The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane

Abstract

Chinese hamster ovary (CHO) cells in suspension were subjected to pulsed electric fields suitable for electrically mediated gene transfer (pulse duration longer than 1 ms). Using the chemiluminescence probe lucigenin, we showed that a generation of reactive-oxygen species (oxidative jump) was present when the cells were electropermeabilised using millisecond pulses. The oxidative jump yield was controlled by the extent of alterations allowing permeabilisation within the electrically affected cell area, but showed a saturating dependence on the pulse duration over 1 ms. Cell electropulsation induced reversible and irreversible alterations of the membrane assembly. The oxidative stress was only present when the membrane permeabilisation was reversible. Irreversible electrical membrane disruption inhibited the oxidative jump. The oxidative jump was not a simple feedback effect of membrane electropermeabilisation. It strongly controlled long-term cell survival. This had to be associated with the cell-damaging action of reactive-oxygen species. However, for millisecond-cumulated pulse duration, an accumulation of a large number of short pulses (microsecond) was extremely lethal for cells, while no correlation with an increased oxidative jump was found. Cell responses, such as the production of free radicals, were present during electropermeabilisation of living cells and controlled partially the long-term behaviour of the pulsed cell.