Claudia Consales

Caterina Merla
email: caterina.merla@enea.it

Barbara Benassi

Tomás Garcia-Sanchez

Adeline Muscat

Franck M André

Carmela Marino

Lluis M Mir
email: luis.mir@cnrs.fr

Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role

Keywords: deposited energy density, extremely low frequency magnetic fields, ultra-short electric pulses, SH-SY5Y, immediate early genes ElectroMagnetic Field ELF-MF: Extremely Low Frequency Magnetic Field MPP + : 1-methyl-4-phenylpyridinium ECT: ElectroChemoTherapy EGT: ElectroGeneTherapy EthyleneDiamine-Tetra-Acetic acid BSA: Bovine Serum Albumin nanosecond miRNA: microRNA MFI: Mean Fluorescence Intensity Egr-1: Early Growth Response 1 TMP: TransMembrane Potential

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Introduction

There is wide interest in understanding electromagnetic field (EMF) effects in biology, both to enable a better comprehension of the interaction mechanisms for protection purposes of the exposed populations, and to establish innovative and advantageous medical treatments [START_REF] Belyaev | EUROPAEM EMF Guideline 2016 for the prevention, diagnosis and treatment of EMF-related health problems and illnesses[END_REF][START_REF] Saliev | Biological effects of non-ionizing electromagnetic fields: Two sides of a coin[END_REF]. A large number of studies, applying very different exposure modalities in terms of amplitudes, waveforms and frequencies (from direct current, up to tens of GHz) have been carried out in many experimental models, both in vitro and in vivo, experimental models, with a wide range of different outcomes. Most of these studies suffer of the lack of a well-characterized mechanisms of interaction for the observed effects [START_REF] Kim | Possible Effects of Radiofrequency Electromagnetic Field Exposure on Central Nerve System[END_REF][START_REF] Gherardini | Searching for the perfect wave: the effect of radiofrequency electromagnetic fields on cells[END_REF][START_REF] Simkó | Quality Matters: Systematic Analysis of Endpoints Related to "Cellular Life" in Vitro Data of Radiofrequency Electromagnetic Field Exposure[END_REF][START_REF] Consales | Electromagnetic fields, oxidative stress, and neurodegeneration[END_REF].

In the bio-electromagnetics research community, considerable attention is devoted to the extremely low frequency (ELF) electric and magnetic fields, due to their widespread distribution in everyday life (e.g. electrical utility power lines, house hold appliances, etc.) and to their use in approved medical treatments, in particular for brain stimulation (e.g. Transcranial Magnetic Stimulation and Deep Brain Stimulation). The therapeutic efficacy of these latter procedures is limited by the lack of knowledge of a clear action mechanism and effector pathways, at the molecular level and at the level of cells and tissues [START_REF] Consales | Electromagnetic fields, oxidative stress, and neurodegeneration[END_REF][START_REF] Lazzaro | A consensus panel review of central nervous system effects of the exposure to low-intensity extremely low-frequency magnetic fields[END_REF][START_REF] Consales | The epigenetic component of the brain response to electromagnetic stimulation in Parkinson's Disease patients: A literature overview[END_REF].

Several studies, aiming at understanding the effect of ELF-magnetic fields (ELF-MFs) on the nervous system, identified cell redox homeostasis as one of the exposure targets [START_REF] Consales | Electromagnetic fields, oxidative stress, and neurodegeneration[END_REF][START_REF] Manikonda | Extremely low frequency magnetic fields induce oxidative stress in rat brain[END_REF][START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF]. We have extensively characterized the response of neuronal-like cells (SH-SY5Y, human neuroblastoma cell line) to 50Hz, 1mT sinusoidal magnetic flux density (B), providing evidence of effects on the cellular redox system and epigenome [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. Indeed, we demonstrated that a 50 Hz, 1 mT MF, over 72 hours of continuous exposure, alters redox homeostasis by inducing an increase of ROS with subsequent protein carbonylation, which sensitizes cells to the neurotoxin 1-methyl-4phenylpyridinium (MPP + ) [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF]. We demonstrated further that the same MF exposure (50 Hz, 1 mT, up to 72 hours) downregulates the expression of microRNA-34b/c in SH-SY5Y cells and in mouse primary neurons by hypermethylating the miR promoter [START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. MicroRNA are short, non-coding and endogenously expressed RNA that play a pivotal role in post transcriptional regulation of gene expression [START_REF] Mohr | Overview of microRNA biology[END_REF]. miR-34b and miR-34c are targets of p53 and are involved in physiological cell differentiation by regulating the cell cycle and apoptosis [START_REF] Hermeking | The miR-34 family in cancer and apoptosis[END_REF][START_REF] Wu | Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis[END_REF]. Considering the pivotal role that these microRNAs play in normal cell homeostasis, it is not surprising that their action is often mis-regulated in pathological processes. In particular, miR-34b/c expression is downregulated in cancer and neurodegenerative diseases [START_REF] Rokavec | The p53/miR-34 axis in development and disease[END_REF].

In addition to these studies oriented toward risks evaluation and individuals protection, another area of active research involves the application of ultra-short (from millisecond down to hundreds of picoseconds) and intense (from few kV/m up to tens of MV/m) electric pulses to cells and tissues. In the clinic today already we find therapeutic procedures that use microsecond electric pulses with fields up to 100 kV/m -electrochemotherapy (ECT) [START_REF] Breton | Microsecond and nanosecond electric pulses in cancer treatments[END_REF][START_REF] Gehl | Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases[END_REF], electrogenetherapy (EGT) [START_REF] Mir | Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future[END_REF], and irreversible electroporation (IRE) [START_REF] Jiang | A review of basic to clinical studies of irreversible electroporation therapy[END_REF]. Nanosecond-pulses-based medical application, with field greater than 1 MV/m, include nerve and muscle stimulation [START_REF] Casciola | Damage-free peripheral nerve stimulation by 12-ns pulsed electric field[END_REF] and new oncologic treatments [START_REF] Nuccitelli | Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death[END_REF].

For these high field amplitude exposure conditions (microsecond and nanosecond electric pulses), the primary effect on cells is believed to be permeabilization of cell membranes. The pulsed external field induces a large voltage across the plasma membrane, and this causes the formation of conductive lipid pores (electroporation). Under the right conditions this is followed by a persistent permeabilization that results from structural and chemical rearrangements of membrane constituents and other membrane-associated species (electropermeabilization) [START_REF] Breton | Investigation of the chemical mechanisms involved in the electropulsation of membranes at the molecular level[END_REF][START_REF] Vernier | Nanoelectropulse-driven membrane perturbation and small molecule permeabilization[END_REF]. This breakdown of the membrane barrier function triggers a set of secondary events, such as cell apoptosis -with or without caspase activation- [START_REF] Beebe | Nanosecond pulsed electric field ablation of hepatocellular carcinoma[END_REF][START_REF] Pakhomova | Two modes of cell death caused by exposure to nanosecond pulsed electric field[END_REF], promotion of intracellular calcium oscillations [START_REF] Hanna | Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions[END_REF][START_REF] De Menorval | Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells[END_REF], alteration of ion conductance through the membrane [START_REF] Hristov | Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field[END_REF][START_REF] Yoon | Enhanced Monitoring of Nanosecond Electric Pulse-Evoked Membrane Conductance Changes in Whole-Cell Patch Clamp Experiments[END_REF], cytoskeleton rearrangements, with concomitant cell blebbing and fusion [START_REF] Salomskaite-Davalgiene | Extent of cell electrofusion in vitro and in vivo is cell line dependent[END_REF], and reactive oxygen species production [START_REF] Bonnafous | The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane[END_REF][START_REF] Pakhomova | Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media[END_REF]. Nanosecond electric pulses with amplitudes under the electroporation threshold may also affect ion channels gating, which would enable electromodulation of cell physiology without porating damage to the cell membrane. For example, by activating voltage-gated sodium channels, low-field electric pulses could induce action potential generation. [START_REF] Casciola | Damage-free peripheral nerve stimulation by 12-ns pulsed electric field[END_REF].

Despite increasing evidence on the effects on cells physiology exerted by nanosecond and microsecond electric pulses that do not cause cell electroporation, the molecular pathways activated by such stimulations have not yet been extensively investigated and are still poorly understood.

Hence, in the study reported in this article, we pursued different objectives. First, we compared the biological effects triggered by nanosecond and microsecond electric pulses in SH-SY5Y human neuroblastoma cells to those elicited by a chronic sinusoidal 50 Hz exposure. Applied electric pulse parameters were selected to deposit the same energy density than the one cumulated during a 24 and 48 hours, 50 Hz, 1 mT, MF exposure [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. In this way, we aimed at clarifying the role of the Deposited Energy Density (DED) in the induction of redox modulation, cell death and epigenetic regulation (miR-34b/c expression) for very different exposure durations. magnetic component of the exposure versus the electric one. This study was carried out by exposing SH-SY5Y cells to an electric field of the same amplitude as that induced by the 50 Hz, 1mT MF.

Finally, we characterized SH-SY5Y cells responses to a single microsecond or nanosecond electric pulse with increasing levels of DED. In other published analyses of electric pulse effects, different DEDs were applied, different effects were observed, and scaling rules were proposed based on the magnitude of a given endpoint (e.g. electroporation achievement) [START_REF] Pakhomov | Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane[END_REF][START_REF] Sano | In-vitro Bipolar Nano-And Microsecond Electro-Pulse Bursts for Irreversible Electroporation Therapies[END_REF][START_REF] Ibey | Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells[END_REF][START_REF] Maček-Lebar | Cell electropermeabilization to small molecules in vitro: control by pulse parameters[END_REF][START_REF] Schoenbach | A scaling law for membrane permeabilization with nanopulses[END_REF]. Here, we use DED as a normalizing physical quantity to enable the comparisons among different electromagnetic exposures.

This study provides a first description of different biological effects in the same cell type produced by different exposure modalities with comparable energies, which we expect will lead to a better understanding of the mechanisms underlying the response of biological systems to magnetic and electric stimulation.

Materials and methods

Chemicals

Culture media, serum and supplements, trypsin-EDTA, were obtained from Gibco (Paris, France). Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), ethylenediamine-tetra-acetic acid (EDTA), MPP + , Propidium Iodide (PI), RNAse A, Trypan blue solution (0.4%) and Triton X-100 were purchased from SIGMA-Aldrich (St Louis, MO, USA).

The fluorescent probes dihydroethidium (hydroethidine, DHE) and 20,70-dichlorofluorescein diacetate (H2-DCFDA) were obtained from Molecular Probes (Thermo Fisher Scientific, Waltham, MA, USA).

Exposure systems

To make a comparison, in terms of effects, between the exposures performed with the magnetic field (1 mT, 50 Hz exposure lasting 24 hour) and the electric pulses, the pulse characteristics were chosen in order to have a deposited total energy density identical to the energy density (around 0.015 J/m 3 ) deposited in the medium during the long magnetic field exposures applied to the same cells in our previous experiments [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. The electromagnetic energy density deposit (J/m 3 ) in the medium containing the cell monolayer was computed by equation ( 1)

𝐷𝐸𝐷 = 𝜎𝜏𝐸 2 , (1).
where σ is the conductivity of the cell culture medium equal to 1.5 S/m and  represents the duration of the exposure expressed in seconds. For the magnetic field stimulation  was equal to 24 hour (86400 s) while it was 100 s or10 ns for the electric pulses exposures.

The electric field indicated with E (eq. 1) is the one induced by the magnetic stimulation and computed by numerical simulations (Comsol Multiphysics v. 5) in the actual experimental conditions as already described in [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF]. The induced electric field amplitude in the cell monolayer (at the bottom of the Petri dish) was 0.3 mV/m (peak value) with an homogeneity larger than 95%. The electric field intensity required when electric pulses were applied was computed with equation (1), as in this case we decided that the total DED should have the same value as the one deposited by the magnetic stimulation. Electric pulses of higher energies were also used in order to globally evaluate the cell responses in a dose dependent manner. The whole set of stimulation parameters is listed in Table 1. 1 The whole set of stimulation conditions for microsecond and nanosecond pulsed electric fields.

Conditions Applied Voltage (V) DED (J/m 3 ) E field (V/cm)
Adherent SH-SY5Y cells were exposed in 6-cm Petri dishes, where a pair of stainless-steel electrodes (1 mm thick each) were placed parallel at a distance of 1 cm from each other (Fig. 1 panel A)., Polydimethylsiloxane (PDMS) holders were fabricated (to get an exposure volume of 5×1×0.5 cm 3 ) and included in the Petri dishes. They served to accurately maintain the chosen electrode distance and the electrodes parallelism in order to assure controlled exposure conditions In Fig. 1A, the fabricated PDMS holders are presented together with photos of the Petri dishes with the metallic electrodes placed into the PDMS spacers for the microsecond and nanosecond pulses exposures. The metallic electrodes were connected to a Cliniporator (IGEA, Carpi, Italy) pulse generator to deliver microsecond pulses (duration of 100 µs, rise/fall times 10 µs) of amplitudes equal to 1000 and 600 V. A Tektronix generator (AFG3251) was used to deliver microsecond pulses (duration of 100 µs) with the lowest amplitudes (10 and 0.1 V). A FID Technology generator (model, FID GmbH, Burbach, Germany) was used to deliver nanosecond pulses (duration of 10 ns, rise/fall time of 1 ns) of 1000 V or 10 V. When nanosecond pulses of 10 V were applied to cells an external high voltage attenuator 245NMFFP-100 Barth Electronics (with a linear attenuation factor of 100) was included in the pulse delivery line between the pulse generator and the exposure device. In Fig. 1 B, examples of signal waveforms delivered to cells are presented to demonstrate the absence of artifacts and spurious reflections. These representative examples of the delivered waveforms demonstrate the suitable matching of the generators to the biological load. This aspect was especially relevant when 10 ns electric pulses were applied to SH-SY5Y cells. In this case, a precise volume (1.3 mL) of culture medium was pulsed during the experiments (height of culture medium of 1.3 mm) to suitably match the impedance of the biological load to the 50 ohm corresponding to the output impedance of the pulse generators. Thus matching was obtained thanks to the choice of the height of the biological solution enclosed between the electrodes, avoiding the use of specific low conductive buffers usually needed to match the biological load at 50 ohm [START_REF] Merla | A 10-High-Voltage Nanosecond Pulse Generator[END_REF][START_REF] Silve | Nanosecond-Duration Electric Pulse Delivery In Vitro and In Vivo: Experimental Considerations[END_REF].

In order to separately study the effects of the application of a continuous electric field equivalent to the one induced by the 1mT sinusoidal (50 Hz) magnetic field in the samples, sinusoidal 50 Hz electric field of very low intensity was directly applied to cells (Fig. 1 C). The setup consisted in a voltage divider (1:1000) made with a precision decade divider (Vishay, CNS 471) and an operational amplifier connected to a waveform generator (Agilent 33250A).

According to the estimations obtained in the electromagnetic simulations [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF], two different 50 Hz electric fields with amplitudes Epeak of 0.6 mV/m and 0.3 mV/m were applied, respectively, for 24 or 48 hours, using stainless steel parallel plate electrodes. A PDMS insert was placed in the Petri dishes to ensure the correct positioning of the electrodes, as schematically shown in the left panel of Fig. 1C. In the central panel a picture of the exposure setup for such kind of signals is also reported as well as the corresponding waveform (right panel). Two days before pulsed electric field stimulation, 3×10 5 cells were plated inside the Polydimethylsiloxane (PDMS) insert. After stimulation, cells were harvested, according to the specific biological experiment to be carried out; in any case, they were maintained in culture for no longer than 48 hours. For each experiment and for each experimental condition, sham samples were prepared by plating the cells in the same and electrodes were placed in the PDMS insert to mimic the exposure as in the exposed samples (at the same temperature and for the same time), but no electric stimulation was applied.

For continuous exposure to sinusoidal electric field, 8×10 5 cells were plated 48 hours before the beginning of the exposure, and ROS were evaluated 24 and 48 hours after the beginning of the exposure.

Cell morphology analysis

Cells were seeded on coverslips put inside PDMS insert two days before the stimulation. Twenty-four hour after the electric field stimulation, culture medium was removed and, after one wash with PBS, cells were fixed for 20 minutes with 4% paraformaldehyde (Sigma) and rinsed with PBS. Just after the washing, cells were incubated for 30 min with wheat germ agglutinin (Alexa Fluor 488 Thermo Fisher, 1:300) to stain the plasma membranes. After PBS wash, cells were permeabilized with 0.1 % Triton X100 (Sigma) for 5 min, washed with PBS, and incubated for 30 min with Alexa

Fluor 568 Phalloidin (1:500, Thermo Fisher Scientific). After several washings with PBS, a mounting solution containing DAPI (Vectashield, Vector Laboratories, USA) was applied to the slides to stain nuclear RNA/DNA, and the slides stored at 4 °C until images acquisition was performed using a confocal microscope (Leica Microsystem SP8).

Flow-cytometric analysis of ROS generation

Flow-cytometry analyses were performed using a C6 flow cytometer (BD Accuri, Becton Dickinson). Forward (FSC-H) and side (SSC-H) scatterings were used to exclude cellular debris from the analysis and to gate the intact/healthy cells.

To quantify the oxidative stress, cells were stained with either DHE (detection of superoxide radicals) or H2-DCFDA (measurement of H2O2) [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF]. Cells were harvested on ice, washed twice in cold PBS, and re-suspended in 5 μM DHE in PBS for 20 min at 37 °C in the dark, or in 5 μM H2-DCFDA in PBS for 20 min at 37 °C in the dark. After a final wash in PBS, cells were immediately transferred into a tube kept on ice until the flow cytofluorometric analysis. In each measurement, a minimum of 10.000 cells were analyzed. Data were acquired and analyzed using the BD Accuri C6 software (Becton Dickinson). For fluorescence data analysis, the mean fluorescence intensity (MFI) was calculated for each sample as the mean fluorescence value in the channel of the probe-labelled cells minus the mean fluorescence value of unstained cells.

Assessment of cell permeabilization

Cell permeabilization was assessed by fluorescent microscopy. The treated SH-SY5Y cells were trypsinized and resuspended in DMEM, and then Yo-Pro-1 (Life Technologies) was added, at a final concentration of 3 μM, 5 minutes before the acquisition of the first image. To test cell permeabilization we delivered different pulse protocols consisting in 1 (Table 1) or in 8 electric pulses of 100 μs duration, at a repetition rate of 1 Hz, with field amplitude of 600 or 1000

V/cm respectively.

Images were acquired at λ = 510 nm using an inverted microscope (AxioVert 100, Zeiss) with an exposure time of 300 ms using a 10× objective). Three independent experiments were performed for each tested condition.

Western Blotting

Cells were re-suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % NP-40, and 12 mM Na-deoxycholate (Sigma) supplemented with protease and phosphatase inhibitors cocktail (Merck, Germany). Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C to discard cellular debris. Protein concentration was determined by the Lowry protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by SDS-PAGE and transferred to 0.2 µm nitrocellulose membranes (GE Healthcare Life Science, Europe) that were probed with anti-PARP1 (Cell signaling, 1:1500) and anti-beta actin (Santa Cruz, 1:2000) antibodies. After immuno-staining with appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (SIGMA), bands were revealed using a Fluorchem Imaging system (Alpha Innotech, San Leandro, CA, USA) and the Amersham ECL detection system (GE Healthcare Life Science), and then quantified by densitometry. As positive control for apoptosis detection by immunostaining, the proteins extracted from cells exposed for 48 hours to etoposide (VP-16) at a final concentration of 5 µM were included in all the gels together with the experimental samples.

MicroRNA Expression Analysis

Analysis of mature miRNA expression was carried out on total RNA extracted with the miRcury RNA isolation kit (Exiqon). Ten nanograms of total RNA were retro-transcribed by the miRcury LNA universal RT microRNA kit (Exiqon) and the cDNA diluted 1:80 and amplified by the miRcury LNA SYBR green master mix and miR-specific LNA PCR primer sets (Exiqon), according to manufacturer's instructions. All reactions were run in quadruplicate and the relative abundance of each specific miR (34b, and 34c) was normalized to small nucleolar RNA (U6) by applying the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. Three independent experiments were performed for each tested condition.

Genes expression

Total RNA was extracted with the miRcury RNA isolation kit (Exiqon). The amount and purity of the extracted RNA was evaluated using an optic fiber spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA) calculating the 230/260 nm and 260/280 nm absorbance ratios. Five hundred nanograms of total RNA were retrotranscribed with random primers into total cDNA by TaqMan® Reverse Transcription Reagent (Applied Biosystems, Thermo Fisher Scientific), according to manufacturer's indications. Analysis of the genes expression was carried out with 1 μL of cDNA using SYBR Green master mix (Thermo Fisher) and a 7500 Real-Time PCR System (Thermo Fisher Scientific). PCR conditions were as follows: 45 cycles at 95 °C for 30 s and 60 °C for 30 s.

All reactions were run in triplicate and the relative abundance of the specific mRNA levels was calculated by normalizing to the 18S rRNA expression using the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. Three independent experiments were performed for each tested condition.

The complete list of and the used primer sequences is displayed in Table 2.

Results

Ineffective DED role in inducing cell redox impairment, cell apoptosis, and miR-34b/c modulation

SH-SY5Y neuroblastoma cells were exposed either to a single 100 µs pulse of 0.1 V (induced electric field in the sample: 0.1 V/cm; DED: 0.015 J/m 3 ) or to a single 10 ns pulse of 10 V (induced electric field in the sample 10 V/cm; DED: 0.015 J/m 3 ). ROS generation was analyzed just after the electric pulse delivery (T0) and at three different time points: 1 hour, 6 hours and 24 hour (respectively T1, T6, T24). ROS were also evaluated in non-pulsed control samples.

The absence of detectable hydrogen peroxide production (fluorescence from H2-DCFDA) indicated that neither the microsecond electric pulse nor the nanosecond electric pulse exposure induced changes in ROS generation, at any considered time point (Fig. 2A,D).

To verify whether the delivery of the nanosecond and/or microsecond electric pulses, as described above, might induce apoptosis in SH-SY5Y cells, PARP-1 cleavage was assessed 24 and 48 hours after the pulse delivery. As shown in Fig. 2B and2E, neither the 100 µs at 0.1 V/cm nor the 10 ns at 10 V/cm pulses triggered an apoptotic process, which was clearly activated in the included positive controls (etoposide administration for 24 or 48 hours).

To evaluate the effect of the applied electric pulses on miRNA-34b and miRNA-34c, their expression level was assessed at 1, 6 and 24 hour after the pulse delivery. At all the time points analyzed, no significant variation was detected in the microRNAs expression (Fig. 2C,F).

50 Hz sinusoidal electric stimulation does not alter SH-SY5Y cells redox homeostasis

As described in [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF], 50 Hz, 1mT MF exposure altered SH-SY5Y redox balance by increasing ROS production and proteins carbonylation. To verify whether this redox effect might be related to the electric field component (induced by the 50 Hz, 1mT MF exposure), SH-SY5Y cells were exposed up to 48 hours to a sinusoidal electric field of 0.3mV/m, an intensity equivalent to that induced by the 50 Hz, 1mT MF exposure. The intensity of the induced electric field in the cell monolayer was numerically computed and fully described in [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF], as also reported in the Materials and Methods section. Cells were also exposed to a higher electric field 0.6 mV/m.

ROS production was evaluated at 24 and 48 hours post exposure, and no increase in ROS production was detected at either time points (Fig. 3) for an electric field intensity of 0.3 mV/m. Similar results were obtained for the higher electric field intensity (data not shown).

Pulsed electric fields affect immediate early genes expression

Since the energy density used in the previous section was not able to induce any biological response in terms of redox balance, cell death induction and microRNA expression modulation, higher energy densities were applied to the neuroblastoma cells, by delivering microsecond and nanosecond electric pulses of higher field intensities, in order to explore if any of the above-mentioned biological effects could be induced.

These higher doses were a single electric pulse of 100 μs, at three different field intensities (10, 600 and 1000 V/cm) or one electric pulse of 10 ns at 1000 V/cm.

We first assessed if these more intense electric pulse conditions were able to permeabilize the cell membrane using YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity. Just after pulse application, a weak intracellular stain was visible when a single 100 μs pulse at 600 V/cm was applied (Supp Fig. 1A). The fluorescence was more evident when a single 100 μs pulse at 1000 V/cm was applied. YO-PRO-1 signal very much increased when 8 repeated 600 or 1000 V/cm were applied, demonstrating a complete and strong permeabilization of the samples. No YO-PRO-1 staining was detected in cells exposed to a single 10 ns pulse at 1000 V/cm. Adherent cells, located in the areas of the cell layer not exposed to the pulses, were negative for the stain (Supp. Fig. 1B).

Supplementary figure 1 YO-PRO-1 staining after the delivery of pulses lasting 100 μs with an intensity of 600 V/cm (A) and 1000 V/cm (B). YO-PRO-1 uptake after a single pulse and 8 repeated electric pulses at 1 Hz is shown in the left and right panels of the figure, respectively. All images were collected with an exposure time of 300 milliseconds Finally, in order to assess the immediate molecular cell response to the electric pulses in all the analyzed conditions (both low and high intensities), the expression of two immediate early genes, c-fos and egr-1 (early growth response protein 1), was evaluated in cells, up to 6 hours after the exposure. As displayed in Fig. 4, all the different pulse protocols were able to induce a significant increase in egr-1 expression, which reached its highest level at 1 hour after the exposure, while at 6 hours the egr-1 expression was comparable or significantly lower than in the not-pulsed controls.

c-fos was significantly increased 1hour post stimulation for all the pulse exposure conditions, except for the lowest microsecond pulse exposure amplitude (0.1 V/cm). Six hours after the pulse application, c-fos mRNA level was again comparable or significantly lower than in the unexposed controls (Fig. 4).

One pulse, 100 μs, 1000 V/cm exposure induces cell membranes fusion and affects cell morphology

Looking at the cells 24 hour after the exposure to the highest energy density condition, we identified some cells which, viewed with phase contrast microscope, appeared fused (data not shown). For this reason, we investigated whether our conditions of pulse stimulation could effectively induce cell fusion.

We evaluated the cells morphology 24 hour after exposure for all pulse conditions. Cells were examined with fluorescence microscopy after staining with fluorescence derivative of wheat germ agglutinin, which binds to glycoproteins of the cell membrane, and phalloidin, a probe for filamentous actin. Cells stimulated with nanosecond pulses did not show any change in morphology compared to the non-pulsed samples (CTRL, Fig. 5). Among the microsecond pulse exposures, only at the highest pulse intensity, 1000 V/cm, we did observe formation of giant cells with fused membranes, and in these cells the cytoskeleton did not merge (Fig. 5, 100 μs, 1000 V/cm).

Fig. 5 Confocal fluorescence microscopy images of SH-SY5Y cells pulsed under all the different conditions and stained

24 hour later for membrane (with wheat germ agglutinin, in red), actin F (with alexa fluor, in green) and DNA (with DAPI, in blue)

Short electric pulses at larger energy densities do not affect redox homeostasis, cell apoptosis or miR-34b and miR 34c expression

To investigate whether exposure conditions higher than 100 µs, 0.1V and 10 ns, 10 V could influence redox homeostasis, ROS production was also evaluated under these exposure conditions, just after the pulse application (T0) and 1, 6, and 24 hour after it.

With nanosecond exposures (10 ns, 1000 V), we observed no variation in ROS production (data not shown). When microsecond pulses were applied, no ROS modulation was observed at T0, T1, and T24 (data not shown). At T6, only at the highest amplitude, a small, not statistically significant, increase in ROS generation was seen (Fig. 6).

Fig. 6 ROS production assessment 6 hours after one 100 µs electric pulse at various field amplitudes. ROS were evaluated using the H2DCFDA probe. Values are the means ± S.D. of three independent experiments Since nanosecond and microsecond pulses applied at the same energy condition as that associated with 50 Hz, 1 mT magnetic field exposures did not activate cell apoptosis, we investigated whether higher energy density conditions could promote such an effect. Analyzing PARP-1 cleavage 24 and 48 hours after exposure of cells to these electric pulses, we observed no apoptotic signal for all the different exposure conditions considered (Fig. 7), even for the strongest one that led to a complete cell permeabilization (i.e., 1 pulse, 100 µs, 1000 V/cm). We also estimated miR-34b and miR-34c expression in cells exposed to one pulse of 100 μs, 1000 V/cm at 1, 6 and 24 hour after the exposure. Such exposure condition is the strongest used in our experiments. Also, in this case no significant modulation of both microRNAs was detectable (Supp. Fig. 2).

Discussion

Role of the energy density and of the electric field amplitude

Despite the large number of in vitro and in vivo experimental studies focused on the effects of ELF-MF exposure, the specific interaction mechanisms underlying such effects, at the cellular and molecular levels, have not been elucidated so far. Several studies have demonstrated that ELF-MFs can modulate cell redox homeostasis [START_REF] Saliev | Biological effects of non-ionizing electromagnetic fields: Two sides of a coin[END_REF][START_REF] Consales | Electromagnetic fields, oxidative stress, and neurodegeneration[END_REF][START_REF] Kıvrak | Effects of electromagnetic fields exposure on the antioxidant defense system[END_REF], but the modalities for triggering such effects are still a matter of debate.

Unraveling how ELF-MFs interact with biological systems has a fundamental importance both for environmental and occupational safety and for the development of new applications. As widely reported, there is a broad public concern about the spread of electromagnetic field sources in the environment and a possible association between ELF-MF exposure and cancer. These fields were classified in the group 2B, "possibly carcinogenic to humans", by the International Agency for Research on Cancer (IARC) in 2002 [START_REF]Nonionizing radiation Part I: static and extremely low frequency (ELF) electric and magnetic fields[END_REF]. Up to date, no experimental data supported this hypothesis. On the other hand, both low and high frequency EMF are increasingly being applied in the field of biomedical therapies, with protocols that have arisen from experience only, without the benefit of optimization guided by a predictive theory. Understanding which component of these fields is responsible for the observed effects and what are the underlying molecular pathways would surely lead to improvements in therapeutic efficacy and likely inspire new applications.

We have previously demonstrated that long-term exposure to a 1mT sinusoidal (50Hz) MF affects the redox homeostasis of SH-SY5Y cells by increasing ROS production and depleting the cell antioxidant reserve, which makes the cells more sensitive to the pro-oxidant neurotoxin MPP + . Furthermore, 24 and 48 hours of such an exposure induces a decrease in expression of both miR-34b and miR-34c [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. miR-34b/c share the same promoter [START_REF] Hermeking | The miR-34 family in cancer and apoptosis[END_REF], and in our experiments we showed that the down-regulation of these two microRNAs was p53-independent and due to another epigenetic event, the hyper-methylation of their promoter.

In the present work, starting from our findings regarding redox balance and epigenome modulation in the neuroblastoma cell line SH-SY5Y following a 50 Hz, 1 mT exposure, we report on the biological effects -on the same cell line-induced by nanosecond or microsecond electric pulses with dose parameters selected to deliver the same energy density as 24 hour, 50 hz, 1 mT magnetic field exposure studied previously [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. We applied electric pulses lasting either 100 μs, (a standard pulse length in electrochemoterapy protocols), or 10 ns, because they are among the shortest electric pulses that can be commercially generated and they are potential modality for further clinical applications [START_REF] Casciola | Damage-free peripheral nerve stimulation by 12-ns pulsed electric field[END_REF][START_REF] Nuccitelli | Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death[END_REF].

The rationale for including nanosecond and microsecond electric pulses in this study came also from curiosity about unexplored biological responses induced by this kind of stimulation. The effects of pulsed electric fields on biological cells and tissues have been studied for more than forty years [START_REF] Sher | On the possibility of nonthermal biological effects of pulsed electromagnetic radiation[END_REF][START_REF] Farndale | Effects of low-amplitude pulsed magnetic fields on cellular ion transport[END_REF], and it has been established that the most common outcome of these exposures is modification of the plasma membrane, making it more permeable to molecules which, otherwise, cannot cross this barrier [START_REF] Breton | Microsecond and nanosecond electric pulses in cancer treatments[END_REF][START_REF] Joshi | Bioelectric effects of intense ultrashort pulses[END_REF][START_REF] Batista Napotnik | Effects of high voltage nanosecond electric pulses on eukaryotic cells (in vitro): A systematic review[END_REF]. Electric pulses of sufficient magnitude and duration induce a transmembrane potential (TMP), that causes nanoscale rearrangements of the membrane. If the applied field is not too intense and does not last too long, the membrane becomes transiently permeable, without affecting cell viability [START_REF] Teissie | Electropermeabilization of the cell membrane[END_REF].

The ability of nanosecond and microsecond electric pulses to modify the permeability of the plasma membrane is routinely used in cell biology to transfer DNA into cells [START_REF] Mir | Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future[END_REF]. Furthermore, their application enables an effective cancer therapy, electrochemoterapy, by increasing anticancer drugs penetration into tumor cells [START_REF] Breton | Microsecond and nanosecond electric pulses in cancer treatments[END_REF]. Cellular modifications induced by microsecond and nanosecond electric pulses under the electroporation threshold dose, however, have not been clearly identified and characterized. A deeper understanding of cellular responses to both magnetic fields and electric pulses would greatly enhance the range of their application in therapeutics.

To compare the biological effects induced by microsecond and nanosecond electric pulses with those observed after exposing SH-SY5Y to a 50 Hz, 1 mT magnetic field, we focused on three biological targets: redox homeostasis, cell viability, and microRNA expression modulation. In our previous work, we observed that an ELF-MF exposure can affect redox balance and miR-34 b/c expression, but contributes to the induction of apoptosis only when the magnetic field is applied before MPP+ neurotoxin administration [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF].

Here, we demonstrate that exposure to a single 10 ns at 10 V/cm pulse or a single 100 µs 0.1 V/cm pulse, with energy densities equivalent to that during 24 hour, 50 Hz, 1 mT magnetic field, does not affect redox homeostasis in SH-SY5Y cells at any of the considered time points (from T0, just after the pulse application, up to 24 hour after the exposure). Furthermore, these pulses do not induce cell death and do not affect miR-34 b/c expression, which is downregulated in SH-SY5Y cells exposed to a 50 Hz, 1 mT magnetic field [START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. Delivery of a certain DED is, therefore, not sufficient to produce the responses seen after the EMF-MF exposure.

One important exposure aspect to be considered is the duration of the exposure itself. The effects detailed in [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF] were obtained for exposures lasting at least 24 hour, while nanosecond and microsecond electric pulse delivery with the same DED lasts only 10 ns and 100 μs, respectively, a duration which could be too short to promote a sustained cell response both in terms of oxidative stress or epigenetic modulation.

Another possible contributing factor is the electric field induced by the magnetic field exposure (Faraday's Law). To investigate the possible electric field involvement, we exposed SH-SY5Y cells to a sinusoidal 50 Hz electric field of the same intensity as that induced by 50 Hz, 1 mT magnetic field. No modulation of ROS production was detected, indicating that the magnetically induced electric field is not responsible for the biological effects elicited by the 50 Hz ELF-MF exposure, and suggests that time-varying magnetic field is the possible causative agent.

Nanosecond and microsecond pulsed electric fields and immediate early genes (IEG) expression

We then explored the effects induced by the delivery of nanosecond and microsecond electric pulses at higher energy densities, up to the dose that results in plasma membrane poration. To search for the molecular responses that are activated by these intermediate-dose electric pulse exposures, where the field amplitude is lower or at the limit of the threshold for the cell membrane permeabilization achievement, cells were exposed to one single pulse at increasing electric field amplitudes up to 1000 V/cm, corresponding to an energy density of 1.5x10 6 J/m 3 . This is the energy density at which membrane poration becomes clearly evident in our SH-SY5Y cell model.

To identify an immediate molecular response induced by these pulses on SH-SY5Y cells, the expression of two IEG, c-fos and egr-1, was measured 1 and 6 hours after the pulses delivery. IEGs can be activated and transcribed within minutes after stimulation, even in the presence of protein synthesis inhibitors, and they are stimulated in response to both cell-extrinsic and cell-intrinsic signals, in particular to stress. IEG expression is usually quick and transient, and protein products are usually unstable and quickly degraded without ubiquitination. IEGs usually code for transcription factors which then can activate secondary response genes [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF].

Both Egr-1 and c-Fos are transcription factors which can be activated by a plethora of stimuli [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF]. In the nervous system, their expression is restricted to the brain, where they modulate synaptic plasticity and neurite outgrowth [START_REF] Duclot | The Role of Early Growth Response 1 (EGR-1) in Brain Plasticity and Neuropsychiatric Disorders[END_REF][START_REF] Okuno | Regulation and function of immediate-early genes in the brain: beyond neuronal activity markers[END_REF][START_REF] Jaworski | c-Fos and neuronal plasticity: the aftermath of Kaczmarek's theory[END_REF]. Electrical stimuli are able to induce the activation of these two genes. In particular, continuous electrical stimulation of neuronal and neuron-like cells was demonstrated to increase neurite outgrowth through the up-regulation of both transcription factors [START_REF] Yan | Electrical stimulation induces calcium-dependent neurite outgrowth and immediate early genes expressions of dorsal root ganglion neurons[END_REF][START_REF] Fields | Regulation of neurite outgrowth and immediate early gene expression by patterned electrical stimulation[END_REF][START_REF] Chang | Electrical stimulation promotes nerve growth factorinduced neurite outgrowth and signaling[END_REF].

Under our experimental conditions, there was a clear stimulation of IEG expression. egr-1 expression was statistically up-regulated 1 hour after exposure for all conditions of the applied pulsed electric field, using microsecond or nanosecond pulses, even for the lowest applied DED. At 6 hours egr-1 expression returned to the basal level, or to levels significantly lower. c-fos transcript displayed a similar statistically significant up-regulation, except when cells were pulsed at the lowest energy (i.e. 100 μs, 0.1 V/cm).

It is very interesting to notice that, for the same energy transferred to the sample, the two pulse conditions 100 μs at 0.1 V/cm and 10 ns at 10 V/cm, produce two different patterns of c-fos expression. This leads to the hypothesis that modulation of c-fos expression by pulsed electric fields is independent of the DED. We can speculate that these two exposure modalities (microsecond and nanosecond electric pulses) elicit different bio-responses because nanosecond electric pulses have bioelectrical properties different from microsecond ones, such as intracellular penetration and larger electric field amplitude, on top of their duration. [START_REF] Nuccitelli | Application of pulsed electric fields to cancer therapy[END_REF]. It is worth pointing out that, since the cells were not electroporated by these pulse doses, the differences in the responses to microsecond and nanosecond pulses cannot be accounted for by different modes of permeabilization.

Our findings demonstrate that the cells respond to an ultrashort electric stimulation below the electroporation threshold. IEG activation in SH-SY5Y cells under, these two stimulation conditions, has not previously investigated, so our results cannot be directly compared to any of the studies conducted so far [25-28, 33, 34]. Both egr-1 and c-fos can be activated by an elevation of intracellular calcium [START_REF] Thiel | Egr-1-A Ca(2+)-regulated transcription factor[END_REF][START_REF] Morgan | Role of ion flux in the control of c-fos expression[END_REF], in particular after cell depolarization, when voltagedependent calcium channels are activated [START_REF] Thiel | Egr-1-A Ca(2+)-regulated transcription factor[END_REF][START_REF] Sheng | Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB[END_REF]. It seems possible then that, for observations reported here, even when nanosecond and microsecond electric pulses are applied below the electroporation threshold in SH-SY5Y cells, calcium channels could be activated, leading to IEG up-regulation.

Nanosecond and microsecond pulsed electric fields and cell morphology

A well-known effect induced by nanosecond and microsecond pulses is cell fusion [START_REF] Salomskaite-Davalgiene | Extent of cell electrofusion in vitro and in vivo is cell line dependent[END_REF][START_REF] Mekid | In vivo cell electrofusion[END_REF][START_REF] Rems | Cell electrofusion using nanosecond electric pulses[END_REF][START_REF] Li | Cell electrofusion based on nanosecond/microsecond pulsed electric fields[END_REF]. The ability of electric pulses to induce membrane fusion has led to applications like hybridoma formation [START_REF] Ke | Electrofusion by a bipolar pulsed electric field: Increased cell fusion efficiency for monoclonal antibody production[END_REF], where this technique is more effective and less toxic than standard methods [START_REF] Ishizaki | High voltage electric pulses efficiently induce fusion of cells in monolayer culture[END_REF]. For electrofusion to occur, cells need to be in contact and in a "fusogenic state" (related to "electropermebilized state"), which can be achieved by electric pulses application; this fusogenic state involves a fractural rearrangement of the lipidic bilayer that is similar to that caused by electroporation [START_REF] Teissié | Fusion of mammalian cells in culture is obtained by creating the contact between cells after their electropermeabilization[END_REF].

In some of our experiments, we observed cell fusion in samples monitored with phase contrast microscopy. We investigated this phenomenon further, under all the different exposure conditions, by staining cells 24 hour post exposure with a fluorescent marker specific for Actin F (phalloidin) and another for cell membrane (wheat germ agglutinin). We saw changes only in the cells pulsed at the highest energy condition (one pulse of 100 μs at 1000 V/cm): in that case, some giant cells with fused membranes, but separate cytoskeleton, were observed.

Together with other aspects, such as the importance of membrane poration, the membrane fusogenic state, the temperature, and the ionic component of the medium in which cells were pulsed, the involvement of the actin/tubulin cytoskeleton rearrangement and cell swelling during cells electrofusion was demonstrated [START_REF] Kandušer | Cell electrofusion: past and future perspectives for antibody production and cancer cell vaccines[END_REF][START_REF] Blangero | Cytoskeletal reorganization during electric-field-induced fusion of Chinese hamster ovary cells grown in monolayers[END_REF][START_REF] Meulenberg | Differential cellular effects of electroporation and electrochemotherapy in monolayers of human microvascular endothelial cells[END_REF]. In SH-SY5Y cells, looking at actin organization, we did not observe its remodeling in fused cells, even though we detected cell swelling, still clearly visible 24 hour after exposure, when cells were plated at high cell density (more than 70% of confluence;

data not shown).

Several features differentiate our experimental conditions from the ones described before, and from other studies published in the literature: 1) our highest pulsed electric field condition was lower than the ones usually applied in the other studies; 2) most of the published papers analyzed the biological effects only during the first hours after the pulses delivery; 3) we pulsed cells in cell growth medium (DMEM/F12) which is rich of ions, in particular Ca++, which have been demonstrated to play a critical role in guiding electrofusion; 4) we pulsed SH-SY5Y cells which, to the best of our knowledge, have never been used before to investigate nanosecond and microsecond electric pulses effects. This is a crucial point, since the response to these pulses and the ability of the cells to fuse are strictly cell type dependent [START_REF] Salomskaite-Davalgiene | Extent of cell electrofusion in vitro and in vivo is cell line dependent[END_REF][START_REF] Mekid | In vivo cell electrofusion[END_REF].

As mentioned before, one of the conditions to trigger fusion is that electroporation occurs. Both the extent of electroporation and the fusion level, can be controlled by the amplitude, duration, and number of the applied pulses;

namely, increasing any of the mentioned pulse parameters leads to a higher level of membrane electroporation and consequently higher number of fused cells, if reversible electroporation is assured [START_REF] Salomskaite-Davalgiene | Extent of cell electrofusion in vitro and in vivo is cell line dependent[END_REF][START_REF] Teissié | Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes[END_REF]. Interestingly, in our experiments, cell fusion was observed only at the pulse condition able to electroporate cells. Despite that, it is worth to remember that the energy applied to the bio-samples was very low, probably one of the lowest ever applied in the literature, which demonstrates that also mild electric pulses exposure can induce cell fusion. This aspect could also be related to the specifically used cell line, the SH-SY5Y cells, a neuroblastoma cell line largely used in literature for its neuronal like features [START_REF] Kovalevich | Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology[END_REF], which also make this cell line particularly sensitive to calcium fluxes [START_REF] Lambert | Second-messenger responses associated with stimulation of neuronal muscarinic receptors expressed by a human neuroblastoma SH-SY5Y[END_REF], one of the principal actors of the electrofusion [START_REF] Ohnishi | Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas[END_REF][START_REF] Suo | Optimal concentration of calcium and electric field levels improve tetraploid embryo production by electrofusion in mice[END_REF].

Nanosecond and microsecond pulsed electric fields and ROS generation, apoptosis, and microRNA

Since with the lowest energy conditions we did not observe redox homeostasis imbalance, apoptosis or miR-34b/c modulation, as previously reported in 50 Hz-exposed cells, we also investigated whether more intense exposure conditions could impact one or several of these parameters. Looking at cell apoptosis none of the conditions considered in our study was able to induce cell death. Similarly, redox homeostasis was not influenced after the cell exposure to any of the analyzed conditions, even though at the highest DED, a slight increase, not statistically significant, in ROS production was observed just after the pulse delivery (T0) and up to 6 hours after the exposure.

After nanosecond and microsecond pulse application, ROS production, associated to phospholipids oxidation [START_REF] Breton | Investigation of the chemical mechanisms involved in the electropulsation of membranes at the molecular level[END_REF],

has been demonstrated, when a specific electric field intensity threshold is reached and plasma membrane is permeabilized [START_REF] Gabriel | Generation of reactive-oxygen species induced by electropermeabilization of Chinese hamster ovary cells and their consequence on cell viability[END_REF]. It is important to highlight that, to observe cytoplasmic ROS increase, the electropermeabilization needs to occur and to be reversible, in order to maintain the cell survival [START_REF] Bonnafous | The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane[END_REF]. Under our experimental conditions, after the delivery of one 100 µs electric pulse, cells started to be permeabilized only at 1000 V/cm, confirming that our exposure conditions were much weaker than the ones described in the papers cited before.

Even under this electrical stimulation condition, delivering the highest DED in our study, no change in miR-34b/c expression was observed. We can conclude that the expression of these two miRNAs is probably not a target of the electric pulses stimulation, only of the magnetic stimulation [START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF], or that the experimental conditions used here to deliver the electric pulses were not able to induce a significant change in miR-34b/c expression.

Conclusion

The present in vitro study tackles the open question related to the role of the DED and the contribution of the induced electric field component to provoke different biological effects observed under or after cells exposure to ELF-MFs.

Responses of SH-SY5Y cells were analyzed after pulsed electric fields at a DED equal to the one delivered by a continuous 24 hour EMF-MF treatment (50 Hz, 1 mT) and also after exposure to a continuous, sinusoidal electric field of the same intensity as the one induced by the ELF magnetic field exposure. None of the tested electric field exposures impaired cellular redox homeostasis, nor did they affect the down-regulation of the miR-34 b/c expression observed following ELF-MF exposure. We can, therefore conclude that neither total DED (delivered from pulsed electric fields instead of a continuous magnetic field) nor the induced electric field associated with the magnetic field exposure are responsible for the biological effects reported in [START_REF] Benassi | Extremely Low Frequency Magnetic Field (ELF-MF) Exposure Sensitizes SH-SY5Y Cells to the Pro-Parkinson's Disease Toxin MPP+[END_REF][START_REF] Merla | Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach[END_REF][START_REF] Consales | Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells[END_REF]. Other components of the ELF-MF exposure that might be considered crucial for the induction of a biological response, such as a direct action of the magnetic field on cells, or the amplitude and the duration of the delivered electric pulses, must be investigated in order to identify the mechanisms of ELF-MFs mediated bio-effects.

It is also important to note that the exploration of the biological effects induced by the pulsed electric fields of different durations and intensities below or at the threshold of cell electroporation on the SH-SY5Y cells revealed the ability of the non-porating electric fields to induce the cell signal transduction through the stimulation of the expression of two immediate early genes (egr-1 and c-fos). Because this stimulation was achieved with no membrane permeabilization, no change in cells morphology and in cell survival, no other apparent perturbation, our results should motivate further studies to disclose possible molecular mechanisms activated by electric pulses exposure, for translation towards new therapeutic and technological applications.

Fig. 1 :

 1 Fig. 1: a) PDMS spacers, left, and picture of the exposure arrangements for microsecond (center) and nanosecond (right) pulses delivery. b) Delivered waveforms for microsecond (left and central panels) and nanosecond pulses (right panel). Figures reporting the maximum delivered voltages are reported as examples. c) Exposure arrangement and waveform of the 50 Hz sinusoidal electric field

Fig. 2 A

 2 Fig. 2 A) and D) ROS assessment, B) and E) apoptosis evaluation, C) and F) miR 34-b/c expression in SH-SY5Y cells after their pulsation with the same energy density, using one pulse of 100 µs at 0.1 V/cm (A, B and C) or one pulse of 10 ns at 10V/cm (D, E and F). ROS data are expressed as relative mean fluorescence intensity (MFI), while the expression of miRNA-34 b/c is related to the standard U6. The dashed line corresponds to a fold change of 1

Fig. 3

 3 Fig. 3 ROS assessment in SH-SY5Y cells exposed to a 0.3 mV/m sinusoidal (50Hz) electric field. ROS production was evaluated after 24 and 48 hours of exposure through the determination of the levels of A) H2O2 (using H2DCFDA) or B) superoxide (using DHE) as detailed in the Materials and Methods. Data are expressed as relative mean fluorescence intensity (MFI). Values are the means ± S.D. of three independent experiments

Fig. 4

 4 Fig. 4 egr-1 and c-fos expression carried out in the six first hours after the exposure of the cells, under all the pulse conditions. The values are means±SD and represent the fold change in the pulsed samples related to the control (nonpulsed), considered as 1 **P ≤0.05, ***P ≤0.001. The dashed line corresponds to a fold change of 1. Values are the means ± S.D. of three independent experiments

Fig. 7

 7 Fig. 7 Cell apoptosis analyzed through western blot analysis of PARP1 cleavage (molecular masses are given in kDa on the right), carried out in SH-SY5Y cells 24 and 48 hours after the exposure to one single electric pulse (conditions reported in the figure). As positive controls, the proteins from cells treated with 5 μM etoposide for 48 hours were included among the experimental samples. The immunoblots are representative of different experiments giving similar results. Values are the means ± S.D. of three independent experiments

Table 2

 2 Sequences of the primers used to study gene expression2.10. Statistical analysisThe variations of the samples values are reported as mean ± S.D. calculated with n ≥ 3 replicates. After assessing the normal distribution of our results from sham and stimulated samples by using the Shapiro-Wilk and Shapiro-Francia normality tests implemented in Matlab (2016a, the Math Works, USA), the significance of nanosecond and microsecond electric pulse stimulation was evaluated using the Student's T test, where the null hypothesis was that there is no significant difference between the means of the two data sets. We used the KailedaGraph program (Synergy Software, Reading PA, USA) by applying the two-sided Student's t test. The precise number of experimental replicates, as well as the experimental groups that have been compared and statistically analyzed, have been detailed in the previous paragraphs. p values < 0.05 were considered statistically significant and indicated as follows:

* p < 0.05; ** p < 0.01; *** p < 0.001.

Acknowledgments

This research was undertaken during a Short Term Scientific Mission funded within the framework of COST Action EMF-MED (COST Action BM1309), supported by COST (European Cooperation in Science and Technology) Programme. The funding support of CNRS, Université Paris-Saclay and Gustave Roussy is also acknowledged. The work was also partially supported by H2020 MSCA-IF OPTIC BIOEM GA-661041.

We are especially grateful to Professor Tom Vernier for the thorough revision of English and the critical reading of the manuscript.

We also want to thank Francesca Pacchierotti for her helpful critical review of the manuscript.

DMSO: dimethyl sulfoxide (DMSO) PI: Propidium Iodide DHE: DiHydroEthidium H2-DCFDA: 2',7'-DiChloroDihydrofluorescein DiAcetate DMEM/F12: Dulbecco's Modified Eagles Medium/Ham's F12 IARC: International Agency for Research on Cancer PDMS: PolyDiMethylSiloxane PBS: Phosphate-Buffered Saline

Supplementary figure 2 miR-34b and miR-34c expression evaluated in SH-SY5Y cells at 1 (pulsed T1), 6 (pulsed T6) and 24 (pulsed T24) hours after the cell exposure to one electric pulse of 100 µs at 1000 V/cm