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 defense and maintenance of the intracellular redox state. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, gamma-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-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1) site. The nuclear factor-kappaB (NF-kappaB) binding site may also regulate the heavy subunit. Increased GSH and gamma-glutamyltranspeptidase 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 expression of GCS subunits. After 3 weeks of TAA treatment, liver GSH level remained unchanged despite significant oxidative stress as measured by the thiobarbituric 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 showed binding to ARE, AP-1, and NF-kappaB probes all increased. These results suggest TAA treatment increased hepatic GCS subunit expression and GCS activity by inducing oxidative stress and increasing the binding to redox-sensitive cis-acting elements important for transcriptional up-regulation of GCS. This is the first in vivo study that examined the effect of a hepatocarcinogen on GCS expression.
Lu
S C
SC
USC Liver Disease Research Center, USC School of Medicine, Los Angeles, California, 90033, USA. shellylu@hsc.usc.edu
Huang
Z Z
ZZ
Yang
H
H
Tsukamoto
H
H
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013853:Thioacetamide / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002273:Carcinogens; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D013853:Thioacetamide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione