Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

Epigenetic dysregulation in various types of cells exposed to extremely low-frequency magnetic fields.

Epigenetic mechanisms regulate gene expression, without changing the DNA sequence, and establish cell-type-specific temporal and spatial expression patterns. Alterations of epigenetic marks have been observed in several pathological conditions, including cancer and neurological disorders. Emerging evidence indicates that a variety of environmental factors may cause epigenetic alterations and eventually influence disease risks. Humans are increasingly exposed to extremely low-frequency magnetic fields (ELF-MFs), which in 2002 were classified as possible carcinogens by the International Agency for Research on Cancer. This review summarizes the current knowledge of the link between the exposure to ELF-MFs and epigenetic alterations in various cell types. In spite of the limited number of publications, available evidence indicates that ELF-MF exposure can be associated with epigenetic changes, including DNA methylation, modifications of histones and microRNA expression. Further research is needed to investigate the molecular mechanisms underlying the observed phenomena.

Long INterspersed element-1 mobility as a sensor of environmental stresses.

Long INterspersed element (LINE-1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a "copy-paste" mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1-RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1-RTP is generally repressed; however, a number of observations collected over about 15 years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1-RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1-RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1-RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, and drugs), physical agents (ionizing and non-ionizing radiations), and experiential factors (voluntary exercise, social isolation, maternal care, and environmental light/dark cycles). These data come from in vitro studies on cell lines and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic.

Effect of electromagnetic field exposure on the transcription of repetitive DNA elements in human cells.

Repetitive DNA (RE-DNA) was long thought to be silent and inert; only recent research has shown that it can be transcribed and that transcription alteration can be induced by environmental stress conditions, causing human pathological effects. The aim of this study was to determine whether exposure to radiofrequency electromagnetic fields (RF-EMF) could affect the transcription of RE-DNA. To this purpose, three different human cell lines (HeLa, BE(2)C and SH-SY5Y) were exposed to 900 MHz GSM-modulated RF-EMF at specific absorption rate of 1 W/kg or to sham. After exposure, mRNA levels of RE-DNA were evaluated through quantitative real-time PCR. The following RE-DNA types were investigated: Long Interspersed nucleotide Element 1, DNA alpha satellite and Human Endogenous Retroviruses-like sequences. When comparing cells exposed to RF-EMF versus control samples, different results were found for the three cell lines evaluated, indicating that RF-EMF exposure can significantly affect RE-DNA transcription and that the effects strongly depend on the cellular context and the tissue type. Further studies are needed to elucidate which molecular mechanisms could be involved.

Long INterspersed nuclear Elements (LINEs) in brain and non-brain tissues of the rat.

Long INterspersed Element-1 (L1) is a transposable element that can insert copies of itself in new genomic locations causing genomic instability. In somatic cells, L1 retrotransposition activity is usually repressed but somatic L1 retrotransposition has recently been observed during neuronal differentiation. In this study, we evaluate whether L1 elements are differentially active in rat tissues during postnatal development. To this purpose, we quantified L1 in genomic DNA extracted from the olfactory bulb (OB), cerebellum (CE), cortex (CO) and heart (H). Each analysis was repeated on rats aged 7, 21 and 60 days. We found that L1 content in OB and CE tissue was significantly higher than H tissue, in rats of all three ages studied, suggesting that L1 activity could be modulated in postnatal development and neurogenesis.

Assessing the combined effect of extremely low-frequency magnetic field exposure and oxidative stress on LINE-1 promoter methylation in human neural cells.

Extremely low frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic", but their genotoxic effects are still unclear. Recent findings indicate that epigenetic mechanisms contribute to the genome dysfunction and it is well known that they are affected by environmental factors. To our knowledge, to date the question of whether exposure to ELF-MF can influence epigenetic modifications has been poorly addressed. In this paper, we investigated whether exposure to ELF-MF alone and in combination with oxidative stress (OS) can affect DNA methylation, which is one of the most often studied epigenetic modification. To this end, we analyzed the DNA methylation levels of the 5'untranslated region (5'UTR) of long interspersed nuclear element-1s (LINE-1 or L1), which are commonly used to evaluate the global genome methylation level. Human neural cells (BE(2)C) were exposed for 24 and 48 h to extremely low frequency pulsed magnetic field (PMF; 50 Hz, 1 mT) in combination with OS. The methylation levels of CpGs located in L1 5'UTR region were measured by MassARRAY EpiTYPER. The results indicate that exposures to the single agents PMF and OS induced weak decreases and increases of DNA methylation levels at different CpGs. However, the combined exposure to PMF and OS lead to significant decrease of DNA methylation levels at different CpG sites. Most of the changes were transient, suggesting that cells can restore homeostatic DNA methylation patterns. The results are discussed and future research directions outlined.

An evaluation of genotoxicity in human neuronal-type cells subjected to oxidative stress under an extremely low frequency pulsed magnetic field.

The possible genotoxicity of extremely low frequency magnetic field (ELF-MF) exposure is still a controversial topic. The most of the reported data suggests that it alone does not affect DNA integrity, but several recent reports have suggested that sinusoidal ELF-MF may increase the effect of known genotoxic agents. Only a few studies deal with non sinusoidal ELF-MF, including pulsed magnetic field (PMF), which are produced by several devices. The aim of this study is to investigate whether PMF exposure can interfere with DNA damage and repair in the presence of a genotoxic oxidative agent in neuronal type cells. To this purpose gamma-H2AX foci formation, which is a sensitive marker of DNA double strand breaks (DSB), was investigated at different points of time (1, 24, 48, 72h) after the H2O2 treatment (300µM for 1h) under PMF exposure (1mT, 50Hz) in human neuroblastoma BE(2)C cells. Moreover, cytotoxicity evaluation, by MTT assay and cell cycle analysis, was performed at various points of time after the treatment. Taken together, results suggest that PMF exposure does not interfere with genotoxicity and cytotoxicity induced by oxidative stress.

ELF magnetic fields tuned to ion parametric resonance conditions do not affect TEA-sensitive voltage-dependent outward K(+) currents in a human neural cell line.

Despite the experimental evidence of significant biological effects of extremely low frequency (ELF) magnetic fields (MFs), the underlying mechanisms are still unclear. Among the few mechanisms proposed, of particular interest is the so called "ion parametric resonance (IPR)" hypothesis, frequently referred to as theoretical support for medical applications. We studied the effect of different combinations of static (DC) and alternating (AC) ELF MFs tuned on resonance conditions for potassium (K(+)) on TEA-sensitive voltage-dependent outward K(+) currents in the human neuroblastoma BE(2)C cell line. Currents through the cell membrane were measured by whole-cell patch clamp before, during, and after exposure to MF. No significant changes in K(+) current density were found. This study does not confirm the IPR hypothesis at the level of TEA-sensitive voltage-dependent outward K(+) currents in our experimental conditions. However, this is not a direct disprove of the hypothesis, which should be investigated on other ion channels and at single channel levels also.

Cell-host, LINE and environment: Three players in search of a balance.

Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship.

Assessing LINE-1 retrotransposition activity in neuroblastoma cells exposed to extremely low-frequency pulsed magnetic fields.

Mobile genetic elements represent an important source of mutation and genomic instability, and their activity can be influenced by several chemical and physical agents. In this research we address the question whether exposure to extremely low-frequency pulsed magnetic fields (EMF-PMF) could affect the mobility of the human LINE-1(RP) retrotransposon. To this purpose, an in vitro retrotransposition assay was used on human neuroblastoma BE(2) cells exposed for 48h to 1mT, 50Hz PMF, or sham-exposed. Moreover, since it is well known that retrotransposition causes DNA double-strand breaks (DSB), an estimation of γ-H2AX foci, which is a marker of DNA DSB, was carried out on PMF- and sham-exposed samples. The results show that PMF-exposed cells had a lower number of both retrotransposition events and DNA DSB compared with sham-exposed samples. These results suggest that exposure to PMF can interfere with retrotransposition activity by inducing a decrease of retrotransposition events.

LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress.

Long interspersed element-1s (LINE-1 or L1s) are abundant retrotransposons that occur in mammalian genomes and that can cause insertional mutagenesis and genomic instability. L1 activity is generally repressed in most cells and tissues but has been found in some embryonic cells and, in particular, in neural progenitors. Moreover, L1 retrotransposition can be induced by several DNA-damaging agents. We have carried out experiments to verify whether L1 retrotransposition is affected by oxidative DNA damage, which plays a role in a range of human diseases, including cancer and inflammatory and neurodegenerative disease. To this purpose, BE(2)C neuroblastoma cells, which are thought to represent embryonic precursors of sympathetic neurons, have been treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay involving an episomal L1(RP) element tagged with enhanced green fluorescent protein. Our results indicate that hydrogen peroxide treatment induces an increase in the retrotransposition of transiently transfected L1(RP) and an increase in the expression of endogenous L1 transcripts. An increase of γ-H2AX foci and changes in the mRNA levels of MRE11, RAD50, NBN and ERCC1 (all involved in DNA repair) have also been found. Thus, oxidative stress can cause L1 dysregulation.

Effect of extremely low frequency magnetic field exposure on DNA transposition in relation to frequency, wave shape and exposure time.

PURPOSE: To examine the effect of extremely low frequency magnetic field (ELF-MF) exposure on transposon (Tn) mobility in relation to the exposure time, the frequency and the wave shape of the field applied. MATERIALS AND METHODS: Two Escherichia coli model systems were used: (1) Cells unable to express ß-galactosidase (LacZ(-)), containing a mini-transposon Tn10 element able to give ability to express ß-galactosidase (LacZ(+)) upon its transposition; therefore in these cells transposition activity can be evaluated by analysing LacZ(+) clones; (2) cells carrying Fertility plasmid (F(+)), and a Tn5 element located on the chromosome; therefore in these cells transposition activity can be estimated by a bacterial conjugation assay. Cells were exposed to sinusoidal (SiMF) or pulsed-square wave (PMF) magnetic fields of various frequencies (20, 50, 75 Hz) and for different exposure times (15 and 90 min). RESULTS: Both mini-Tn10 and Tn5 transposition decreased under SiMF and increased under PMF, as compared to sham exposure control. No significant difference was found between frequencies and between exposure times. CONCLUSIONS: ELF-MF exposure affects transposition activity and the effects critically depend on the wave shape of the field, but not on the frequency and the exposure time, at least in the range observed.

Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements.

Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C.

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response.

In our earlier experiments, we found that extremely low frequency magnetic fields (ELF-MF) affect heat shock protein (HSP) expression in wild type Escherichia coli cells. In the present work we investigate the ability of ELF-MF exposure to trigger an increase of DnaK and GroEL protein levels also in E. coli cells not exhibiting the classic heat shock response (HSR) when subjected to a 42 degrees C heat stress. We find that these cells, although lacking a HSR to heat shock treatment, show an enhancement of DnaK and GroEL protein levels after 30 or 90 min sinusoidal ELF-MF exposure (50 Hz, 1 mT). This result suggests that the HSP induction pathway triggered by ELF-MF exposure could be different from that elicited by heat shock treatment.

Synthesis of DnaK and GroEL in Escherichia coli cells exposed to different magnetic field signals.

The effects of extremely low frequency magnetic field (ELF-MF)(1 mT, 50 Hz) on the heat shock protein (HSP) synthesis in Escherichia coli were investigated. Two magnetic field signals were studied: sinusoidal (SMF) and pulsed square wave (PMF). It was found that bacteria exposed to SMF showed a significantly higher level of DnaK and GroEL proteins as compared to sham-exposed bacteria as revealed by Western blot, whereas a lower level was observed after PMF exposure. Similar results were obtained when bacterial cells were exposed to heat shock (HS) after ELF-MF exposure: again SMF and PMF resulted in an increase and in a reduction of HSP amount in comparison with sham control, respectively. In conclusion, the MF influences the synthesis of HSPs in E. coli in a way that critically depends on the signal characteristics.