Monitoring an oxidative stress mechanism at a single human fibroblast

Abstract

Easily oxidizable substances inside human diploid fibroblast cell strains were monitored amperometrically with a platinized carbon-fiber microelectrode. The experiment involved positioning a microelectrode over a single biological cell, forcing the electrode tip into the cell via micromanipulator control, and measuring the transient current corresponding to the complete electrolysis of electroactive species released by the cell. A second series of experiments involved puncturing a hole into the cell with a micropipet and measuring the transient current corresponding to the complete electrolysis of electroactive species emitted by the cell with an electrode positioned above the cell. The selectivity of both amperometric measurements was demonstrated through the use of known hydrogen peroxide scavengers (added catalase or intracellular peroxidase + added o-dianisidine) to the media bathing the cells. The abolition of the amperometric signal under these conditions suggested that hydrogen peroxide was the primary substance detected. The magnitude and the time course of the transient current measured implied that the hydrogen peroxide detected was not only that initially present in the cell before its membrane was pierced but represented mostly an oxidative stress response of the cell to its injury.

Nitrogen monoxide and oxidative stress: composition and intensity of cellular oxidative bursts cocktail. A study through artificial electrochemical synapses on single human fibroblasts

“…In this review, we wish therefore to summarize the results achieved so far in our laboratory through precise electroanalytical investigations of the nature, intensity and kinetics of oxidative stress [32][33][34][35]. To complete and strengthen this view, we wish also to include some recent data on the effect of inhibitors which have not been reported so far [36].…”

“…The exposed carbon disk was beveled and polished, and platinized so as to increase its electrochemical activity towards small oxygen-containing molecules expected to be released in oxidative bursts (vide supra). The electrode was placed in the desired position with a micromanipulator under optical microscopy control [33].Oxidative bursts were stimulated by the fast pricking of the cell membrane with a sealed patch-clamp micropipette (ca. 1 µm radius) moved with a second micromanipulator [33].…”

“…The electrode was placed in the desired position with a micromanipulator under optical microscopy control [33].…”

“…All these undesirable effects are prevented when the potential range examined is restricted to any values above 0.275 V vs. SSCE whenever the electrode is close to the cell (i.e., within micrometers) or when, as in the experiments shown in figure 1c, the electrode is far away from the cell [33]. However, in this second situation, collection efficiencies are small, and moreover several shortlived species may not survive the several seconds-long diffusional flights required to reach the electrode surface (vide infra).Because of the dendritic platinization required to obtain decent electrochemical responses [33], the ultramicroelectrode capacitances are excessively large and so are capacitive currents. On the other hand, pricking the cell membrane creates a connection between its cytosol and the extracellular fluid where the electrode is positioned.…”

Nanoelectrochemistry of mammalian cells

“…Only a few examples of intracellular electrochemical measurements have been reported to date (21)(22)(23). One of the main obstacles here is the electrode size and geometry; even a sharp, conical electrode puncturing a lipid membrane may cause a solution leakage (20).…”