Pulsed power treatment has been used to induce regulated cell death (RCD) in cells or ablate tumors in animals. A subset of pulsed power as electroporation with high voltage and pulse duration of milliseconds is used for biomedical treatment to induce pores in the plasma membrane of cells. Nanosecond Pulsed Electric Fields (nsPEFs)– an extension of electroporation, uses waveforms with pulse durations on the order of 10-900 nanoseconds. nsPEF treatment has demonstrated intracellular effects for potential biomedical applications. In this work, nsPEF treatment is used to demonstrate changes that affect viability, plasma membrane permeability ROS (Reactive Oxygen Species) in the cytosol and mitochondria, and Electron Transport Chain (ETC) in cell cultures. The preferential effectiveness of nsPEF on cultured cancerous versus non-cancerous breast epithelial cells is shown. The nsPEF treatment compromised the integrity of the plasma membrane and outer mitochondrial membrane (OMM), without affecting the integrity of the inner mitochondrial membrane (IMM). Also, nsPEF - changes in redox proteins such as NADH dehydrogenase both in the plasma membrane and in Complex I. A reduced cellular oxygen consumption after nsPEFs treatment indicates an alteration of the ETC at Complex I in intact and permeabilized cells as well as in isolated hepatocyte mitochondria This evidence suggests a new paradigm for considering nsPEF effects on cell function. The combination of nsPEF and rotenone synergistically enhanced ROS production in intact cells suggesting that nsPEF and rotenone act at different Complex I sites. These studies establish how nsPEF impacts several intracellular mechanisms that disrupt mitochondrial function directly on Complex I and IV and indirectly by Ca2+- and ROS-mediated mechanisms that cooperate to collapse the mitochondrial membrane potential and cause cell death. Together, these observations provide new evidence for mechanisms triggered by nsPEF treatment for inducing cancer cell death and changing cellular metabolism in ways useful for treating cancer and other diseases in clinical settings
