Glutathione‐dependent reductive stress triggers mitochondrial oxidation and cytotoxicity

<jats:p> To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss‐ and gain‐of‐function GSH content <jats:italic>in vitro</jats:italic> . We monitored the redox events in the cytosol and mitochondria using reduction‐oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> or the Trx reductase inhibitor 1‐chloro‐2,4‐dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, <jats:italic>E</jats:italic> <jats:sub>hc</jats:sub> (GSSG/2GSH), respectively. In contrast, <jats:italic>N</jats:italic> ‐acetyl‐L‐cysteine (NAC) treatment in H9c2 cells, or overexpression of either the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) or GCL modifier subunit (GCLM) in human embryonic kidney 293 T (HEK293T) cells, led to 3‐ to 4‐fold increase of GSH and caused 7 or 12 mV more reducing <jats:italic>E</jats:italic> <jats:sub>hc</jats:sub> , respectively. This condition paradoxically increased the level of mitochondrial oxidation, as demonstrated by redox shifts in mitochondrial roGFP and Trx2. Lastly, either NAC treatment (EC <jats:sub>50</jats:sub> 4 mM) or either GCLC or GCLM overexpression exhibited increased cytotoxicity and the susceptibility to the more reducing milieu was achieved at decreased levels of ROS. Taken together, our findings reveal a novel mechanism by which GSH‐dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.—Zhang, H., Limphong, P., Pieper, J., Liu, Q., Rodesch, C. K., Christians, E., Benjamin, I. J. Glutathione‐dependent reductive stress triggers mitochondrial oxidation and cytotoxicity. <jats:italic>FASEB J.</jats:italic> 26, 1442–1451 (2012). <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.fasebj.org">www.fasebj.org</jats:ext-link> </jats:p>