Effects of pulsed electric fields on DNA of human lymphocytes

The effects of pulsed electric fields of low frequency (50 Hz) on DNA of human lymphocytes were investigated. The influence of additional external factors, such as hydrogen peroxide (H₂O₂) and γ-irradiation, as well as the repair efficiency in these lymphocytes, was also evaluated. The comet assay, a very sensitive and rapid method for detecting DNA damage at the single cells level was the method used. A significant amount of damage was observed after exposure to the electric fields, compared to the controls. After 2 h incubation at 37^°C, a proportion of damage was repaired. H₂O₂ and γ-irradiation increased the damage to lymphocytes exposed to pulsed electric fields according to the dose used, while the amount of the repair was proportional to the damage.     

Nanosecond pulsed electric fields cause melanomas to self-destruct

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.     

Time-varying magnetic fields of 60 Hz at 7 mT induce DNA double-strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.     

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. CONCLUSIONS: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.     

A reporter mouse for in vivo detection of DNA damage in embryonic germ cells

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1‐mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell‐specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB‐inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose‐ and time‐dependent manner. The dynamic time‐sensitive and dose‐sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.     

System for the exposure of cell suspensions to power-frequency electric fields

A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line. In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated. Electric fields in the range 0.02–3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5%. The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 μT. The temperatures of the experimental and control cell suspensions are held fixed within ±0.1°C by a water bath. Special chambers were developed to hold cell cultures during exposure and sham exposure. Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16–17 h, approximately the same as for CHO cells grown under standard cell-culture conditions.     

Effects of localised,low-voltage pulsed electric fields on the development and inhibition of Pseudomonas aeruginosa biofilms

Abstract

This work describes the use of low-voltage (0.5 – 5 V) pulsed electric fields to prevent Pseudomonas aeruginosa biofilm development. Interdigitated electrodes (IDEs) with 29-μm spacing between 22-μm-wide electrodes, were used as a platform where the effect of localised, high-strength electric fields could be tested. Alternating current, square-wave pulses were applied to the IDEs in 1 sec intervals. A two-level, three-variable factorial design experiment was used to detect the effects of applied voltage, frequency, and pulse duty ratio (i.e. percentage of pulsing time over one cycle) on the inhibition of biofilm formation. The observations indicated that a pulse configuration of 1% duty ratio, 5 V, and 200 Hz frequency reduced the area of the electrodes covered by biofilm by 50%. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage were observed to have less influence on biofouling.     

General properties of the interaction between animals and ELF electric fields

An analysis is given of the interaction between extremely low-frequency (ELF) electric fields and animals of arbitrary body shape. This analysis is based on three approximations which are valid in the ELF range: In living tissues, capacitive (displacement) currents are negligible compared to conduction currents; effects resulting from the finite velocity of propagation of electromagnetic fields are negligible; skin effect in living tissues is negligible. Major conclusions of the analysis are: (a) The electric field outside the body, the induced charge on the surface of the body, and the total current crossing any section through the body (eg, through the neck or limbs) are completely determined by the characteristics of the applied ELF electric field, the shape of the body, its location relative to ground and other conductors, and any conduction currents from the body to ground or other conductors. (b) All of the quantities in (a) can be measured using conducting animal models. (c) The magnitudes of the electric field outside the body and the induced charge density on the surface of the body are independent of frequency, in the ELF range, when the body is either insulated from or shorted to ground (and any other conductors in the system). (d) The only quantities affected by the electrical properties of the tissues comprising the body are the current density and electric field inside the body. (e) The electric field outside and inside a body will be unchanged by a scaled change in its size.     

Controversial cytogenetic observations in mammalian somatic cells exposed to extremely low frequency electromagnetic radiation: a review and future research recommendations

During the years 1990-2003, a large number of investigations were conducted using animals, cultured rodent and human cells as well as freshly collected human blood lymphocytes to determine the genotoxic potential of exposure to nonionizing radiation emitted from extremely low frequency electromagnetic fields (EMF). Among the 63 peer reviewed scientific reports, the conclusions from 29 studies (46%) did not indicate increased damage to the genetic material, as assessed from DNA strand breaks, incidence of chromosomal aberrations (CA), micronuclei (MN), and sister chromatid exchanges (SCE), in EMF exposed cells as compared with sham exposed and/or unexposed cells, while those from 14 investigations (22%) have suggested an increase in such damage in EMF exposed cells. The observations from 20 other studies (32%) were inconclusive. This study reviews the investigations published in peer reviewed scientific journals during 1990-2003 and attempts to identify probable reason(s) for the conflicting results. Recommendations are made for future research to address some of the controversial observations.     

Microwaves from UMTS/GSM mobile phones induce long-lasting inhibition of 53BP1/gamma-H2AX DNA repair foci in human lymphocytes

We have recently described frequency-dependent effects of mobile phone microwaves (MWs) of global system for mobile communication (GSM) on human lymphocytes from persons reporting hypersensitivity to electromagnetic fields and healthy persons. Contrary to GSM, universal global telecommunications system (UMTS) mobile phones emit wide-band MW signals. Hypothetically, UMTS MWs may result in higher biological effects compared to GSM signal because of eventual "effective" frequencies within the wideband. Here, we report for the first time that UMTS MWs affect chromatin and inhibit formation of DNA double-strand breaks co-localizing 53BP1/gamma-H2AX DNA repair foci in human lymphocytes from hypersensitive and healthy persons and confirm that effects of GSM MWs depend on carrier frequency. Remarkably, the effects of MWs on 53BP1/gamma-H2AX foci persisted up to 72 h following exposure of cells, even longer than the stress response following heat shock. The data are in line with the hypothesis that the type of signal, UMTS MWs, may have higher biological efficiency and possibly larger health risk effects compared to GSM radiation emissions. No significant differences in effects between groups of healthy and hypersensitive subjects were observed, except for the effects of UMTS MWs and GSM-915 MHz MWs on the formation of the DNA repair foci, which were different for hypersensitive (P < 0.02[53BP1]//0.01[gamma-H2AX]) but not for control subjects (P > 0.05). The non-parametric statistics used here did not indicate specificity of the differences revealed between the effects of GSM and UMTS MWs on cells from hypersensitive subjects and more data are needed to study the nature of these differences.     

Biological effects of oscillating electric fields: Role of voltage-sensitive ion channels

An alternating component of potential across the membrane of an excitable cell may change the membrane conductance by interacting with the voltagesensing charged groups of the protein macromolecules that form voltage-sensitive ion channels. Because the probability that a voltage sensor is in a given state is a highly nonlinear function of the applied electric field, the average occupancy of a particular state will change in an oscillating electric field of sufficient magnitude. This “rectification” at the level of the voltage sensors could result in conformational changes (gating) that would modify channel conductance. A simplified two-state model is examined where the relaxation time of the voltage sensor is assumed to be considerably faster than the fastest changes of ionic conductance. Significant changes in the occupancy of voltage sensor states in response to an applied oscillating electric field are predicted by the model.     

Comparison of the coupling of grounded humans,swine and rats to vertical, 60-Hz electric fields

Published and new data for grounded humans, swine, and rats exposed to vertical, 60-Hz electric fields are used to determine field strengths at the surfaces of the bodies and average components of induced-current density along the axes of the bodies. At the tops of the bodies, surface electric fields are increased (enhanced) over the unperturbed field strength present before the subjects entered the field by factors of 17,7, and 4 for humans, swine, and rats, respectively. For an unperturbed field strength of 10 kV/m, average induced axial current densities in the neck, chest, abdomen, and feet are: 550, 190, 250, and 2000 nA/cm², respectively, for humans; 40, 13, 20, and 1100 nA/cm², respectively, for swine; and 28, 16, 2, and 1400 nA/cm², respectively, for rats. These data are used to show that the actual electric fields experienced by animals depend strongly on the shape of the body and its orientation relative to the electric field and ground plane. This fact must be taken into account if biological data obtained with laboratory animals are to be used for the assessment of possible hazards to humans exposed to 60-Hz electric fields.     

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical‐free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5–100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10⁵–1.4 ± 0.2 × 10⁵ cell/mL range. Our results suggest that IDPEFs may prove useful as a non‐chemical method for fibroblast density control in human wound healing. Biotechnol. Bioeng. 2013; 110: 1759–1768. © 2013 Wiley Periodicals, Inc.     

Influence of 50 Hz electric and magnetic fields on the human heart

This investigation studied the effect of 50 Hz electric and magnetic fields on the human heart. The electrocardiograms of 27 transmission-line workers and 26 male volunteers were recorded with a Holter recorder both in and outside the fields. The measurements took from half an hour to a few hours. The electric field strength varied from 0.14 to 10.21 kV/m and the magnetic flux density from 1.02 to 15.43 μT. Analysis of the ECG recordings showed that extrasystoles or arrhythmias were as frequent outside the field as in the field. In some cases a small decrease in heart rate was observed after field exposure. © 1993 Wiley-Liss, Inc.     

BRCA2 is needed for both repair and cell cycle arrest in mammalian cells exposed to S23906, an anticancer monofunctional DNA binder

Repair of DNA-targeted anticancer agents is an active area of investigation of both fundamental and clinical interest. However, most studies have focused on a small number of compounds limiting our understanding of both DNA repair and the DNA damage response. {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 is an acronycine derivative that shows strong activity toward solid tumors in experimental models. {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 forms bulky monofunctional DNA adducts in the minor groove which leads to destabilization of the double-stranded helix. We now report that {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 induces formation of DNA double strand breaks that are processed through homologous recombination (HR) but not Non-Homologous End-Joining (NHEJ) repair. Interestingly, {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 exposure was accompanied by a higher sensitivity of BRCA2-deficient cells compared to other HR deficient cell lines and by an S-phase accumulation in wild-type (wt), but not in BRCA2-deficient cells. Recently, we have shown that {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906-induced S phase arrest was mediated by the checkpoint kinase Chk1. However, its activated phosphorylated form is equally induced by {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 in wt and BRCA2-deficient cells, likely indicating a role for BRCA2 downstream of Chk1. Accordingly, override of the S phase arrest by either 7-hydroxystaurosporine (UCN-01) or AZD7762 potentiates the cytotoxic activity of {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 in wt, but not in BRCA2-deficient cells. Together, our findings suggest that the pronounced sensitivity of BRCA2-deficient cells to {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 is due to both a defective S-phase arrest and the absence of HR repair. Tumors with deficiencies for proteins involved in HR, and BRCA2 in particular, may thus show increased sensitivity to {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906, thereby providing a rationale for patient selection in clinical trials.     

Genome Instability in Ataxia Telangiectasia (A-T) Families: Camptothecin-Induced Damage to Replicating DNA Discriminates between Obligate A-T Heterozygotes,A-T Homozygotes and Controls

Previously we used the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells primarily by inducing double strand breaks (DSBs) in replication forks, to show that ataxia telangiectasia (A-T) fibroblasts are defective in the repair of this particular subclass of DSBs. CPT treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges, viewed as prematurely condensed G2 chromosomes (G2 PCC), compared with normal cells where aberrations are mostly chromatid breaks. Here we show that A-T lymphoblastoid cells established from individuals with different mutations in the ATM gene also exhibit increased levels of chromosomal exchanges in response to CPT, indicating that the replication-associated DSBs are misrepaired in all these cells. From family studies we show that the presence of a single mutated allele in obligate A-T heterozygotes leads to intermediate levels of chromosomal exchanges in CPT-treated lymphoblastoid cells, thus providing a functional and sensitive assay to identify these individuals.     

γH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products

Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as γH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that γH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring γH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that γH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that γH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.