Activation of the NADPH oxidase in human fibroblasts by mechanical intrusion of a single cell with an ultramicroelectrode

Abstract

We present here a real-time and single cell study of an oxidative stress mechanism in human fibroblasts. Hydrogen peroxide released by a single normal or SV40-transformed human fibroblast was detected at the surface of an ultramicroelectrode while puncturing the cell membrane with the ultramicroelectrode tip itself or with a micropipette. This mechanical intrusion induced the emission of large quantities (10-15-10-14 mol) of H2O2 by the cell with a very short time delay (< 0.5 s). We show that this H2O2 production was an active neo-production by fibroblasts when the membrane was stressed by the cellular puncture and is a model which could mimic similar effects as particle (virus, bacteria, etc.) intrusion into the cell. Cell incubations in the presence of some inhibitors of the different NADPH oxidase enzymes, using ultramicroelectrode measurements of the short time effects (< 20 min) let us believe that an NADPH oxidase-like enzyme may be implicated in this induced-H2O2 generation. Phenylarsine oxide (PAO), a specific NADPH oxidase inhibitor, at concentrations between 0.5-50 μM seemed to quickly kill the transformed cells preferentially to the normal cells, pointing out for the future a possible anti-cancerous chemotherapic use.