Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1,2-dithiole-3-thione

Background: The induction of phase 2 enzymes by dithiolethiones such as olt ipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expressio n of at least some of these protective enzymes is mediated through the anti oxidant response element (ARE) found in the upstream regulatory region of m any phase 2 genes. The transcription factor Nrf2, which binds to the ARE, a ppears to be essential for the induction of prototypical phase 2 enzymes su ch as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductas e (NQO1) in vivo. Materials and Methods: In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and ch emopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-d isrupted mice by Northern blot hybridization and reverse transcriptase-poly merase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 f unction also affects the induction of a broader representation of phase 2 a nd antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, el ectromobility shift assay, and Western blot analysis. Results: Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. Howe ver, changes in levels of RNA transcripts following D3T treatment of nrf2-d isrupted mice were multidirectional, dependent upon the particular gene exa mined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hyd rolase and gamma -glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increa ses in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transf erase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were indu cible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B-1 aldehyde reductase and catalase d ecreased following D3T treatment in the mutant mice in the absence of any i nductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within severa l hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine G ST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed i ncreased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 su pershifted the DNA binding bands of these nuclear extracts. Moreover, immun oblot analysis indicated accumulation of Nrf2, in extracts prepared from he patic nuclei of D3T-treated mice at the same time points. Conclusions: Nrf2 plays a central role in the regulation of constitutive an d inducible expression of multiple phase 2 and antioxidative enzymes by che moprotective dithiolethiones in vivo, although patterns of response vary am ong different genes. Knowledge of the factors controlling the specificity o f actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective chemoprotective agents.