Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

Pakhomova, Olga N.; Khorokhorina, Vera A.; Bowman, Angela M.; Rodaitė‐Riševičienė, Raminta; Saulis, Gintautas; Xiao, Shu; Pakhomov, Andrei G.

doi:10.1016/j.abb.2012.08.004

Cited by 163 publications

(109 citation statements)

References 49 publications

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“…The integrated fluorescence was significantly decreased in all nsPEF-treated groups, although less so in the saline+sucrose. The generation and diffusion of reactive oxygen species is a common cause of bleaching of fluorescent dyes [42], and the recent observations of ROS generation by nsPEF [22, 43] can provide a reasonable explanation to the observed bleaching of tagged actin. If this is indeed the case, then the protection from swelling rendered by sucrose has also reduced the intracellular ROS generation, e.g., by diminishing the damage to mitochondria (which would therefore also be a downstream consequence of swelling).…”

Section: Resultsmentioning

confidence: 99%

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Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

Pakhomov

Xiao

Pakhomova

et al. 2014

Bioelectrochemistry

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Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

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Section: Resultsmentioning

confidence: 99%

“…It is also being debated if permeabilization of membranous structures is the only primary effect of nsPEF. For example, a recent study established ROS formation by nsPEF in both living cells and culture media [22], although its biological significance has yet to be explored.…”

Section: Introductionmentioning

confidence: 99%

Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

Pakhomov

Xiao

Pakhomova

et al. 2014

Bioelectrochemistry

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“…Changes in the whole-cell conductance and cell morphology triggered by nsEP are similar to those of a free radical-induced necrosis [8, 17, 58]. ROS formation was demonstrated in nsEP-treated media even in the absence of cells [59]. A recently proposed hypothesis separates the short-lived electroporation of the membrane from its long-lasting permeabilization due to the oxidation of phospholipids [60].…”

Section: Discussionmentioning

confidence: 99%

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity

Pakhomov

Semenov

Xiao

et al. 2014

Cell. Mol. Life Sci.

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Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca2+ mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1–60), and amplitudes (15–60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-μs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

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“…These pathways interplay with cellular respiration and, at low levels, may stimulate the cell. Additionally, recent publications have also pointed to reactive oxygen species (ROS) generation both around and within cells exposed to nsPEF [22]. The generation of ROS has been shown to increase cellular respiration, suggesting that both calcium and ROS generation are playing a role in the observed OCR increase.…”

Section: Number Of Pulsesmentioning

confidence: 93%

Investigation of a direct effect of nanosecond pulse electric fields on mitochondria

et al. 2014

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The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few µL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.

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Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

Cited by 163 publications

References 49 publications

Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity

Investigation of a direct effect of nanosecond pulse electric fields on mitochondria

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