The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (<2 nm) are created in the plasma membrane in contrast to larger diameter pores (>2 nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.
The cellular response to subtle membrane damage following exposure to nanosecond electric pulses (nsEP) is not well
understood. Recent work has shown that when cells are exposed to nsEP, ion permeable nanopores (< 2nm) are created
in the plasma membrane in contrast to larger diameter pores (> 2nm) created by longer micro and millisecond duration
pulses. Macroscopic damage to a plasma membrane by a micropipette has been shown to cause internal vesicles
(lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis.
Formation of large pores in the plasma membrane by electrical pulses has been shown to elicit lysosomal exocytosis in a
variety of cell types. Our research objective is to determine whether lysosomal exocytosis will occur in response to
nanopores formed by exposure to nsEP. In this paper we used propidium iodide (PI) and Calcium Green-1 AM ester
(CaGr) to differentiate between large and small pores formed in CHO-K1 cells following exposure to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm. This information was compared to changes in membrane organization
observed by increases in FM1-43 fluorescence, both in the presence and absence of calcium ions in the outside buffer. In
addition, we monitored the real time migration of lysosomes within the cell using Cellular Lights assay to tag LAMP-1,
a lysosomal membrane protein. Both 1 and 20 pulses elicited a large influx of extracellular calcium, while little PI
uptake was observed following a single pulse exposure. Statistically significant increases in FM1-43 fluorescence were
seen in samples containing calcium suggesting that calcium-triggered membrane repair may be occurring. Lastly,
density of lysosomes within cells, specifically around the nucleus, appeared to change rapidly upon nsEP stimulation
suggesting lysosomal migration.
Several international organizations establish minimum safety standards to ensure that workers and the general
population are protected against adverse health effects associated with electromagnetic radiation. Suitable standards are
typically defined using published experimental data. To date, few experimental studies have been conducted at Terahertz
(THz) frequencies, and as a result, current THz standards have been defined using extrapolated estimates from
neighboring spectral regions. In this study, we used computational modeling and experimental approaches to determine
tissue-damage thresholds at THz frequencies. For the computational modeling efforts, we used the Arrhenius damage
integral to predict damage-thresholds. We determined thresholds experimentally for both long (minutes) and short
(seconds) THz exposures. For the long exposure studies, we used an in-house molecular gas THz laser (υ= 1.89 THz,
189.92 mW/cm2, 10 minutes) and excised porcine skin. For the short exposure studies, we used the Free Electron Laser
(FEL) at Jefferson Laboratory (υ= 0.1-1.0 THz, 2.0-14.0 mW/cm2, 2 seconds) and wet chamois cloths. Thresholds were
determined using conventional damage score determination and probit analysis techniques, and tissue temperatures were
measured using infrared thermographic techniques. We found that the FEL was ideal for tissue damage studies, while
our in-house THz source was not suitable to determine tissue damage thresholds. Using experimental data, the tissue
damage threshold (ED50) was determined to be 7.16 W/cm2. This value was in well agreement with that predicted using
our computational models. We hope that knowledge of tissue-damage thresholds at THz frequencies helps to ensure the
safe use of THz radiation.
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