Characterization of the rat glutathione S-transferase Yc2 subunit gene, GSTA5: identification of a putative antioxidant-responsive element in the 5′-flanking region of rat GSTA5 that may mediate chemoprotection against aflatoxin B1

Abstract

We have isolated and characterized genomic DNA encoding the rat glutathione S-transferase Yc2 subunit. This protein is now referred to as rGSTA5 and is noteworthy because of its high activity towards aflatoxin B1-8,9-epoxide, its marked inducibility by chemoprotectors, its sex-specific regulation, and its overexpression in hepatoma and preneoplastic nodules. The rGSTA5 gene, which was isolated on two overlapping bacteriophage λ clones, is approx. 12 kb in length and, unlike other class Alpha genes described to date, it comprises six exons. The transcription start site has been identified 228 bp upstream from the ATG translational initiation codon, and is situated 51 bp downstream from a consensus TATA-box. Deletion analysis, using luciferase reporter constructs, has shown that the region between -177 bp and +65 bp from the transcriptional start site contains a functional promoter. Computer-assisted analysis of the upstream sequence has indicated the presence of an antioxidant-responsive element (ARE), and several elements thought to be required for tissue-specific expression of the enzyme. In addition, several putative oestrogen-responsive half sites were observed in both upstream and intronic sequences.

Oltipraz Is a Bifunctional Inducer Activating Both Phase I and Phase II Drug-Metabolizing Enzymes via the Xenobiotic Responsive Element

“…The locations of ARE (Ϫ430 to Ϫ422) and XRE (ϩ437 to ϩ456) sequences (Pulford and Hayes, 1996) are shown in the promoter map (Fig. 1A).…”

“…XRE is present in many phase II detoxifying genes, such as rGSTA2 (Rushmore et al, 1990), rGSTA5 (Pulford and Hayes, 1996), and rQR (Favreau and Pickett, 1991), and it is also present in some phase I genes, such as CYP1A1 (Corchero et al, 2001). Therefore, we hypothesize that oltipraz exposure can affect both phase I and II drug-metabolizing enzymes.…”

“…Moreover, rGSTA5 is of critical importance in detoxifying the hepatocellular carcinogen AFB1, because it exhibits approximately 180-fold greater activity toward AFB1-8,9-epoxide than rGSTA3 and 1000-fold greater activity than rGSTA1 and rGSTA2 (Hayes et al, 1991;Pulford and Hayes, 1996).…”

“…Rat GSTA5 stands out from other drug-metabolizing enzymes in that it consistently shows a high level of inducibility by chemopreventive agents such as oltipraz, and is highly efficient at metabolizing AFB1 (Hayes et al, 1991). Both ARE and XRE homologous sequences are present in the promoter area of rGSTA5 (Pulford and Hayes, 1996). We found that the ARE of rG-STA5 is not fully functional and has lost responsiveness to oltipraz activation, whereas the XRE, located downstream of the transcription initiation site, functions as the cis-acting regulatory element responsible for oltipraz-induced expression of rGSTA5.…”

Expression of the Aflatoxin B1-8,9-Epoxide-Metabolizing Murine GlutathioneS-Transferase A3 Subunit Is Regulated by the Nrf2 Transcription Factor through an Antioxidant Response Element

“…However, at 24.6 kb, mGSTA3 is relatively large, in that other characterized class ␣ GST genes span between 11 and 15 kb. Interestingly, the rGSTA5 gene is predicted to comprise only six exons (Pulford and Hayes, 1996). It apparently lacks the first exon equivalent to that encoding the 5Ј-untranslated sequence in other class ␣ GST genes.…”