Hypoxic reduction in cellular glutathione levels requires mitochondrial reactive oxygen species

1 Abramson Family Cancer Research Institute and 2 Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Submitted 27 April 2004 ; accepted in final form 27 May 2004 When exposed to hypoxia (1.5% O 2 ), several cell types have been shown to increase production of reactive O 2 species derived from the mitochondrial electron transport chain (mtROS). The general physiological consequences of hypoxic mtROS production are not completely understood, although several groups have demonstrated that mtROS promote the stabilization and activity of hypoxia inducible factor-1 (HIF-1 ) transcription factor, alter cardiac myocyte contractility, and modulate Na + -K + -ATPase activity. To investigate the effects of hypoxia-induced mtROS on general cellular oxidative metabolism, we measured the levels of glutathione, a major cellular antioxidant, in response to hypoxic treatment. Our data indicate that HEK293 and Hep3B cells exposed to 1.5% O 2 exhibit a time-dependent decrease in cellular glutathione stores and concomitant inhibition of glutathione biosynthesis, which correlates to impaired transport of the substrate cystine. Using a combination of ROS scavengers, mitochondrial electron transport inhibitors, and mitochondrial DNA-deficient 0 cells, we demonstrate that this decrease in cellular glutathione levels is mediated by hypoxia-induced mtROS. Intriguingly, this effect is also inhibited by cyclohexamide but is not dependent on HIF-mediated transcription. Overall, these data suggest a novel HIF-independent role for mitochondrial ROS in regulating glutathione synthesis, and hence cellular oxidative homeostasis, during hypoxic exposure. hypoxia; mitochondria; hypoxia-inducible factor-1 ; cystine Address for reprint requests and other correspondence: B. Keith, 438 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104 (E-mail: bkeith{at}mail.med.upenn.edu ).