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Title:
Generation of of hypochlorous acid by high-voltage pulses and its influence on the cell plasma membrane
Authors:
Saulis, Gintautas - Vytautas Magnus University, Kaunas, Lithuania
Rodaite, Raminta - Vytautas Magnus University, Kaunas, Lithuania
Sventoraitiene, Jurgita - Lithuanian University of Health Sciences, Kaunas, Lithuania
Dainauskaite, Viktorija - Vytautas Magnus University, Kaunas, Lithuania
Batiuskaite, Danute - Vytautas Magnus University, Kaunas, Lithuania
Golberg, Alex - Tel Aviv University, Tel Aviv, Israel
Saule, Rita - Vytautas Magnus University, Kaunas, Lithuania
Abstract:
Cell electroporation is widely used in cell biology, biotechnology, and medicine. However, pulses of strong electric field (up to 300 kV/ cm) utilized for cell membrane permeabilization, also causes a range of of electrolysis reactions at the electrode–solution interfaces. Tissues as well as solutions used for cell electroporation 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 powerfull oxidant – it can react with a wide variety of biomolecules including DNA, RNA, fatty acid groups, cholesterol, and proteins [2]. In many practical applications of electroporation, it is important to avoid any contamination of tissues or samples. For example, when using electroporation to extract RNA and proteins (e-biopsy) [3], any damage to these molecules is undesirable.
The aim of this work was to study the formation of hypochlorous 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. To estimate the formation of hypochlorous acid, fluorescent indicator of hypochlorite 3’-p-Aminophenyl fluorescein (APF) [4] was used 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 aluminium 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. Incubation of erythrocytes with HOCl led to haemolysis of erythrocytes. HOCl-induced haemolysis was mediated by increased permeability of the cell plasma membrane to ions and small molecules. The estimated radius of permeable structures, which appeared in the plasma membrane of erythrocytes as a result of the exposure to hypochlorous acid, 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].
It can be concluded that hypochlorous acid can be formed as a result of electrolysis during high-voltage pulses, which are usually utilized for cell electroporation. Hypochlorous acid can increase permeability of the cell plasma membrane to ions and small molecules, which can cause the reduction of the cell viability.
Keywords:
hypochlorous acid, reactive oxygen species, electrochemical reactions, pore size, electroporation
Refs:
[1] G. Saulis, R. Rodaite-Riseviciene, V.S. Dainauskaite, R. Saule, Electrochemical processes occurring during high-voltage electric pulses and their importance for the technology of food processing by pulsed electric fields, in: Ravishankar Rai, V. (Eds.), John Wiley & Sons, West Sussex, 2016, pp. 575-591.
[2] C.M.C. Andres, J.M. Perez de la Lastra, C.A. Juan, F.J. Plou, E. Perez-Lebena, Int. J. Mol. Sci. 23 (2022) 10735.
[3] A. Golberg, J. Sheviryov, O. Solomon, L. Anavy, Z. Yakhini, Sci. Rep. 9 (2019) 15750.
[4] K. Setsukinai, Y. Urano, K. Kakinuma, H.J. Majima, T. Nagano, J. Biol. Chem. 278 (2003) 3170-3175.
[5] I.R. Freshney, Culture of animal cells: a manual of basic techniques, John Wiley & Sons, Inc., New York, 2000
[6] G. Saulis, Biomed. Sci. Instrum. 35 (1999) 291-296.
Topic 1:
1. Biological responses (molecular, subcellular, cellular and intercellular)
Topic 2:
2. Biophysics and biochemistry of interaction mechanisms
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