US EPA

4325229 

Journal Article 

Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity 

Zhang, H; Limphong, P; Pieper, J; Liu, Q; Rodesch, CK; Christians, E; Benjamin, IJ 

2012 

Yes 

FASEB Journal
ISSN: 0892-6638
EISSN: 1530-6860 

26 

4 

1442-1451 

English 

22202674 

10.1096/fj.11-199869 

WOS:000302359700007 

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 in vitro. 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(2)O(2) 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, E(hc) (GSSG/2GSH), respectively. In contrast, N-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 E(hc), 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(50) 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. 

redox potential; thioredoxin; roGFPs; reactive oxygen species