Leukemic cell intracellular responses to nanosecond electric fields

Intense, nanosecond (ns) pulsed electric fields (PEFs) are known to affect the intracellular structures of cells. The probability of preferentially inducing subcellular effects increases with decreasing pulse length while effects on the plasma membrane are diminished. This has been demonstrated by applying electrical pulses of 60 and 10 ns duration with electric field intensities of up to 6.5 MV/m to HL-60 cells. Using confocal microscopy, PEF-induced changes in the integrity of the plasma membrane and nucleus were measured by recording fluorescence changes with propidium iodide (PI) and acridine orange (AO), respectively. Results suggest that high voltage, nsPEFs target the nucleus and modify cellular functions while plasma membrane effects are delayed and become smaller as pulse duration is shortened. Cell viability was not affected by these pulses. In spite of the high pulsed electric fields, thermal effects can be neglected because of the ultrashort pulse duration. The results suggest application of this ultrashort pulse technology to modulate nuclear structure and function for potential therapeutic benefit.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system.The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field.The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field.In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions,the increase of [Ca2+]i was still observable.It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

Dynamic changes of [Ca~(2 )]_i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca2 concentration ([Ca2 ]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca2 ]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca2 ]i depend on the intensity of external electric field. In the presence of Ca2 chelant EGTA or Ca2 channels blocker La3 in the pulsation solutions, the increase of [Ca2 ]i was still observable. It was also observed that [Ca2 ]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2 ]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca²⁺]_i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca²⁺]_i depend on the intensity of external electric field. In the presence of Ca²⁺ chelant EGTA or Ca²⁺ channels blocker La³⁺ in the pulsation solutions, the increase of [Ca²⁺]_i was still observable. It was also observed that [Ca²⁺]_i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca²⁺]_i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Current densities measured in human models exposed to 60-Hz electric fields

This paper gives current densities measured in homogeneous grounded human models exposed to vertical, 60-Hz electric fields. The methods used for these measurements were validated by measuring the current densities induced in a grounded hemisphere and in a grounded prolate hemispheroid; agreement between measurement and theory was good. For an unperturbed field strength of 10 kV/m, current densities measured in the human chest were in the range 125-300 nA/cm2. A strong horizontal current-density enhancement was observed in the axillae, with peak values of about 400 nA/cm2. The vertical current density in the arms, when held downward, was in the opposite direction to that in the chest. Current densities in the abdomen, pelvis, and legs were a strong function of whether the body was grounded through one or both feet. With one foot grounded, the horizontal current density in the lower pelvic region, just above the crotch, was 770 nA/cm2. This value was the largest of those measured in the head, arms, or torso of the human model. Scaling factors derived from these data and similar data for animals will provide a quantitative basis for comparing animal and human exposure to 60-Hz electric fields. In addition, current-density data given in this paper can be directly extrapolated to higher frequencies, at least to 1 MHz. These extrapolated data may be useful to individuals and groups involved in the determination of safety standards for the lower radiofrequency region.     

Relationship between field strength and arousal response in mice exposed to 60-Hz electric fields

White-footed mice, Peromyscus leucopus, were exposed to 60-Hz electric fields to study the relationship between field strength and three measures of the transient arousal response previously reported to occur with exposures at 100 kV/m. Five groups of 12 mice each were given a series of four 1-h exposures, separated by an hour, with each group exposed at one of the following field strengths: 75, 50, 35, 25, and 10 kV/m; 8 additional mice were sham-exposed with no voltage applied to the field generator. All mice were experimentally naive before the start of the experiment, and all exposures occurred during the inactive (lights-on) phase of the circadian cycle. The first exposure produced immediate increases in arousal measures, but subsequent exposures had no significant effect on any measure. These arousal responses were defined by significant increases of gross motor activity, carbon dioxide production, and oxygen consumption, and were frequently recorded with field strengths of 50 kV/m or higher. Significant arousal responses rarely occurred with exposures at lower field strengths. Responses of mice exposed at 75 and 50 kV/m were similar to previously described transient arousal responses in mice exposed to 100-kV/m electric fields. Less than half of the mice in each of the field strength groups below 50 kV/m showed arousal responses based on Z (standard) scores, but the arousals of the mice that did respond were similar to those of mice exposed at higher field strengths. Polynomial regression was used to calculate the field strength producing the greatest increases for each of the arousal measures. The results show that the amplitude of the transient arousal response is related to the strength of the electric field, but different measures of arousal may have different relationships to field strength.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Progesterone concentrations during estrous cycle of dairy cows exposed to electric and magnetic fields

Sixteen multiparous nonpregnant lactating Holstein cows (each weighing 662 ± 65 kg in 150.4 ±40 day of lactation) were confined to wooden metabolic cages with 12:12 h light:dark cycle during the experiment. The cows were divided into two sequences of eight cows each and exposed to electric and magnetic fields (EMF) in an exposure chamber. This chamber produced a vertical electric field of 10 kV/m and a uniform horizontal magnetic field of 30 μT at 60 Hz. One sequence was exposed for three estrous cycles of 24 to 27 days. During the first estrous cycle, the electric and magnetic fields were off; during the second estrous cycle, they were on; and during the third estrous cycle, they were off. The second sequence was also exposed for three 24 to 26 days estrous cycles, but the exposure to the fields was reversed (first estrous cycle, on; second estrous cycle, off; third estrous cycle, on). The length of each exposure period (21 to 27 days) varied according to the estrous cycle length. No differences were detected in plasma progesterone concentrations and area under the progesterone curve during estrous cycles between EMF nonexposed and exposed periods (2.28 ±0.17 and 2.25 ± 0.17; and 24.5 ± 1.9 vs. 26.4 ± 1.9 ng/ml, respectively). However, estrous cycle length, determined by the presence of a functional corpus luteum detected by concentrations of progesterone equal to or more than 1 ng/ml plasma, was shorter in nonexposed cows than when they were exposed to EMF (22.0 ± 0.9 vs. 25.3 ± 1.4 days). Bioelectromagnetics 19:438–443, 1998. © 1998 Wiley-Liss, Inc.     

Quantitative phase imaging of cells in a flow cytometry arrangement utilizing Michelson interferometer‐based off‐axis digital holographic microscopy

We combined Michelson‐interferometer‐based off‐axis digital holographic microscopy (DHM) with a common flow cytometry (FCM) arrangement. Utilizing object recognition procedures and holographic autofocusing during the numerical reconstruction of the acquired off‐axis holograms, sharply focused quantitative phase images of suspended cells in flow were retrieved without labeling, from which biophysical cellular features of distinct cells, such as cell radius, refractive index and dry mass, can be subsequently retrieved in an automated manner. The performance of the proposed concept was first characterized by investigations on microspheres that were utilized as test standards. Then, we analyzed two types of pancreatic tumor cells with different morphology to further verify the applicability of the proposed method for quantitative live cell imaging. The retrieved biophysical datasets from cells in flow are found in good agreement with results from comparative investigations with previously developed DHM methods under static conditions, which demonstrates the effectiveness and reliability of our approach. Our results contribute to the establishment of DHM in imaging FCM and prospect to broaden the application spectrum of FCM by providing complementary quantitative imaging as well as additional biophysical cell parameters which are not accessible in current high‐throughput FCM measurements.     

Detection and controlled depletion of cancer cells using photothermal phase microscopy

We present a dual‐modality technique based on wide‐field photothermal (PT) interferometric phase imaging and simultaneous PT ablation to selectively deplete specific cell populations labelled by plasmonic nanoparticles. This combined technique utilizes the plasmonic reaction of gold nanoparticles under optical excitation to produce PT imaging contrast by inducing local phase changes when the excitation power is weak, or ablation of selected cells when increasing the excitation power. Controlling the entire process is carried out by dynamic quantitative phase imaging of all cells (labelled and unlabelled). We demonstrate our ability to detect and specifically ablate in vitro cancer cells over‐expressing epidermal growth factor receptors (EGFRs), labelled with plasmonic nanoparticles, in the presence of either EGFR under‐expressing cancer cells or white blood cells. The latter demonstration establishes an initial model for depletion of circulating tumour cells in blood. The proposed system is able to image in wide field the label‐free quantitative phase profile together with the PT phase profile of the sample, and provides the ability of both detection and selective cell ablation in a controlled environment.

Quantitative phase imaging with molecular specificity and specific cell depletion. ( a ) Label‐free quantitative phase profiles of mixed population of EGFR⁺/EGFR^– cancer cells. ( b ) When weak modulated PT excitation is applied, selective phase contrast is generated in the modulation frequency only for the EGFR⁺ cancer cells labelled with plasmonic nanoparticles. ( c ) When stronger modulated PT excitation is applied, selective ablation of the EGFR⁺ cancer cells labelled with plasmonic nanoparticles occurs. White scalebars represent 10 µm upon sample.     

Low electric field parameters required to induce death of cancer cells

Irreversible electroporation (IRE) is a novel technique that deals with killing undesirable cells, mainly cancer cells, directly without using any cytotoxic drugs. Commonly in this technique very high electric field up to 1000?V/cm is used but for very short exposure time (nanoseconds). Low electric fields (LEFs) are used before to internalize molecules and drugs inside the cells (electroendocytosis) but mainly not in killing the cells. The aim of this work is to determine the ability of using LEFs to kill cancer cells (Hela cells). The Physics idea is in making LEFs energy equivalent to IRE energy. Four IRE protocols were selected to represent very high, high, moderate and mild voltages IRE, then we make equivalent energy for each of these protocols using different LEFs’ parameters of different amplitudes (7, 10, 14 and 20?V), different pulse numbers (40, 80, 160 and 320 pulses), different frequencies from 0.5 to 106.86?Hz and different pulse widths from 9.38 to 2000?ms. Each of the calculated LEF equivalent to IRE was applied on Hela cell line. The results show complete destruction of the cancer cells for all the tested exposure protocols. This damage was not due to thermal effect because the measured temperature was not changed before and after the exposure. The possible effect mechanism is discussed. It was concluded that the lethal effect on the cancer cells can be achieved using LEFs if the same energy equivalent to IRE is used. This work will help in using low-risk drug-free techniques in cancer treatment.     

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.     

Prolonged Ca2+ transients in ATP-stimulated endothelial cells exposed to 50 Hz electric fields

Human umbilical vein endothelial cells were exposed to sinusoidal electric fields of 0.3 or 30 kV/m, 50 Hz, for 24 h. Changes in intracellular calcium concentration ([Ca(2+)](i)) induced by ATP-stimulation in the absence of extracellular Ca(2+) were observed in individual cells. No differences were observed between the exposure and sham-exposure groups in [Ca(2+)](i) resting level before ATP-stimulation, or in the [Ca(2+)](i) peak levels induced by stimulation. However, the duration of the initial transients in [Ca(2+)](i) following an ATP stimulus was significantly prolonged by exposure to a 30 kV/m field. The inositol trisphosphate receptor inhibitor, xestospongin C, inhibited the ATP-induced elevation in [Ca(2+)](i) in both the exposure and sham-exposure groups. The ATP-receptor P2Y appeared to play an important role in the increase of [Ca(2+)](i). The present results suggest that an extremely low-frequency electric field affects the function of vascular endothelial cells by a mechanism involving activation of P2Y.     

Neuroendocrine parameters in the rat exposed to 60-Hz electric fields

This study was designed to assess the neuroendocrine response of male Long-Evans rats to sustained or intermittent 60-Hz electric fields when exposed for 1 or 3 h at 100 kV/m. No significant differences were noted in corticosterone, prolactin, or thyrotropin levels between exposed and sham-exposed rats. A statistically significant increase (P less than .01) in growth hormone was noted in rats exposed to intermittent electric fields for 3 h. Emphasis was placed on good experimental design and the need to avoid standard laboratory stressors (excessive handling, temperature extremes, transportation, noise, etc.) known to be present in many biomedical studies. The importance of avoiding reactions due to extraneous factors in experiments predicated on investigating physiological function in relation to electric field exposure is discussed.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Hematologic and serum chemistry studies in rats exposed to 60-Hz electric fields

Numerous hematologic and serum chemistry variables were examined in rats exposed to unperturbed 60-Hz electric fields at 100 kV/m for 15, 30, 60, or 120 days. Each study was replicated once. Rigorous statistical evaluations of these data did not detect any consistent effect of the electric field for exposures of up to 120 days. It was, however, not unusual in any individual study to detect certain variables that were significantly different between the exposed and sham-exposed animals. This emphasizes the need for replicate designs and appropriate statistical analyses when investigating chemical or physical insults that may have minimal influence on biologic function.     

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.     

DEXA analysis on the bones of rats exposed in utero and neonatally to static and 50 Hz electric fields

Effects of the electromagnetic fields on living bodies, bones in particular, are among the relevant issues of contemporary life. In this study, we report the influences of 50 Hz and 0 Hz (static) electric fields (EF), on intact rat bones, as evaluated by dual energy X-ray absorbtion (DEXA) measurements on bone content and density when these animals (n = 27) are continuously exposed in utero and neonatally to EFs (10 kV/m) 14 days before and 14 days after their birth, for 28 days in total. Differences between 50 Hz EF and static EF groups are found to be significant (95% confidence level) for total bone mineral content (BMC), TBMC (P = .002). Differences between 50 Hz and control groups are found to be significant for total bone mineral density (BMD), TBMD (P = .002), lumbar BMC, LBMC (P = .023), and TBMC (P = .001). Differences between static EF and control groups are found to be significant for femoral BMD, FBMD (P = .009), TBMD (P = .002), LBMC (P = .001), and TBMC (P = .001). Note that TBMC parameters are jointly significant for all differences between the three groups of test animals. These results have shown that both static and 50 Hz EFs influence the early development of rat bones. However, the influence of static EFs is more pronounced than that of the 50 Hz field.