Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance. The rate-limiting enzyme in the de novo synthesis of glutathione is ␥-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing catalytic and regulatory functions, respectively. Exposure of HepG2 cells to ␤-naphthoflavone (␤-NF) resulted in a time-and dose-dependent increase in the steady-state mRNA levels for both subunits. In order to identify sequences mediating the constitutive and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5 -flanking region (؊3802 to ؉465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells. Constitutive expression was maximally directed by sequences between ؊202 and ؉22 as well as by elements between ؊3802 to ؊2752. Glutathione (L-␥-glutamyl-L-cysteinyl-glycine, GSH), 1 a nonprotein sulfhydryl compound present in millimolar concentrations in virtually all cells, serves a myriad of cellular functions and plays a prominent role as an intracellular protectant (1, 2). GSH is an effective oxygen radical scavenger and serves as a critical co-factor in peroxide detoxification via a reaction catalyzed by glutathione peroxidase. Furthermore, conjugation with GSH is an integral step in the detoxification and elimination of diverse classes of toxic chemical compounds. The formation of hydrophilic glutathionyl conjugates is catalyzed by glutathione S-transferases, a family of isozymes that mediate the conjugation reaction in a substrate-dependent fashion (3). Long the object of interest from a toxicology perspective, the protective properties of GSH have assumed even further significance since GSH not only plays a critical role in protection of normal cells, but it has recently been implicated in protection of neoplastic cells from a number of chemotherapeutic agents that exert their cytotoxic effects via generation of reactive oxygen species or production of electrophilic intermediates (4, 5). The augmentation of GSH and GSH-related detoxification systems has also engendered considerable interest as a possible approach for the chemoprevention of cancer. Many chemical chemopreventive agents have been shown to exert an effect on GSH homeostasis or on other elements of GSH detoxification pathways (6 -8).Exposure of cells to a number of xenobiotic agents has been demonstrated to result in an increase in the total intracellular GSH content. In several cases (9 -16) where it has been examined, the increase in GSH has been attributed to an

Transactivation of phase II detoxification enzymes and antioxidant proteins is mediated by the Cap'NCollar transcription factor, Nrf2, which is sequestered in the cytoplasm by the actin-binding protein Keap1. Mutation of a conserved serine (S104A) within the Keap1 BTB/POZ domain disrupts Keap1 dimerization and eliminates the ability of Keap1 to sequester Nrf2 in the cytoplasm and repress Nrf2 transactivation. Disruption of endogenous Keap1 dimerization using BTB/POZ dominant negative proteins also inhibits the ability of Keap1 to retain Nrf2 in the cytoplasm. Exposure to an electrophilic agent that induces Nrf2 release and nuclear translocation disrupts formation of a Keap1 complex in vivo. Collectively, these data support the conclusion that Keap1 dimerization is required for Nrf2 sequestration and transcriptional repression. Furthermore, exposure to inducing agents disrupts the Keap1 dimerization function and results in Nrf2 release.

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Exposure of HepG2 cells to ␤-naphthoflavone (␤-NFMutational analyses indicate that basal expression of the GCS l gene is directed by a consensus AP-1-binding site located 33 base pairs upstream of a consensus electrophile responsive element (EpRE) sequence; both ciselements are capable of supporting ␤-NF inducibility. Elimination of the inducible response requires simultaneous mutation of both sequences, however, in the presence of an intact EpRE the upstream AP-1 site is irrelevant to induction. Regulation of expression of both human GCS subunit genes in response to ␤-NF is therefore mediated by cis-elements satisfying the consensus core EpRE motif.

Gamma-glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5'-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-kappaB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery.