Effect of thioacetamide on the hepatic expression of gamma-glutamylcysteine synthetase subunits in the rat

Glutathione (GSH) is the main nonprotein thiol important in antioxidant def ense and maintenance of the intracellular redox state. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, g amma-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 stress-induced transcriptional up-reg ulation of both subunits are antioxidant response element (ARE) and activat or protein-1 (AP-1) site. The nuclear factor-kappa B (NF-kappa B) binding s ite may also regulate the heavy subunit. Increased GSH and gamma-glutamyltr anspeptidase are often observed in preneoplastic hepatocyte nodules and may be important in hepatocarcinogenesis. The current work examined the effect of a commonly used hepatocarcinogen, thioacetamide (TAA), on the expressio n of GCS subunits. After 3 weeks of TAA treatment, liver GSH level remained unchanged despite significant oxidative stress as measured by the thiobarb ituric acid reactive substance assay. The mRNA levels of GCS-HS and GCS-LS increased six- and fourfold, respectively, and the protein level of GCS-HS and GCS activity all increased. Electrophorectic mobility shift assay showe d binding to ARE, AP-1, and NF-kappa B probes all increased. These results suggest TAA treatment increased hepatic GCS subunit expression and GCS acti vity by inducing oxidative stress and increasing the binding to redox-sensi tive cis-acting elements important for transcriptional upregulation of GCS. This is the first in vivo study that examined the effect of a hepatocarcin ogen on GCS expression, (C) 1999 Academic Press.