Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

Abstract

In this study, we determined the LD50 (50% lethal dose) for cell death, and the ED50 (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

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

“…have shown that acute uptake of PI and externalization of phosphatidylserine are observed at or near the exposures used in this paper, suggesting that the plasma membrane is likely breached and calcium levels within the cell are elevated [17,18]. Future work will couple the results presented in this paper with flow cytometry data at similar time points to correlate membrane damage with OCR increase.…”

“…This data strongly suggests that a calcium-dependent mechanism is at play. Previous data published by our group has shown that modulation of the extracellular calcium during exposure caused deleterious effects in Jurkat cells, suggesting that influx of extracellular calcium magnified loss of cell survival [17]. The data was collected at high pulse numbers (100-300), but show that calcium indeed plays a role in cell response to nsPEF.…”

“…The 10-ns exposure system was nearly identical to the exposure system described in previous publications [17][18][19]. In short, a custom Blumlein line pulser that utilizes a chamber containing pressurized sulfur hexafluoride (SF 6 ) gas was used to deliver the nsPEF.…”

“…Multiple publications have reported a difference in sensitivity to nsPEF exposure between Jurkat and U937 cells, with Jurkat cells being more sensitive (i.e., show changes in viability at lower doses) [17][18][19]. Figure 3A shows the resulting OCR measurements made in Jurkat cells following exposure to 50 kV/cm pulses.…”

Dose dependent translocations of fluorescent probes of PIP2hydrolysis in cells exposed to nanosecond pulsed electric fields

“…In cells without G q/11 receptors, nsPEF initiated PIP 2 hydrolysis bypassing known physiological mechanisms and, presumably, acting directly on the plasma membrane. Recently, we reported electric field dose-dependent thresholds of nsPEF-induced plasma membrane disruption and nanopore formation in the multiple cell lines, including primary cultured neurons [4,16]. Neuronal cells are especially sensitive to regulation by intracellular phosphoinositide signaling [10,12] and the ability to selectively tune the degree of signaling would be highly beneficial.…”