Generation of Hypochlorous Acid by High-Voltage Pulses and its Influence on the Cell Plasma Membrane

Abstract

Pulses of strong electric field utilized for cell electropermeabilization, also cause electrolysis reactions at the electrode–solution interfaces. Tissues and solutions usually contain high amounts of chloride ions. As a result of electrolysis, Cl2 gas can be formed at the anode [1]. Chloride ions react with the water molecules and form hypochlorous acid (HOCl), which is a powerful oxidant – it can react with a wide variety of biomolecules including DNA, RNA, cholesterol, and proteins [2]. For practical applications of electroporation, e.g, when using electroporation to extract DNA, RNA or proteins (e-biopsy) [3], it is important to avoid any contamination of samples. The aim of this work was to study the formation of HOCl acid as a result of electrolysis during high-voltage pulses, as well as its influence on the viability of cells and the barrier function of the cell plasma membrane. The formation of hypochlorous acid was evaluated with fluorescent indicator of hypochlorite 3’-p-Aminophenyl fluorescein (APF) [4] along with the scavengers of various reactive oxygen species (ROS). The viability of Chinese hamster ovary (CHO) cells was determined by a colony-forming assay [5]. The size of the pores created in human erythrocytes was estimated by studying the protective action of xylitol (152 Da), mannitol (182 Da), and sucrose (342 Da) against colloid-osmotic lysis [6]. It has been obtained, that during high–voltage electric pulses, ROS are generated. In cell–free media, micro–millisecond pulsed electric field increased fluorescence of hypochlorite indicator APF proportionally to the pulse number and amplitude. APF fluorescence was reduced by both vitamin C and mannitol. Also, it has been shown that ROS formation was more intensive in the case of stainless–steel electrodes comparing to the aluminum ones. The results of this work can be useful for optimizing the electroporation technology used in biotechnology, medicine, and food industry. The influence of hypochlorous acid on the viability of Chinese hamster ovary (CHO) cells in vitro was evaluated. HOCl caused the reduction of CHO viability. Less than 50 % of CHO cells survived, when the concentration of hypochlorous acid in the cell growth medium was 0.8 mM. The influence of HOCl on the plasma membrane of human erythrocytes was also studied. HOCl increased the permeability of the cell plasma membrane to ions and small molecules, what caused haemolysis of erythrocytes. The estimated radius of permeable structures, which appeared in the plasma membrane of erythrocytes, was about 0.3–0.5 nm. This is close to the size of the pores generated by the exposure of cells with pulses of strong electric field [6]. Conclusion: hypochlorous acid can be formed due to electrolysis during high-voltage pulses. It can increase permeability of the cell plasma membrane to ions and small molecules, which can cause the reduction of the cell viability.