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Abstract
The use of chemilumigenic probes to monitor the production of chemically induced reactive oxygen species in situ has been hampered by commonly occurring interference of test compounds with radical intermediates of the probe and by the lack of suitable on-line detection systems. In this study we have explored the ability of the cell-permeable agent luminal to assess reactive oxygen species generated from redox-cycling compounds in cultured rat hepatocytes. Luminol-derived chemiluminescence was induced by exposure of hepatocytes to the 1,4-naphthoquinone drug menadione in a concentration-dependent manner. The luminescence signal was quantitated by integrating the light emission over time using a temperature-controlled luminescence plate reader. The signal was peroxidase-dependent and catalase-sensitive, indicating that extracellular hydrogen peroxide was detected. It was possible to rank a series of cytotoxic quinones according to their potential to undergo redox cycling. This assay includes rigorous controls for possible interference of quinone-containing compounds with light emission from the luminol reaction and for chemical quenching due to a reaction between the compound and a luminol radical intermediate. Taken together, this method, which we developed for use in multiwell plates, offers a fast and reliable tool for detecting the redox-cycling compound-derived production of reactive oxygen species in cultured hepatocytes.