Cytotoxicity of a Cell Culture Medium Treated with a High-Voltage Pulse Using Stainless Steel Electrodes and the Role of Iron Ions.

High-voltage pulses applied to a cell suspension cause not only cell membrane permeabilization, but a variety of electrolysis reactions to also occur at the electrode-solution interfaces. Here, the cytotoxicity of a culture medium treated by a single electric pulse and the role of the iron ions in this cytotoxicity were studied in vitro. The experiments were carried out on mouse hepatoma MH-22A, rat glioma C6, and Chinese hamster ovary cells. The cell culture medium treated with a high-voltage pulse was highly cytotoxic. All cells died in the medium treated by a single electric pulse with a duration of 2 ms and an amplitude of just 0.2 kV/cm. The medium treated with a shorter pulse was less cytotoxic. The cell viability was inversely proportional to the amount of electric charge that flowed through the solution. The amount of iron ions released from the stainless steel anode (>0.5 mM) was enough to reduce cell viability. However, iron ions were not the sole reason of cell death. To kill all MH-22A and CHO cells, the concentration of Fe3+ ions in a medium of more than 2 mM was required.

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

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13 kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.

Increase of the roughness of the stainless-steel anode surface due to the exposure to high-voltage electric pulses as revealed by atomic force microscopy.

The changes of the stainless-steel electrode surface morphology occurring due to dissolution of the anode under the action of electric pulses which are commonly utilized in cell electromanipulation procedures, have been studied by using atomic force microscopy. The surface of the polished electrode was rather smooth--the average roughness was 13-17 nm and the total roughness 140-180 nm. After the treatment of the chamber filled with 154 mM NaCl solution to a series of short (about 20 mus), high-voltage (4 kV) pulses, the roughness of the surface of the anode has increased, depending on the total amount of the electric charge that has passed through the unit area of the electrode, and exceeded 400 nm for the dissolution charge of 0.24 A s/cm(2). No changes of the cathode surface were detected. Well-defined peaks with the width of 1-2 mum and the height of over 400 nm have appeared. These peaks create local enhancements of the electric field at the interface between the solution and the electrode surface which can lead to the non-homogeneity treatment of cells by electric pulses and can facilitate the occurrence of the electrical breakdown of the liquid samples.