Glutathione (GSH) is important in antioxidant defense. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, ␥-glutamylcysteine synthetase (GCS). A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally. Cis-acting elements important for the oxidative stressinduced transcriptional up-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1). Nuclear factor-B (NF-B) may also regulate the heavy subunit. Chronic ethanol ingestion causes oxidative stress, increases AP-1 expression, and depletes hepatic GSH. Data conflict regarding GSH synthesis and are lacking regarding GCS subunit gene expression. We examined the effect of chronic ethanol ingestion on ARE, AP-1, and NF-B activity and GCS subunit expression. Male Wistar rats were fed an ethanol and high-fat (28.7% cal) diet intragastrically for 9 weeks. Liver GSH level fell by 40%, although GCS activity doubled. GCS-HS mRNA level doubled, whereas GCS-LS mRNA level remained unchanged. Electrophoretic mobility shift assay (EMSA) showed that binding to ARE, AP-1, and NF-B probes all increased. In conclusion, chronic ethanol ingestion increased GCS-HS expression and GCS activity by activating cis-acting elements important for transcriptional up-regulation of GCS-HS. GCS-LS mRNA level remained unchanged despite activation of ARE and AP-1, suggesting that negative transcriptional factors may be involved or the mRNA may be unstable. Despite induction in GCS activity, GSH level fell because of alterations in the other factors important in determining the steady-state GSH level. (HEPATOLOGY 1999;30:209-214.) Glutathione (GSH) is a tripeptide, ␥-glutamylcysteinylglycine, which defends against toxins and free radicals. 1 The cellular GSH level is determined by a balance of the rate of its synthesis and the rates of its utilization (conjugation) and loss (export). 2 Synthesizing GSH from its constituent amino acids involves two adenosine triphosphate-requiring enzymatic steps: forming ␥-glutamylcysteine from glutamate and cysteine, and forming GSH from ␥-glutamylcysteine and glycine. The rate of GSH synthesis is determined by the availability of cysteine and the activity of the rate-limiting enzyme, ␥-glutamylcysteine synthetase (GCS), which is subject to feedback-competitive inhibition by GSH (K i ϭ 2.3 mmol/L). 1-3 The GCS enzyme is composed of a heavy (Mr ϳ 73,000) and a light (Mr ϳ 30,000) subunit that are encoded for by different genes and dissociate under reducing conditions. 4,5 The heavy subunit (HS) exhibits all of the catalytic activity of the isolated enzyme as well as the feedback inhibition by GSH. 6 The light subunit (LS) is enzymatically inactive but plays an important regulatory function by lowering the K m of GCS for glutamate and raising the K i for GSH. 5,7 Recent studies have shown that both GCS subunits are up-regulated transcriptionally by oxidative stress. 1,[8][9][10][11][12][13] The 5Ј-flanking region of both human GCS subunits ha...

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