Effects of Nanosecond Pulsed Electric Fields on the Intracellular Function of HeLa Cells As Revealed by NADH Autofluorescence Microscopy

Abstract

The fluorescence lifetime of the endogenous fluorophore of reduced nicotinamide adenine dinucleotide (NADH) in HeLa cells is affected by the application of nanosecond pulsed electric fields (nsPEFs). In this study, we found that after nsPEF application, the fluorescence lifetime became longer and then decreased in a stepwise manner upon further application, irrespective of the pulse width in the range of 10–50 ns. This application time dependence of the NADH fluorescence lifetime is very similar to the time-lapse dependence of the NADH fluorescence lifetime following the addition of an apoptosis inducer, staurosporine. These results, as well as the membrane swelling and blebbing after the application of nsPEFs, indicate that apoptosis is also induced by the application of nsPEFs in HeLa cells. In contrast to the lifetime, the fluorescence intensity remarkably depended on the pulse width of the applied nsPEF. When the pulse width was as large as 50 ns, the intensity monotonically increased and was distributed over the entire cell as the application duration became longer. As the pulse width of the applied electric field became smaller, the magnitude of the field-induced increase in NADH fluorescence intensity decreased; the intensity was reduced by the electric field when the pulse width was as small as 10 ns. These results suggest that the mechanism of electric-field-induced apoptosis depends on the pulse width of the applied nsPEF.

Effects of Nanosecond Pulsed Electric Field on Intracellular NADH Autofluorescence: A Comparison between Normal and Cancer Cells

“…The observed pulse-width dependence of the electric field effect on the intensity and lifetime of NADH fluorescence in these normal and cancer cells is the same as the one observed in HeLa cells, 25 implying that these field effects are common in live cells. In HeLa cells, it was suggested that the so called mitochondrial permeability transition pore complex, which may induce apoptosis, is generated by the application of nsPEF, which has a pulse width as large as 50 ns.…”

“…In fact, some morphological changes were detected after application of nsPEF in our study in both the cell types, depending on the amplitude and pulse width of the applied electric field, as shown in Figures S5 and S6 . The detection of apoptosis using the fluorescence lifetime of NADH has a great advantage of confirming apoptosis with label-free detection, and this method has been applied in our previous study regarding the nanosecond pulsed field-induced apoptosis of HeLa cells 25 and also in a recent study by another group. 30 …”

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells

“…As shown in our previous reports of pulsed electric field effects on autofluorescence lifetime in different cell lines such as HeLa cells, WFB and W31 cells, and MRC‐5 and H661 cells, NADH fluorescence lifetime became longer after application of nanosecond pulsed electric field, that is, the nonradiative decay rate at the emitting state of NADH is decreased by applied electric fields. Therefore, there is also a possibility that the difference in the nonradiative decay rate of NADH and FAD in cancerous and normal cells might be induced by the effects of intracellular electric fields.…”

“…It has been recognized that NADH participates in the O 2 − generation process, as an electron donor . The short lifetime indicates the large nonradiative decay rate of NADH, which may be mainly attributed to a large electron transfer rate in cancerous cells through the generation of excessive O 2 − , compared to that in normal cells . Along with these reactions, oxidation of NADH increases the concentration of NAD + in cells, which may in turn affect the interaction between the isoalloxazine ring and the adenine ring of FAD .…”

“…These fluorophores which serve as electron donor and acceptor in cellular energy metabolism might reflect the microenvironment status and could become sensitive indicators not only for differentiating cancerous and normal cells and tissues, but also for monitoring the dynamics of apoptosis cell death, intracellular pH, cellular heterogeneities and oxidative stress. As a result, staining of exogenous fluorescence dyes or expressing fluorescent proteins are not necessary for observation, and subcellular activities along with biochemical and physiological changes can be monitored using the autofluorescence intensity and lifetime measurement techniques . The most commonly accepted and/or used optical method for detecting metabolic changes has been the fluorescence redox ratio by considering the different oxidation–reduction rates in normal and cancerous cells.…”