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

Abstract

High expression of the aflatoxin B 1 (AFB 1 )-8,9-epoxide-conjugating glutathione S-transferase A3 (mGSTA3) subunit in mouse liver confers intrinsic resistance to AFB 1 hepatocarcinogenesis. It is not known how the gene encoding this protein is regulated. The murine mGSTA3 gene has been identified using bioinformatics. It localizes to mouse chromosome 1 (A3-4), spans approximately 24.6 kilobases (kb) of DNA, and comprises seven exons. High levels of mGSTA3 mRNA are present in organs associated with detoxification. Expression of mGSTA3 in Hepa1c1c7 mouse hepatoma cells was found to be inducible by sulforaphane, an organic isothiocyanate that can transcriptionally activate genes through the antioxidant response element (ARE). Sulforaphane also induced transcription of a luciferase reporter containing a 1.5 kb fragment of the mGSTA3 5Ј-upstream region. A putative ARE, with sequence 5Ј-TGACATTGC-3Ј, was identified within this fragment, approximately 150 base pairs upstream of exon 1. Mutation of this sequence abrogated both basal and sulforaphane-inducible reporter activity. Overexpression of the basic-region leucine zipper Nrf2 transcription factor augmented activity of the mGSTA3-luciferase reporter through this ARE. Electrophoretic mobility shift assays demonstrated that Nrf2 binds the mGSTA3 ARE. Measurement of mGSTA3 mRNA levels in tissues isolated from both wild-type and nrf2-null mice revealed that loss of the Nrf2 transcription factor is associated with a reduction in basal expression of mGSTA3. Collectively, these data demonstrate a role for Nrf2 and the ARE in regulating transcription of mGSTA3.

Glutathione Peroxidase 2, the Major Cigarette Smoke–Inducible Isoform of GPX in Lungs, Is Regulated by Nrf2

“…To confirm the specificity of the inhibition, the siCONTROL nontargeting siRNA 1 (SS siRNA) with microarray-defined signature was used as a negative control. Cells in the exponential growth phase were plated in 6-well plates at a density of 5 ϫ 10 5 cells/well, grown for 12 h, and then transfected with 20 pmol 24,48, and 72 h after transfection using assay on demand primers and probe sets from Applied Biosystems. Assays were performed using the ABI 7000 Taqman system (Applied Biosystems).…”

Dual effects of phloretin on aflatoxin B1 metabolism: Activation and detoxification of aflatoxin B1

“…Therefore, ARE was not found to be a major determinant in the expression of GST isozyme genes induced by phloretin in AML 12 cells. Jowsey et al reported that the loss of the Nrf2 transcription factor was associated with the reduced expression of mGSTA3 mRNA [49]. The expression of mGSTA3 in Nrf2-null liver was reduced to 45% that of the levels in wild-type tissue.…”

Activation of antioxidant defense during dehydration stress in the African clawed frog

“…To assess this, the expression of selected proteins from two families (GSTs and AKRs) that are known to be transcriptionally activated by Nrf2 were assessed (Chanas et al, 2002;Jowsey et al, 2003;Kang et al, 2007;Nishinaka and Yabe-Nishimur, 2005;Ellis et al, 2003). The multiclass family of GST enzymes functions to detoxify xenobiotic compounds.…”