Determination of glutathione, mitochondrial transmembrane potential, and cytotoxicity in H9c2 cardiomyoblasts exposed to reactive oxygen and nitrogen species.

Abstract

Quantitative measurement of cellular oxidative stress (COS) and cytotoxicity are important to establish their significance in pathophysiologic conditions and disease states. So far, ample methods have been described to determine these processes based on spectrophotometric analysis. The application of simple, rapid, and sensitive fluorescence methods to determine the cytotoxicity and COS is described in the present chapter. Murine H9c2 cells were exposed to various free radical and non-free radical oxidants through use of diethylamine NONOate, 3-morpholinosydnonimine (SIN-1), and a synthetic preparation of peroxynitrite (PN). The viability of control and the treated H9c2 cells was measured based on the reduction of resazurin to resorufin which generates a fluorescent signal. The mitochondrial membrane potential was quantified by determining the cellular uptake of a fluorescent dye, (5,5('),6,6(')-tetrachloro-1,1(')-3,3(')-tetraethylbenzimidazolcarbocyanine iodide (JC-1)) and its segregation in the mitochondrial fraction. The intracellular GSH was determined by assaying the glutathione-S-transferase (GST)-catalyzed conjugation of GSH to monochlorobimane. This chapter describes the feasibility and potential of the above-described fluorescence approach as simple alternative methods to determine reactive oxygen and nitrogen species-induced cytotoxicity and oxidative stress using H9c2 cardiomyoblasts as a model system.