The cytotoxicity of menadione on hepatocytes was studied by using the substrate generation͞tip collection mode of scanning electrochemical microscopy by exposing the cells to menadione and detecting the menadione-S-glutathione conjugate (thiodione) that is formed during the cellular detoxication process and is exported from the cell by an ATP-dependent pump. This efflux was electrochemically detected and allowed scanning electrochemical microscopy monitoring and imaging of single cells and groups of highly confluent live cells. Based on a constant flux model, Ϸ6 ؋ 10 6 molecules of thiodione per cell per second are exported from monolayer cultures of Hep G2 cells.electrochemistry ͉ SECM ͉ oxidative stress ͉ ultramicroelectrode ͉ glutathione C ytotoxic effects of menadione on hepatocytes were investigated by scanning electrochemical microscopy (SECM), a technique that allows one to detect electroactive species at an ultramicroelectrode tip that can be positioned with high resolution. Here, the substrate generation͞tip collection mode of SECM was used to monitor the concentration of thiodione from both highly confluent and isolated Hep G2 cells. Thiodione is a biological metabolite that is actively exported out of the cells by a glutathione S-conjugate pump after the application of oxidative stress by menadione on the hepatocytes (Fig. 1). The efflux of thiodione from both isolated and highly confluent (75-100%) cells can be imaged by SECM, and time profiles of the export can also be obtained after exposure to a cytotoxic concentration of menadione. A simplified model was used to treat the concentration of thiodione from highly confluent cells and to estimate the flux per cell. A similar model has previously been applied to an electrochemical study of doxorubicin transport from Chinese hamster ovarian cells (1).SECM has previously been used with mammalian and other cells and has provided useful information about the permeability of cellular membranes to a wide variety of redox couples. The regeneration reaction of different mediators at human breast cells (2) and Rhodobacter sphaeroides (3) was monitored by the feedback mode of SECM, allowing one to distinguish between normal and malignant cells. A theoretical kinetic treatment of these processes is reported in ref. [4][5][6][7][8][9]) also carried out live-cell studies and monitored respiration rate changes from oxygen reduction profiles for different cell types. In these experiments, oxygen present in solution is consumed by the living organism, so that its concentration is lower near the cell surface. This consumption leads to a lower measured oxygen reduction current when the tip is in close proximity to the cells. These SECM studies monitoring efflux from cells used the feedback approach. Menadione (10) has been used in previous studies because it readily enters the cell through the cell membrane, and we have used it in a study closely related to this one involving the use of SECM to monitor the behavior of yeast cells by using a substrate generation͞tip coll...