QUINONE-INDUCED OXIDATIVE STRESS ELEVATES GLUTATHIONE AND INDUCES GAMMA-GLUTAMYLCYSTEINE SYNTHETASE-ACTIVITY IN RAT LUNG EPITHELIAL L2 CELLS

Glutathione (GSH) is one of the most important physiological antioxida nts involved in detoxification of hydrogen peroxide and lipid hydroper oxide. Previous studies have shown that cells can maintain and even in crease cellular GSH content in response to sublethal oxidative stress. We hypothesized that gamma-glutamylcysteine synthetase (gamma GCS), t he rate-limiting enzyme in de novo GSH synthesis, could be induced by oxidative stress. Rat lung epithelial L2 cells were challenged with 2, 3-dimethoxy-1,4 naphthoquinone (DMNQ), which generates O-2(radical ani on) and H2O2 continuously through redox cycling. Exposure of confluent L2 cells with sublethal doses of DMNQ caused sustained elevation of c ellular GSH levels over a 24-h period (to 2.5-fold with 10 mu M). DMNQ caused increases in gamma GCS activity (70% at 24 h with 10 mu M), th e gamma GCS catalytic heavy subunit (gamma GCS-HS) protein level, and gamma GCS-HS mRNA content (similar to 4 fold after 6 h with 10 mu M). The elevation of gamma GCS-HS mRNA by DMNQ was eliminated by co-incuba tion with actinomycin D. Nuclear run-on experiments demonstrated that the transcriptional rate of the gamma GCS-HS gene was increased by 3- or 6 h exposure to 10 mu M DMNQ. Our results suggested that the induct ion of de novo GSH synthesis by naphthoquinone-induced oxidative stres s is associated with the transcriptional activation of the gamma GCS-H S gene and the subsequent elevation in gamma GCS activity. Unlike simp ler quinones, DMNQ cannot form a GSH conjugate. Thus, the induction of gamma GCS-HS gene transcription does not require formation of an elec trophile-glutathione conjugate.