress and/or cell activation

Phagocytic cells can produce large amounts of ROS, as an oxida-tive burst. ROS generation is due to activation of NAD(P)H-oxidase,xanthine oxidase, lipoxygenase or cyclooxygenase. NAD(P)H-oxidase is the most important one in immune cells, where itcatalyses an univalent reduction of O2 to generate the superoxideanion (Vignais, 2002). Bacterial endotoxins (e.g., LPS) and cytokines(including IL-1� and IFN-�) can induce an oxidative burst by acti-vating NAD(P)H-oxidase (Griendling et al., 2000; Vignais, 2002).

ROS at low concentrations can also act as second messengers andactivate signalling cascades which in turn can lead to physiologicalresponses such as gene expression, cell proliferation, and apoptosis(for reviews see Allen and Tresini, 2000; Brookes et al., 2002). It hasbeen shown that free radical mediated mitogenic signals lead tothe activation of NF-�B, and also activation of different antioxidantenzymes (Dalton et al., 1999). NF-�B is one of the transcriptionfactors which directly respond to oxidative stress.

Recently it was shown that primary brain-derived neural pro-genitors, which are proliferative, self-renewing and multipotent,maintained a high ROS status and were highly responsive to ROSstimulation (Le Belle et al., 2011).

Reactive oxygen or nitrogen species, called prooxidants arecounteracted by antioxidants. Antioxidants can be producedendogenously or from exogenous sources. They include enzymeslike superoxide dismutase (SOD), catalase (CAT), glutathione perox-idase (GPx), reduced glutathione (GSH) and glutathione reductase,but also minerals like Se, Mn, Cu and Zn, and vitamins such as vita-min A, C and E. Antioxidant activity includes also glutathione andflavonoids as well as other compounds (Droge, 2002). In a healthybody, prooxidants and antioxidants are in equilibrium. If this ratiois out of balance with a shift towards prooxidants, oxidative stress ispresent. The oxidative stress can be mild or strong depending on theextent of shift, leading to different cellular effects like the inductionof cell signalling, but also lipid peroxidation or DNA damage.

1.3. Chronic inflammation and neurodegenerative diseases

1.3.1. Neurodegenerative diseases (NDD)A Delphi consensus study concludes that epidemiological evi-

dence suggest that 4.6 million new NDD cases appear globallyevery year (Ferri et al., 2005). The estimated 24.3 million demen-tia cases in 2005 will accordingly double every 20 years and reach81 million by 2040. Alzheimer’s disease is regarded as the mostcommon of the dementias, contributing to about 75% of all cases.The pathogenesis of this disease is characterized by accumulationof extracellular depositions of amyloid-� (A�) that triggers neu-ronal dysfunction and death in the brain, as well as intracellularneurofibrillary tangles of the microtubule-associated protein Tauin a hyperphosphorylated form. The presence of A� aggregates issupposed to trigger inflammation, which further accelerates theneurotoxic effects of A� and Tau (e.g., Ballard et al., 2011; Qiu et al.,2009, for excellent recent reviews).

There is a recognized genetic component in AD, influencing ca.70% of the cases (Ballard et al., 2011). However, the over-all riskincrease conferred by a single gene is small. Dominant mutantalleles of the genes APP, PSEN1, and PSEN2, which are the only rec-ognized genes responsible for early onset of AD, are present in ca5% of patients. Other genes such as the �4 allele of the Apolipopro-tein E (APOE) are furthermore found in many cases (Vergheseet al., 2011). Although there is a strong over-all genetic component,environmental exposures to metals such as aluminium, lead, zincand copper have also been implicated (Cannon and Greenamyre,2011). Other risk factors include midlife obesity, smoking, diabetes,midlife hypertension, midlife hypercholesterolaemia, stroke andpossibly MF exposure (Ballard et al., 2011; Qiu et al., 2009).

1.3.2. Inflammation and Alzheimer’s diseaseNDDs are featured by progressive dysfunction and death of cells

in selected areas of the nervous system. The loss of neurons seemsto be associated with conformational changes in proteins, mak-ing it reasonable to consider NDD to be proteinopathies, or proteinmisfolding diseases (Gregersen, 2006; Jellinger, 2003). The reason-ing is that damaged proteins will cause accumulation of proteinoligomers that are exhibiting neurotoxic effects, either directly orindirectly via “neuroinflammation”. This neuroinflammatory pro-cess can persist, even when the inflammation-provoking stimulusis eliminated. This is valid for NDD in general (cf. Amor et al., 2010;Khandelwal et al., 2011 for recent reviews), and also for AD (Galaskoand Montine, 2010; Grammas, 2011). Swardfager et al. (2010) haverecently published a meta-analysis of cytokines in AD, as indicatorsof chronic inflammation. The study included 40 separate studiesmeasuring blood cytokine concentrations, and 14 studies wherecytokine levels in cerebrospinal fluid (CSF) were investigated. Anumber of inflammatory markers were increased in blood from ADpatients compared to blood from healthy individuals. Particularlystrong evidence was found for IL-6, IL-12, and IL-18, whereas TGF-�was significantly increased in CSF. The authors concluded that ADis accompanied by a peripheral immune response which may notspecifically reflect inflammatory activity within the CNS.

A key player in inflammatory conditions in the brain is themicroglia cell (Saijo and Glass, 2011). Microglia expresses surfacemarkers like macrophages and is considered to be functional equiv-alents to macrophages in the brain (Saijo and Glass, 2011). Thesecells perform important protective and repair functions in the brainalthough when “activated” they can cause chronic inflammationand contribute to the development of NDD (Heneka et al., 2010).In AD, glial cells are activated to release cytokines which start theinflammatory process (Van Eldik et al., 2007). Abnormal endoge-nous proteins such as A� aggregates and the heat shock proteinHsp70 cause such activation (Nakamura, 2002; van Rossum andHanisch, 2004). The activation includes production of free radicals,including ROS, which then directly or indirectly damage neuronalcells (e.g., Park et al., 2008; Wang et al., 2006; Wu et al., 2003).

Long-term use of non-steroidal anti-inflammatory drugs(NSAIDs) may protect subjects carrying one or more copies of the�4 allele of APOE against the onset of AD. However, newer studiessuggest that the chronic use of NSAIDs is beneficial only in the veryearly stages of the AD process. When the A� deposition processis already started, NSAIDs are no longer effective (Imbimbo, 2009;Imbimbo et al., 2010).

2. Methods

Information has primarily been obtained from reports published in internationalpeer-reviewed scientific journals in the English language, obtained from PubMedsearches (http://www.ncbi.nlm.nih.gov/pubmed). Analysed articles pertaining toELF-MF effects on immune responses and radical homeostasis have primarily beenpublished from 2007 until March 2012 due to that several comprehensive reviewswere published in the period 2004–2007 (Simkó and Mattsson, 2004; Simkó, 2004,

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2007). Due to the scarcity of studies and due to that there are no published reviewsfocussing on ELF-MF and NDD, no time-limits regarding information retrieval wasfollowed. Not all available studies have been included in this article. Rather, mostlyhigh-qualitative studies that add to the weight of evidence have been used. Inaddition, some studies were excluded since they did not fulfill quality criteria espe-cially regarding detailed exposure and dosimetry protocols. Other studies that wereexcluded had either deficit in description of assay and evaluation protocols and/orinsufficient or inappropriate statistical methods.

3. Results

An overview of the main effects on redox homeostasis, immuneresponses and NDD is given in Table 1. Detailed information regard-ing individual studies is found in Tables 2–5. All studies have used50 Hz MF exposure unless otherwise indicated.

3.1. Extremely low frequency magnetic fields and inflammation

Since several decades the biological effectiveness of ELF-MF hasbeen investigated in vivo and in vitro. It has been shown that manycellular systems, components and processes can be affected byEMF-exposure such as genotoxicity, cell proliferation, apoptosis,cell cycle regulation, cell differentiation, metabolism, and variousphysiological characteristics of cells. Also changes in gene and pro-tein expression have been reported, reflecting the interference ofEMF with living systems. There is no evident mechanism knownand an explanation for such interference is still missing. Sinceseveral studies have shown that the free radical homeostasis isinfluenced by the fields, this mechanism is discussed as an indirectway for several biological responses (Santini et al., 2009; Simkó,2004, 2007; Simkó and Mattsson, 2004).

3.1.1. In vivo studiesOnly a few in vivo studies were conducted in recent years in

order to investigate the oxidant system and immune relevant end-points in the whole organisms after ELF-MF exposure (see Table 2for details).

The antioxidant system was investigated in rats after exposureto MF which caused a decrease in GSH concentration in the heartand liver or kidney and SOD activity was lowered in the plasmaof MF exposed animals. However, no differences in CAT activityand thiobarbituric acid-reactive substances (TBARS) levels amongall the experimental groups were detected, showing that no induc-tion of lipid peroxidation took place (Martinez-Samano et al., 2010).Another group showed that exposure to 40 Hz, 7 mT, 30 min/day for14 days did not modify the concentration of TBARS, H2O2, totalfree SH groups, GSH and total antioxidant capacity of plasma.However, 60 min exposure per/day for 2 weeks caused significantincrease in TBARS and H2O2 concentration and a decrease in GSHand total free SH groups in rat heart homogenates, showing adecreased plasma antioxidant capacity (Ciejka and Goraca, 2009;Goraca et al., 2010). Canseven et al. (2008) investigated the fieldeffects on the activities of pro- and antioxidant system in heartand liver tissues of guinea pigs. The authors detected alterationsof MDA, NO, GSH and myeloperoxidase depending on field densityand exposure duration showing the interference of the field withthe antioxidant system.

Pulsed electromagnetic fields (PEMF) are more and more usedfor therapeutic purposes. Therefore PEMF-exposure was used toinvestigate nerve regeneration in mice (Baptista et al., 2009). Inter-estingly they reported that the PEMF-exposure caused a trendtowards decreased nerve regeneration and an increased free radi-cal level, in the presence of a non-changed TGF-�1 level. Oxidativestress was not leading to loss of function recovery. Using PEMF-exposure of arthritis induced rats already having elevated levelsof lipid peroxides and depleted GSH, GPx, CAT and SOD as well asincreased prostaglandin E2 levels resulted in the restoration of the

altered parameters after exposure, showing an anti-inflammatoryeffect (Selvam et al., 2007).

In summary the few recent in vivo studies have shown thatdifferent ELF-MF exposures using different intensities (from 4 �T(PEMF) to 7 mT), time schedules (single or repetitive) and durations(min to days) influences the redox homeostatic system toward apro-oxidative shift. Since this oxidative stress seems to be rela-tively mild, no or very moderate cellular/tissue damages such aslipid peroxidation were detected.

3.1.2. In vitro studiesROS is a valid measurable endpoint for cellular interaction both

in in vivo and in vitro studies. However the first target of interactionbetween ELF-MF and cells remains unknown.

As a second target of interaction with magnetic fields a compre-hensive pathway has been suggested (Lupke et al., 2006), wherebyELF-MF influences immune cells by membrane-associated compo-nents leading to ROS release and changes in radical homeostasis.This in turn causes down-stream events including changes in geneexpression leading to the activation of the alternative pathway ofhuman monocytes.

The effects of the same MF-exposure conditions on protein lev-els were shown by Frahm et al. (2010), where the modulation ofexpression levels of important proteins acting in redox regula-tory processes (clathrin, adaptin, PI3-kinase, protein kinase B (PKB)and PP2A, gp91phox, Hsp70 and Hsp110) was accompanied by anincreased level of ROS. The used positive controls, LPS and TPA,caused increases in ROS release as well. However, it seems like ELF-MF interacts with other cellular constituents than these chemicals,although induced pathways at least partially converge.

Falone et al. (2007) reported changes in the redox and differenti-ation status in neuroblastoma cells after short term MF-exposure.The results suggested the redox status modulation of these cells,without any oxidative damage. A positive modulation of antiox-idant enzyme expression and a significant increase in GSH levelwas observed. However, MF-exposure for 96 h enhanced the H2O2-induced ROS production and DNA strand breaks. Morabito et al.(2011, 2010) showed that 0.1–1.0 mT ELF-MF did not affect theproliferation and neuritogenesis in PC12 cells. Acute exposure ofundifferentiated PC12 cells increased ROS levels, decreased CATactivity, and affected the spontaneous intracellular Ca2+ levels.However, 7 days exposure increased CAT activity and the basalintracellular Ca2+ concentration. Using muscle cells (C2C12 cells)the authors detected the induction of ROS already at 0.1 mT andthe decrease in mitochondrial membrane potential, activated cel-lular detoxification system, increasing CAT and GTx activities, andaltered intracellular Ca2+ homeostasis (Morabito et al., 2010).

Differentiation of K562 cells after ELF-MF exposure at differenttime courses was investigated by Ayse et al. (2010). While a singleexposure resulted in a decrease in differentiation, 1 h/day duringfour days of exposure caused an increase in differentiation. Anincrease of ROS production was measured in the presence of heminonly. The authors concluded that the time-course of exposure appli-cation is an important parameter determining the physiologicalresponse of cells to ELF-MF. In activated (by bacteria, LPS or IFN-�) monocytic leukaemia cells (THP-1), MF-exposure induced NOrelease and a slight decrease in iNOS levels. Hsp70 expression wasdoubled after 4 h of exposure (Akan et al., 2010). In another studythe same group showed (Garip and Akan, 2010) that the effectof MF in K562 cells depends on the cell status. ROS and Hsp70levels were increased in the presence of H2O2. Mannerling et al.(2010) reported that MF-exposure causes a significant and tran-sient increase in Hsp70 and superoxide radical anion levels in K562cells. Interestingly, addition of free radical scavengers (melatoninor 1,10-phenantroline) inhibited the MF-induced increase in Hsp70and ROS suggesting that the ROS production seems to cause the

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Table 1Summary of direction of changes of important parameters investigated in in vivo and in vitro studies of ELF-MF effects on radical homeostasis and NDD relevant end-points.

Redox homeostasis Immune responses NDD

ROS Anti-oxidants Pro-inflammatory markers Anti-inflammatory markers Studied endpoints

In vivo ↑ ↓ Not sufficiently studied Not sufficiently studied Redox-homeostasis ↑ ↓

In vitro↑ ↓ ↓ ↑ Neural differentiation ↑

Viability ↑No effects on glia cells

Hsp70 induction. The authors summarized that there is a flux den-sity threshold where 50 Hz MF exerts its effects on K562 cells (at orbelow 0.025 mT).

ELF-MF increased iNOS and eNOS expression levels in humankeratinocyte HaCaT cells accompanied by increased NOS activityand production. In addition, higher levels of AP-1 expression aswell as a higher cell proliferation rate were associated with ELF-MFexposure. However, COX-2 expression, PGE2 production, CAT activ-ity and superoxide radical production were decreased (Patrunoet al., 2010). A study showed (Vianale et al., 2008) that MF-exposuresignificantly increased the growth rates of the HaCaT cells after48 h. After 72 h of exposure, the release of chemokines (RANTES,MCP-1, MIP-1� and IL-8) was significantly reduced. The NF-�Blevel was not detectable after 1 h exposure. The authors concludedthat the fields enhance cell growth and decrease pro-inflammatorychemokine production through the inhibition of the NF-�B sig-nalling leading to the inhibition of inflammatory processes. Inanother study (Reale et al., 2006) the effects of over-night expo-sure MF on human monocytes was investigated. Whereas iNOS wasdown-regulated both at the mRNA level and at the protein level, themonocyte chemotactic protein-1 (MCP-1) was up-regulated.

The role of adenosine analogues and MF-stimulation for PGE2release and COX-2 expression in bovine synovial fibroblasts (SFs)has been investigated (De Mattei et al., 2009). The MF-exposureinhibited PGE2 production in the absence of adenosine agonistsand increased the effects of other agonists. Changes in PGE2 levelswere associated with modification of COX-2 expression. This studysupports the anti-inflammatory activity of adenosine receptors andEMFs in bovine SFs. In a later study (Ongaro et al., 2011) the inflam-matory activity of human SFs from osteoarthritis patients wastreated with IL-1� and EMF to investigate a possible involvementof adenosine receptors (ARs). EMF-exposure induced a selectiveincrease in A2A and A3 ARs which were associated with changes

in cAMP levels, indicating that ARs were functionally active. Addi-tional data showed that in the presence of adenosine agonistsand antagonists, EMF inhibited the release of PGE2 and the pro-inflammatory cytokines IL-6 and IL-8, while stimulating the releaseof the anti-inflammatory cytokine IL-10, which is in part mediatedby the adenosine pathway, specifically by the A2A and A3 activation.

An interesting study (Lin and Lin, 2011) investigated co-culturesof osteoblasts (7F2) and macrophage cells (RAW 264.7) exposedto PEMF for 9 h. The cells were LPS stimulated to yield ROS andNO release. In the presence of a significantly elevated NO level,PEMF-exposed osteoblasts showed enhanced cell proliferation, via-bility, and COL I mRNA expression compared to the controls but lessALP activity was measured. In a comprehensive study (de Kleijnet al., 2011) human peripheral blood mononuclear cells (PBMCs)from healthy volunteers were stimulated with TLR2 or TLR4 lig-ands, or with different microorganisms before exposure to MF witha mixture of frequencies (20–5000 Hz). However, the 5 �T expo-sure did not cause significant differences in cytokine production(IL-1�, IL-6, TNF�, IL-8 and IL-10) in the presence of activation. Non-activated cells were not used. Gomez-Ochoa et al. (2011) exposedfibroblast-like cells derived from PBMC, to PEMF for 15 min ondays 7, 8 and 9 of cell culture. The pro-inflammatory cytokinesIL-1� and TNF-� were significantly decreased on days 14 and21 of the culture, whereas IL-10 was significantly increased onday 21. In another study, the effects of 45 mT PEMF were inves-tigated on cytokine production in PBMC from healthy donors andfrom Crohn’s disease patients (CD). Exposed and stimulated PBMCsfrom CD patients showed a decreased IFN-� pro-inflammatoryand an increased IL-10 anti-inflammatory cytokine productionwhereas PEMF-exposure had minimal effect on PBMCs from con-trols (Kaszuba-Zwoinska et al., 2008).

These in vitro studies (see Table 3) are in accordance withthe outcome of the in vivo studies, namely that MF-exposure

Table 2In vivo studies on redox homeostasis and immune response after MF-exposure.

Endpoint/result/release, gene or proteinexpression

Exposure Animal strain and tissue Reference

GSH in heart, liver, kidney ↓Plasma SOD activity ↓No effects: CAT, TBARS, lipid peroxidation

2 h, 60 Hz, 2.4 mT Wistar rats, liver, heart, kidney andplasma of

Martinez-Samano et al. (2010)

30 min exp./day:No effects: TBARS, H2O2, total free SH groups,GSH, total antioxidant capacity of plasma60 min exp. per/day:TBARS, H2O2 ↑, in heart: GSH, total free SHgroups ↓

30 or 60 min/day for 14 days,40 Hz, 7 mT

Sprague-Dawley rats, heart, plasmaMale Wistar rats

Ciejka and Goraca (2009) andGoraca et al. (2010)

MDA, NO, GSH and myeloperoxidase werealtered depending on field densities andexposure time

4 h/day and 8 h/day for 5 days,50 Hz MFs of 1, 2 and 3 mT

Guinea pigs, heart and liver Canseven et al. (2008)

Nerves regeneration ↓ROS ↑ no effects: TGF-�1

30 min/day five days a week forthree weeks, PEMF 72 Hz, 0.2 mT

Male Swiss mice Baptista et al. (2009)

Restoration of altered lipid peroxides,antioxidant enzymes, prostaglandin E(2)

90 min, PEMF 5 Hz, 4 �T Induced arthritis of rats Selvam et al. (2007)

3-Nitrotyrosine (3-NT) ↑ in female ratsno effects: MDA levels

4 h/day, 50 Hz, 1 mT, Wistar rats, liver Erdal et al. (2008)

No effects: MDA, SOD, GPX, CAT, andglucose-6-phosphate dehydrogenase

8 h day for 90 days, PEMF 50 Hz,2 mT

Female albino mice, blood at 45,60, and 90 days

Eraslan et al. (2007)

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Table 3In vitro studies on redox homeostasis and immune response after MF-exposure.

Endpoint/result/release, gene or proteinexpression

Exposure Cell type Reference

ROS ↑ in the presence of H2O2

GST, GPX, �-GCS, GSH ↑GPX1 transcript ↑Bcl-2, p53, GAP-43 neuron-specific enolase(NSE) and PPAR� proteins ↑No effects: SOD, CAT, glutathione reductase

96 h, 50 Hz, 1 mT Human neuroblastoma cell line Falone et al. (2007)

ROS ↑Modulations in several redox regulatoryproteins

45 min, 50 Hz, 1 mT Mouse macrophages Frahm et al. (2010)

ROS ↑CAT ↓ after 30 minCAT ↑ after 7 days

30 min to 7 days, 50 Hz,0.1–1.0 mT

PC12 cells undifferentiated Morabito et al. (2011)

ROS ↑CAT, GPX, peroxidase ↑

50 Hz, 0.1 and 1 mT C2CI2 muscle cells Morabito et al. (2010)

ROS ↑Differentiation (1 h) ↓Differentiation (1 h/4days) ↑

1 h, 1 h for 4 days, 50 Hz, 5 mT K562 cells Ayse et al. (2010)

NO ↑Hsp70 ↑

4 h, 50 Hz, 1 mT THP-1 cells Akan et al. (2010)

ROS ↑Hsp70 ↑

3 h, 50 Hz, 1 mT K562 cells Garip and Akan (2010)

ROS ↑Hsp70 ↑

1 h, 50 Hz, 0.025–0.1 mT K562 cells Mannerling et al. (2010)

7F2: proliferation, viability, geneticexpressions of type I collagen (COL I) ↑ alkalinephosphatase (ALP) activity ↓

9 h, PEMF 75 Hz, 1.5 mT Co-culture 7F2 (osteoblasts)and RAW 264.7 (macrophages)

Lin and Lin (2011)

IFN � ↓IL-10 ↑

45 min, PEMF 50 Hz, 5 mT PBMC from Crohn diseasepatients

Kaszuba-Zwoinska et al. (2008)

PGE (2) ↓COX (2) modifications

24 h, 75 Hz, 1.5 mT Bovine synovial fibroblasts De Mattei et al. (2009)

PG receptor ↑IL-10↑PGE(2) ↓IL-6, IL-8 ↓

24 h, 75 Hz, 1.5 mT Human synovial fibroblastsfrom osteoarthritis patients

Ongaro et al. (2011)

iNOS, eNOS, NO ↑COX 2, PGE2, CAT, superoxide radical

3, 18 and 48 h, 50 Hz, 1 mT Human keratinocyte cell lineHaCaT

Patruno et al. (2010)

No effects on different cytokines 30 min, 20–5000 Hz, 5 �T, PBMC de Kleijn et al. (2011)IL-1�, TNF � ↓IL-10 ↑

15 min on day 7,8,9 PEMF50 Hz, 2.5 mT

Fibroblast like cells Gomez-Ochoa et al. (2011)

RANTES, MCP-1, MIP-1, IL-8 ↓NF-�B ↓

72 h, 50 Hz, 1 mT Human keratinocytes (HaCaTcells)

Vianale et al. (2008)

MCP-1 ↑iNOS ↓

Over night, 50 Hz, 1 mT Human monocytes Reale et al. (2006)

Table 4In vivo studies on neurodegeneration relevant endpoints after MF-exposure.

Endpoint/result/release, gene or protein expression Exposure Animal strain and tissue Reference

Reversible DNA damageNo effects: hsp70

1 or 7 days, 15 h/day, 50 Hz,1.0 mT

Adult male CD mice, brain andliver

Mariucci et al. (2010)

Neurogenesis ↑pro-neural gene expression ↑Ca2+-channel expression ↑Functional integration of new neurons

1–7 h/day, 4 or 7 days, 50 Hz,1.0 mT

C57BL/6 mice, Dentate gyrus ofthe hippocampus

Cuccurazzu et al. (2010)

In aged rats: glutathione-related enzymes, CAT ↓In young rats: SOD2, GR activity ↑No effects: NGF, TrkA proteins

10 days, 50 Hz, 0.1 mT Sprague-Dawley female ratbrain cortex

Falone et al. (2008)

Dose-dependent effects:Oxidative stress markers ↑Antioxidant systems ↓No effects: brain morphology, gliosis, caspase-3

2 h/day, 10 months, 50 Hz MF,0.1 and 0.5 mT

Male Sprague-Dawley brains Akdag et al. (2010)

Synthesis rate of DA and 5HT in frontal cortex. ↑No effects: DA, 5HT, or metabolite

1 h/day, 14 days, 1.8–3.8 mTwithin exposure chamber

Male Wistar rat frontal cortexand corpus striatum

Sieron et al. (2004)

Irritability, oral activity, catalepsy ↓ 1 h/day, 14 days, 1.8–3.8 mTwithin exposure chamber

Male Wistar rat Sieron et al. (2001)

5HT-affinity for 5-HT2A receptor ↓ (prefrontal cortex)5-HT2A density ↑No effects: D1 or D2 receptors in striatum

1, 3, 7 days, 50 Hz MF, 0.5 mT Male Wistar rat, Transmitteractivities in prefrontal cortexand striatum

Janac et al. (2009)

Superoxide anion (cortex, basal forebrain, striatum,hippocampus, brain stem, cerebellum) ↑SOD (basal forebrain) ↑NO (cortex, basal forebrain, hippocampus, brain stem) ↑Lipid peroxidation (cortex, basal forebrain) ↑

7 days continuous exposure,50 Hz MF, 0.5 mT

Male Wistar rat brain (regionalanalysis)

Jelenkovic et al. (2006)

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Table 5In vitro studies on neurodegeneration relevant endpoints after MF-exposure.

Endpoint/result/release, gene or proteinexpression

Exposure Cell type Reference

Differentiation earlier in NGF-induced cells 48 h, 50 Hz, 2 mT AtT20 D16V cells Lisi et al. (2006b)Differentiation: �-3 tubulin ↑MAP2 ↑

Time, 50 Hz, 1 mT Mouse cortical embryonal stem cells Lisi et al. (2005)

Differentiation: MAP2 ↑Spike frequencies ↑GFAP ↓ (glial marker)

Up to 21 days, 50 Hz, 10 mT P19 cell Saito et al. (2009)

Neuronal differentiation ↑ (neuronal markers,Ca2+-channel)

Up to 12 days, 50 Hz, 1.0 mT Primary cortical newborn mouse neurons Piacentini et al. (2008)

No effects:APP695 mRNA

4 h at days 2,10,16; 50 Hz, 50,100, 200 �T

IMR-32 neuroblastoma cells Rao et al. (2002)

No effects:nACH-R

16 or 48 h, 50 Hz, 1 or 2 mT SH-SY5Y neuroblasoma cells Antonini et al. (2006)

No effects:GFAPGlial proliferation

10 s, PEMF 10 Hz, 0.1–0.63 T Cultured astrocytes Chan et al. (1999)

No effects:Glial proliferationHsp70Several proteins

1 h or 11 days, 50 Hz, 1 mT Cultured astrocytes Bodega et al. (2005)

Amyloid � ↑ 18 h, 50 Hz, 3.1 mT Human neuroglioma cell line Del Giudice et al. (2007)Viability ↑Apoptotic DNA-fragmentation ↓BDNF, TrkA ↑ IL-1� ↑No effects: redox status, MDA, GSH, SOD, CAT,GR, GST, �-GCSEffects at 1.0 mT but not at 0.1 mT

7 days, 50 Hz, 0.1 and 1.0 mT Rat cortical neurons Di Loreto et al. (2009)

AChE activity inhibition (27%) at 0.74 mT andhigher, at several frequencies (50–75 Hz,200 Hz, 350 Hz, and 475 Hz), effects after1 minexposure, reversible after exposure

Minutes, 10–650 Hz,0.2–2.0 mT

Mouse cerebellar synaptosomes Cell-freesystem

Ravera et al. (2010)

is interfering with cellular systems. The main mechanism(s) arestill not known. However it seems that MF (from 0.025 mT andhigher) are able to activate cell systems to release moderateamounts of ROS, which leads to the consumption of the intracel-lular antioxidants. Depending on the time point of measurement,an oscillation of the levels of these proteins can appear (up ordown regulation), which can be the reason for contradictory find-ings (increase vs. decrease due to MF-exposure). The few studiesinvestigating cytokine release indicate similar effects. Whereaspro-inflammatory factors are down-regulated, anti-inflammatorycytokines are up-regulated being a result of the moderate oxidativestress. There is a flux density threshold where, e.g., pre-activated(pre-treated) cells are not affected by the fields, whereas higherfield strengths lead to cell activation even in the presence of pre-activation. In summary, MFs are activating cells by shifting themtowards pro-oxidative states which in turn induces the activationof the antioxidant system.

3.2. EMF and NDD

Previous epidemiological findings suggesting a possible asso-ciation between occupational MF-exposure and in particular ADhave been strengthened during the last decade (Hug et al., 2006;Garcia et al., 2008). These meta-analyses concluded that availabledata indicated an increased risk for AD and senile dementia. Huget al. (2006) also pointed to that recent studies strengthen previ-ous associations between “electrical” occupations and welding, andALS. No recent studies or reviews found support for increased PDrisk in occupations with MF-exposure (Hug et al., 2006; Wirdefeldtet al., 2011).

More recent work seems to strengthen the evidence for acorrelation between occupational MF-exposure and AD. Thus, datafrom the Study of Dementia in Swedish Twins (Andel et al., 2010)suggest that AD onset before the age of 75 was increased in malesubjects subjected to medium and high MF-exposures (according

to a job-exposure matrix) in their main life-time occupation. Astudy of Swiss railway workers found an increased risk for AD inemployees with higher MF-exposure (train drivers; on average21 �T) compared to less exposed groups (on average less than0.5 �T). Regarding residential MF-exposure, studies are rare, but arecent article (Huss et al., 2009) showed increased NDD mortality(especially AD) in residents living close (50 m or less) to 220–380 kVpower-lines. The findings also suggested a dose–responserelationship between exposure duration and mortality.

3.2.1. In vivo studiesAnimal studies on pathological changes relevant for human neu-

rodegenerative diseases in the context of ELF-MF exposures are few(see Table 4). Their usefulness for assessing a possible human healthrisk from the MF-exposure is furthermore limited by extrapolationissues, time-frame of exposures, and also investigated end-points.Several of the relevant studies have been focusing on the effectsof ELF-MF on radical homeostasis, and to some extent on viabilityparameters. Falone et al. (2008) studied female Sprague-Dawleyrats that were continuously exposed for ten days to a 0.1 mT MF.Exposed animals were either young at the onset of exposure (3months of age) or aged (19 months old). Antioxidant enzyme activ-ities were increasing with age, irrespective of exposure. In the agedrats, a decrease in the activities of glutathione-related detoxify-ing enzymes and CAT was seen after the exposure. There were noeffects on certain neurotrophins (NGF and its receptor TrkA) onmRNA or protein levels. In contrast, the brains from exposed younganimals displayed increased SOD2 and glutathione reductase activ-ities. These results seem to indicate that the MF-exposure causesreduction in oxidative defence systems in the brain which is underespecially high stress in aged individuals. However, this study isbased on relatively few individuals in each group and there is noreal sham exposure.

Akdag et al. (2010) documented the influence of a long-term exposure (0.10 and 0.50 mT) on male Sprague-Dawley rats.

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A proper sham exposure was included and 10 animals wereassigned to each group. Markers for oxidative stress (total anti-oxidative capacity, total oxidant status, oxidative stress index) wereincreased whereas antioxidant systems (CAT, MDA, myeloperoxi-dase) decreased, in a dose-dependent manner. No morphologicalchanges, including gliosis, or effects on the apoptosis markercaspase-3 were seen. The exposure effect was significantly strongerat 0.50 mT than at 0.10 mT, suggesting a positive dose–responserelationship.

In a study from Jelenkovic et al. (2006), adult male Wistar ratswere continuously (seven days) exposed or sham exposed. Afterexposure, dissected brains were analysed regionally for markers ofoxidative stress. The superoxide anion radical, SOD activity, NO, andlipid peroxidation were increased in a regional manner. These datasuggest that the basal forebrain is the most sensitive region, fol-lowed by frontal cortex. The superoxide ion production increasedin all regions, but the most harmful effect, lipid peroxidation wasseen only in frontal cortex and basal forebrain. Since the exposure(seven days) is relatively long-term it might indicate that a possi-ble chronic MF effect is exhaustion of defences against superoxideproduction.

For the development and also manifestation of certain NDDs,the status of neurotransmitter systems including their synthesis,release, receptor interactions and reuptake/inactivation is impor-tant. Only a few animal studies investigating the effects of ELF-MFsare published. Thus, repeated exposures to a 10 Hz sinusoidal MFduring 14 days did not change the levels of either dopamine (DA)or 5HT or their metabolites in rat frontal cortex or striatum, butdid cause increased turnover of the transmitters in the frontal cor-tex (Sieron et al., 2004). A previous study from the same groupshowed that this MF-exposure also caused DA receptor deactiva-tion (Sieron et al., 2001). In contrast, work from Janac et al. (2009)on Wistar rats exhibited no effects on D1 or D2 ligand binding instriatum, whereas the 5-HT affinity for 5-HT2A receptors decreasedin prefrontal cortex.

Cuccurazzu et al. (2010) exposed adult male C57BL/6 mice foreither four or seven days (various number of hour per day) andinvestigated neurogenesis in the dentate gyrus part of the hip-pocampus. The exposure caused proliferation of the neuronal stemcell population in the investigated area, where longer exposuretimes caused more proliferation. The newly formed cells devel-oped into neurons, expressing neuronal and not glial markers.The exposure stimulated increased gene expression typical forneuronal differentiation and the voltage gated Ca2+-channel Cav.Twenty-eight days after exposure, the newly formed neurons weresurviving and functionally integrated in the hippocampus.

There are few available animal studies of relevance for NDD.The exposures are typically at high flux densities (0.10–1.0 mT),for shorter (days) to longer (weeks–months) periods of time.Longer exposures at least seem to cause increased oxidative stress(increased ROS, NO, lipid peroxidation) and possibly reduction inoxidative defence systems. It is unclear if there are replicable effectson neurotransmitter systems including receptor-interactions andtransmitter synthesis, release and reuptake/inactivation. In addi-tion, no study has reported morphological changes. Interestingly,one study employing short-term exposure (days) could report posi-tive effects on differentiation, where neurogenesis (proliferation ofstem cells, differentiation, physiological integration) was inducedby MF-exposure.

3.2.2. In vitro studiesThere are few dedicated in vitro model systems for any of the

NDDs, including AD. Therefore, in vitro studies that are using nervecells (primary cultures or cell lines) or glial cells (microglia or astro-cytes) are appropriate. In addition, relevant end-points include cellsurvival and cell death, cell differentiation, radical homeostasis,

expression of inflammation markers, synaptic transmission, andfunctionality of the blood–brain barrier.

A few studies have focussed on differentiation into the neuronalphenotype of undifferentiated or lowly differentiated precursorsof nerve cells. Lisi et al. (2006a) found that the neurosecretory dif-ferentiation of AtT20 D16V cells induced by NGF occurred earlierwhen MF-exposed, compared to controls. In another study from thesame group (Lisi et al., 2005), primary cultures of mouse corticalembryonal stem cells were stimulated to differentiate (increasedexpression of �-3-tubulin and MAP2) when exposed. In a study bySaito et al. (2009), P19 embryonal carcinoma cells were induced todifferentiate if exposed to 10 mT, but not at 1 mT. The expressionof MAP2 and spike frequencies increased, whereas the glial markerGFAP decreased. Primary cultures of newborn mouse cortical neu-ronal stem cells were stimulated to increase their differentiationrate after continuous exposure for up to twelve days (Piacentiniet al., 2008). The differentiation was seen as enhanced expressionof neuronal markers and enhanced Cav-channel expression andactivity.

Rao et al. (2002) could not find any effects of 60 Hz MF-exposureon the levels of APP695 mRNA (as a marker for amyloid production)in IMR-32 neuroblastoma cells at various MF-levels for differentperiods of time. The nicotinic cholinergic receptor subunit familywas studied on the mRNA level by Antonini et al. (2006). Thesereceptors are the ones most affected in AD and thus of relevanceto study. There were no effects of exposure in a neuroblastoma cellline.

No significant effects on GFAP levels and glial cell proliferationwere seen in two studies on cultured astrocytes that were exposedto either a PEMF (Chan et al., 1999) or to 50 Hz at 1 mT (Bodega et al.,2005). In the latter study, no effects of exposure on various proteins,including some heat shock proteins such as HSP70 were seen. Ina study from Di Loreto et al. (2009) primary cultures of embry-onal rat cortical neurons were used. The cells were exposed forseven days to a 50 Hz MF (0.1 or 1.0 mT). The higher exposure levelhad stronger effects, if effects occurred at all. The 1.0 mT exposurecaused increased vitality and decreased apoptosis, possibly due toenhancement of neurotrophic support. This seems to be indepen-dent of radical homeostasis disturbances, since redox status, MDAlevels, and enzymatic activities were unaffected by exposure. Thestudy did not include any positive control(s).

The in vitro studies are mostly acute or short-term (with expo-sures ranging from minutes to a few days) and also limited bythat they almost always only include one cell type, primary cul-tures of neuronal precursors or established cell lines. The studiesdo not allow any conclusions regarding a possible effect of ELF-MF exposure on NDD development, but offer some results that areinteresting and possibly worthwhile following up, including thenoted positive effects on differentiation. Otherwise, there are noin vitro findings documenting effects on disease markers or trans-mitter systems.

4. Discussion

The over-all picture from the current analysis of ELF-MF effectson redox homeostasis and immune responses is that both in vivoand in vitro studies suggest positive effects on ROS production andreduction of antioxidant activities. Data from in vivo studies onimmune responses are not sufficient for any conclusion, but thein vitro studies suggest that exposure decreases pro-inflammatorycytokines and increases anti-inflammatory markers. It needs alsoto be mentioned that effects on ROS production are modest and farfrom any “oxidative burst”. It thus seems that the ELF-MF exposureis capable of causing a mild oxidative stress and possibly stimu-lating anti-inflammatory processes in the short-term perspective.

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Fig. 1. Hypothetical interaction between ELF-MF and living systems. ELF-MF interacts with cellular receptors and/or the inner or outer site of the membrane and activatesspecific molecular pathways leading to a change of the redox homeostatic capacity of the cell. This can be due to the release of free radicals triggering cell signalling thatinfluences cell activation, release of intermediates, and/or the activation of cell proliferation. Cell signalling can also lead to the activation of differentiation and cell survival,being a helpful effect in therapeutical use. On the other hand, signalling can also trigger the immune response. In a short term perspective, it seems that anti-inflammatoryresponses are activated, which could be a useful tool for treatment of inflammation. Long term effects however, could trigger pro-inflammatory pathways causing theamplification of or the development of diseases.

Long-term effects of exposure are difficult to evaluate due to lack ofproper studies. The hypothetical interaction between ELF-MF andliving system is outlined in Fig. 1.

There is a definitive lack of experimental studies that haveaddressed the question of ELF-MF exposure and NDD. At least theshort-term studies analysed here suggest that the exposure is notleading to neuronal cell death or impaired neuronal function, orto activation or proliferation of glial cells. On the contrary, somedata suggest positive effects on neuronal survival and differentia-tion. However, the largest weakness is that there is a lack of studieswith sufficient exposure duration, and at exposure levels similar tothe ones encountered in the environment.

Another difficulty is the heterogeneity of the performed stud-ies. There are several parameters which are influencing the results:the exposure duration, the flux density, the biological endpoint butalso the cell type and the time point of investigation. Since thereare many possibilities to combine all these parameters, it followsthat results are very heterogeneous. One study has shown, e.g.,that there is a flux density threshold (0.025 mT) for MF reactiv-ity in vitro using K562 cells (Mannerling et al., 2010), whereas thisthreshold and even this effect is absent using other cell types. Ina recent review (Santini et al., 2009) it has been clearly demon-strated, that different cell types are differently affected by differentflux density ELF-MF. Depending on the investigated cell type effectscan reach from negative to positive changes in cell/tissue func-tions. For example, cell proliferation in wound healing is a positiveresult, however in cancer it is definitively not. It is apparent, thatthe experimental approach has clearly to address the hypothesisby using the appropriate cell types for the investigated endpoint.Also the use of proper controls and blinded protocols is essen-tial. In many studies positive controls are not used. Proper sham

exposures are also preferable. In some studies pre-activated bloodcells were exposed to very low flux densities, but unfortu-nately non-activated cells were not included in the experimentalsequence. It seems, that by using very low flux densities (in therange of �T) to expose pre-activated cells (de Kleijn et al., 2011),it does not lead to an ELF-MF effect. However, it is not clear if ahigher flux density and/or the absence of pre-activation is leadingto a different result. Enzyme oscillations within cells are knownto occur. Therefore, the phenomenon of up or down-regulation ofcertain proteins after MF-exposure can depend on the time pointof measurement represented by the up or down-regulated antiox-idants.

Possible neutral or even positive effects of exposure duringshort-term experiments do not necessary imply that long-termexposures are innocent. Even a mild chronic inflammation canhave negative consequences, which is seen in chronic diseases likecancer, cardiovascular disease, arthritis, diabetes and obesity thatall have inflammatory background (Prasad et al., 2011). A meta-analysis by Swardfager et al. (2010) showed that AD patients haveinflammatory cytokines in the circulation, which possibly also canhave at least indirect effects in the CNS. If MF exposures thus havea systemic effect, it may influence the status of the brain. Further-more, chronic peripheral inflammation is also having a disruptiveeffect on the blood–brain barrier integrity (Huber et al., 2001). Dam-age to the blood–brain barrier by neurotoxic agents has also beenimplicated in NDD (Cannon and Greenamyre, 2011).

The microglia population in the brain has a dual role, whereit performs important protective assignments and may also causechronic inflammation and promote NDD (Heneka et al., 2010).These cells can be activated and subsequently generate localinflammation, by various stimuli, including A� and apoptotic rest

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products from neurons (Glass et al., 2010). Their role, if any, in apossibly MF-mediated NDD scenario is unknown.

Co-exposures of several factors may have a significant influenceon development of NDD. Examples include various insecticides andPD (Heusinkveld and Westerink, 2012) and copper together withaluminium in the case of AD (Becaria et al., 2003). Regarding MF,the available epidemiological studies have focussed on the occu-pational setting, and directly or indirectly provided data on theMF exposure. There is however, a lack of knowledge regarding thepossible interactions and synergisms of MF-exposures and expo-sures to other agents. It is not unlikely that MF exposures are alsofound together with metal exposures, and also together with pos-sible chemical pollutants that have been implicated in onset anddevelopment of NDD (Cannon and Greenamyre, 2011; Wirdefeldtet al., 2011). Another area of MF-co-exposure is where ultrafine andnanoparticles are found, e.g., in welding (as discussed by Simkó andMattsson, 2010). Whether or not these examples impose a higherrisk for AD is unknown but of relevance to study.

Many studies have investigated the positive influence of ELF-MFon cells and organisms after short-term exposure. The intention isnot to give an overview about the therapeutic use of the MF, how-ever, it has to be mentioned that this area is reaching from woundand bone healing over pain relief to transcranial magnetic stimula-tion (TMS). In the latter technique, neurons are actively stimulatedby MF-induced electric fields (see Hiscock et al., 2008). TMS is usedas an antidepressant (Zyss et al., 2010), against migraine (Dodicket al., 2010) and also to enhance motor functions (Hiscock et al.,2008), and it can interfere with human behaviour and also with cog-nitive tasks. The cellular mechanisms underlying all these magneticstimulations remain unclear. Interestingly, albeit the positive influ-ence of the fields is more and more recognized and used in thera-peutic applications, the general effectiveness is still controversial.

There are obvious knowledge gaps that make a conclusion ofthe risk for NDD due to ELF-MF exposure very difficult. Experimen-tal research efforts should include a proper long-term perspective,possibly as life-long animal studies. Comprehensive and system-atic studies regarding cytokine release and threshold identificationas well as studies with non-activated and pre-activated cells couldgive more insight into the mode of action of field exposure and cells.Where appropriate, alternative animal models for, e.g., AD shouldbe considered, since such alternatives as Drosophila, Caenorhabdi-tis elegans and the zebrafish Danio rerio all have the advantage ofbeing susceptible to genetic modifications, have relatively short lifetimes, and are offering tools for high-throughput screening (Link,2005; Newman et al., 2007; Wentzell and Kretzschmar, 2010).In addition, their nervous system, although far simpler than thehuman one, is still complex enough to exhibit neurodegenerativetraits, including the signs of AD.

Beside the time perspective, another important research avenuewould be to pursue studies of the activating capacity of MF onmicroglia. This cell type is important for development of NDD ingeneral (Glass et al., 2010), and responding to A� with releaseof inflammatory cytokines (Cameron and Landreth, 2010). It isalso shown that environmental exposure-related neuroinflamma-tion caused by agents such as diesel exhaust particles act viamicroglia activation (Kraft and Harry, 2011). Co-exposure studiesusing microglia would thus be appropriate. As noted earlier in thisreview, there are studies indicating that at least short-term ELF-MFexposure has positive effects on neuronal survival and differentia-tion. Such findings need replication and extension but would if theyare correct indicate an important therapeutic potential of ELF-MF.

5. Conclusion

Our hypothesis has been that ELF-MF exposure can promotethe development of NDD via pro-inflammatory actions (see Fig. 1).

The investigated evidence do neither support nor contradict thishypothesis, since both in vivo and in vitro studies employ-ing MF-exposure rather indicate mild oxidative stress (modestROS increases and changes in antioxidant levels) and possiblyanti-inflammatory processes (decrease in pro-inflammatory andincrease in anti-inflammatory cytokines). A caveat is that availablestudies have not employed real long-term (e.g., life-long) exposuresat low field strengths, which would be a more realistic exposurescenario.

Accordingly, the few studies that specifically have investigatedNDDs or NDD relevant endpoints are also not supporting our initialhypothesis. Rather, effects of exposure are either lacking or indicat-ing positive effects on neuronal viability and differentiation. Onceagain, studies with realistic long-term exposures are lacking.

However, it cannot be ruled out at present that long-term MF-exposures, causing mild oxidative stress, are involved in promotionof NDD, especially AD. The existing experimental studies are notadequate for answering if there is a causal relationship betweenMF-exposure and AD, which is suggested in epidemiological stud-ies.

Conflict of interest

The authors declare that there are no conflicts of interest.

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