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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

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Abstract

Background

The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (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 chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility 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. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and γ-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases 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-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive 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 several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points.

Conclusions

Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective chemoprotective agents.

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Abbreviations

D3T:

3H-1,2-dithiole-3-thione

ARE:

antioxidant response element, NF-E2, erythroid-specific nuclear factor

NFE2:

NF-E2 consensus sequence

GST:

glutathione S-transferase

NQO1:

NAD(P)H:quinone oxidoreductase

UGT1A6:

UDP-glucuronyltransferase 1A6

mEH:

microsomal epoxide hydrolase

gGCSr:

g-glutamylcyteine synthetase regulatory chain

AFAR:

aflatoxin B1 aldehyde reductase

HO-1:

heme oxygenase-1

MnSOD:

manganese superoxide dismutase

EMSA:

electromobility shift assay

βNF:

β-naphthoflavone

EQ:

ethoxyquin

HEPES:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

PMSF:

phenylmethylsulfonylfluoride

References

  1. Wattenberg LW, Bueding E. (1986) Inhibitory effects of 5-(2-pyrazyinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) oncarcinogenesis induced by benzo[a]pyrene, diethylnitrosamine and uracil mustard. Carcinogenesis 7: 1379–1388.

    Article  CAS  PubMed  Google Scholar 

  2. Kensler W, Egner PA, Dolan P, Groopman JD, Roebuck BD. (1987) Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazyinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones. Cancer Res. 47: 4271–4277.

    PubMed  CAS  Google Scholar 

  3. Kensler TW, Groopman JD, Sutter TR, Curphey TJ, Roebuck BD. (1999) Development of cancer chemopreventive agents: Oltipraz as a paradigm. Chem. Res. Toxicol. 12: 113–126.

    Article  CAS  PubMed  Google Scholar 

  4. Rushmore TH, King RG, Paulson KE, Pickett CB. (1990) Regulation of glutathione S-transferase Ya subunit gene expression: Identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. Proc. Natl. Acad. Sci. U.S.A. 87: 3826–3830.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Friling RS, Bensimon A, Tichauer T, Daniel V. (1990) Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proc. Natl. Acad. Sci. U.S.A. 87: 6258–6262.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Okuda A, Imagawa M, Maeda Y, Sakai M, Muramatsu MJ. (1989) Structural and functional analysis of an enhancer GPE1 having a phorbol 12-O-tetradecanoate-13-acetate responsive element-like sequence found in the rat glutathione transferase P gene. J. Biol. Chem. 264: 16919–16926.

    PubMed  CAS  Google Scholar 

  7. Li Y, Jaiswal AK. (1992) Regulation of human NAD(P)H: quinone oxidoreductase gene: Role of AP-1 binding site contained within human antioxidant response element. J. Biol. Chem. 267: 15097–15104.

    PubMed  CAS  Google Scholar 

  8. Favreau LV, Pickett CB. (1995) The rat quinone reductase antioxidant response element: Identification of the nucleotide sequence required for basal and inducible activity and detection of antioxidant response element-binding proteins in hepatoma and non-hepatoma cell lines. J. Biol. Chem. 270: 24468–24474.

    Article  CAS  PubMed  Google Scholar 

  9. Primiano T, Gastel JA, Kensler TW, Sutter TR. (1996) Isolation of cDNAs representing dithiolthione-responsive genes. Carcinogenesis 17: 2297–2303.

    Article  CAS  PubMed  Google Scholar 

  10. Moinova HR, Mulcahy RT. (1998) An electrophile Responsive element (EpRE) regulation of β-naphthoflavone induction of the human γ-glutamylcysteine synthetase regulatory subunit gene; Constitutive expression is mediated by an adjacent AP-1 site. J. Biol. Chem. 273: 14683–14689, 1998.

    Article  CAS  PubMed  Google Scholar 

  11. Prestera T, Talalay P, Alam J, Ahn YI, Lee PJ, Choi AM. (1995) Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: Regulation by upstream antioxidant-responsive elements (ARE). Mol. Med. 1: 827–837.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Jaiswal AK. (1994) Antioxidant response element. Biochem. Pharmacol. 48: 439–444.

    Article  CAS  PubMed  Google Scholar 

  13. Friling RS, Bergelson S, Daniel V. (1992) Two adjacent AP-1-like binding sites from the electrophile-response element of the murine glutathione S-transferase Ya subunit gene. Proc. Natl. Acad. Sci. U.S.A. 89: 668–672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xie T, Belinsky M, Xu Y, Jaiswal AK. (1995) ARE- and TRE-mediated regulation of gene expression: Response to xenobiotics and antioxidants. J. Biol. Chem. 270: 6894–6900.

    Article  CAS  PubMed  Google Scholar 

  15. Favreau LV and Pickett CB. (1993) Transcriptional regulation of the rat NAD(P)H quinone reductase gene: Characterization of a DNA-protein interaction of the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol-13-acetate. J. Biol. Chem. 268: 19875–19881.

    PubMed  CAS  Google Scholar 

  16. Nguyen T, Rushmore TH, Pickett CB. (1994) Transcriptional regulation of rat liver glutathione S-transferase Ya subunit gene: Analysis of the antioxidant response element and its activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. J. Biol. Chem. 269: 13656–13662.

    PubMed  CAS  Google Scholar 

  17. Wang B, Williamson G. (1996) Transcriptional regulation of the human NAD(P)Hquinone oxidoreductase (NQO1) gene by monofunctional inducers. Biochim. Biophys. Acta. 1307: 104–110.

    Article  PubMed  Google Scholar 

  18. Wasserman WW, Fahl WE. (1997) Comprehensive analysis of proteins which interact with the antioxidant responsive element: Correlation of ARE-BP-1 with the chemoprotective induction response. Arch. Biochem. Biophys. 344: 387–396.

    Article  CAS  PubMed  Google Scholar 

  19. Wang B, Williamson G. (1994) Detection of nuclear protein which binds specifically to the antioxidant responsive element (ARE) of the human NAD(P)H:quinone oxidoreductase gene. Biochim. Biophys. Acta. 1219: 645–652.

    Article  PubMed  Google Scholar 

  20. Venugopal R, Jaiswal AK. (1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H: quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. U.S.A. 93: 14960–14965.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Andrews NC, Kotkow KJ, Ney PA, Erdjument-Bromage H, Tempst P, Orkin SH. (1993) The ubiquitous subunit of erythroid transcription factor NF-E2 is a small basic-leucine zipper protein related to the v-maf oncogene. Proc. Natl. Acad. Sci. U.S.A. 90: 11488–11492.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Igarashi K, Itoh K, Motohashi H, et al. (1995) Activity and expression of murine small maf family protein MafK. J. Biol. Chem. 270: 7615–7624.

    Article  CAS  PubMed  Google Scholar 

  23. Chan JY, Han XL, Kan YW. (1993) Cloning of Nrf1, and NF-E2-related transcription factor, by genetic selection in yeast. Proc. Natl. Acad. Sci. U.S.A. 90: 11371–11375.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Moi P, Chan K, Asunis I, Cao A, Kan YW. (1994) Isolation of NF-E2-related factor (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP-1 repeat of the β-globin locus control region. Proc. Natl. Acad. Sci. U.S.A. 91: 9926–9930.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kobayashi A, Ito E, Toki T, et al. (1999) Molecular cloning and functional characterization of a new Cap’n Collar family transcription factor Nrf3. J. Biol. Chem. 274: 6443–6452.

    Article  CAS  PubMed  Google Scholar 

  26. Anrew NC, Erdjumant-Bromage H, Davidson MB, Tempst P, Orkin SH. (1993) Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature 362: 722–728.

    Article  Google Scholar 

  27. Itoh K, Chiba T, Takahashi S, et al. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme gene through antioxidant response elements. Biochem. Biophys. Res. Commun. 236: 313–322.

    Article  CAS  PubMed  Google Scholar 

  28. Alam J, Stewart D, Touchard C, Boinapally S, Choi AMK, Cook JL. (1999) Nrf2, a Cap’n Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J. Biol. Chem. 274: 26071–26078.

    Article  CAS  PubMed  Google Scholar 

  29. Egner PA, Kensler TW, Prestera T, et al. (1994) Regulation of phase 2 enzyme induction by oltipraz and other dithiolethiones. Carcinogenesis 15: 177–181.

    Article  CAS  PubMed  Google Scholar 

  30. Buetler TM, Eaton DL. (1992) Complementary DNA cloning, messenger RNA expression, and induction of α-class glutathione S-transferase in mouse tissues. Cancer Res. 52: 314–318.

    PubMed  CAS  Google Scholar 

  31. Prochaska HJ, Santamaria AB, Talalay P. (1992) Rapid detection of inducers of enzymes that protect against carcinogens. Proc. Natl. Acad. Sci. U.S.A. 89: 2394–2398.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Habig WH, Pabst MJ, Jakoby WB. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 219: 7130–7139.

    Google Scholar 

  33. Chomczynski P, Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.

    Article  CAS  PubMed  Google Scholar 

  34. Kim ND, Kwak MK, Kim SG. (1997) Inhibition of cytochrome P-450 2E1 expression by 2-(allylthio)pyrazine, a potential chemoprotective agent: Hepatoprotective effects. Biochem. Pharmacol. 53: 261–269.

    Article  CAS  PubMed  Google Scholar 

  35. Dignam JD, Lebovitz RM, Roeder RG. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11: 1475–1489.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Otieno MA, Kensler TW, Guyton KZ.(2000) Chemoprotective 3H-1,2-dithiole-3-thione induces antioxidant genes in vivo. Free Radic. Biol. Med. 28: 944–952.

    Article  CAS  PubMed  Google Scholar 

  37. Buetler TM, Gallagher EP, Wang C, Stahl DL, Hayes JD, Eaton DL. (1995) Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. Toxico. Appl. Pharmacol. 135: 45–57.

    Article  CAS  Google Scholar 

  38. Knight LP, Primiano T, Groopman JD, Kensler TW, Sutter TR. (1999) cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3. Carcinogenesis 20: 101–108.

    Article  Google Scholar 

  39. Monroe DH, Eaton DL. (1987) Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse. Toxicol. Appl. Pharmacol. 90: 401–409.

    Article  CAS  PubMed  Google Scholar 

  40. Wild AC, Moinova HR, Mulcahy RT. (1999) Regulation of γ-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J. Biol. Chem. 274: 33627–33636.

    Article  CAS  PubMed  Google Scholar 

  41. Wilkinson J, Venugopal Radjendirane IV, Pfeiffer GR, Jaiswal AK, Clapper ML. (1998) Disruption of c-Fos leads to increased expression of NAD(P)H:quinone oxidoreductase1 and glutathione S-transferase. Biochem. Biophys. Res. Commun. 253: 855–858.

    Article  CAS  PubMed  Google Scholar 

  42. Yoshioka K, Deng T, Cavigelli M, Karin M. (1995) Antitumor promotion by phenolic antioxidants: Inhibition of AP-1 activity through induction of Fra expression. Proc. Natl. Acad. Sci. U.S.A. 92: 4972–4976.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wild AC, Gipp JJ, Mulcahy T. (1998) Overlapping antioxidant response element and PMA response element sequences mediate basal and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase catalytic subunit gene. Biochem. J. 332: 373–381.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Montano M, Katzenellenbogen BS. (1997) The quinone reductase gene: A unique estrogen receptor-regulated gene that is activated by antiestrogens. Proc. Natl. Acad. Sci. U.S.A. 94: 2581–2586.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Montano M, Jaiswal AK, Katzenellenbogen BS. (1998) Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-a and estrogen receptor-β. J. Biol. Chem. 273: 25443–25449.

    Article  CAS  PubMed  Google Scholar 

  46. Chan K, Lu R, Chang JC, Kan YW. (1996) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc. Natl. Acad. Sci. U.S.A. 93: 13943–13948.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Dalton TP, Shertzer HG, Puga A. (1999) Regulation of gene expression by reactive oxygen. Annu. Rev. Pharmacol. Toxicol. 39: 67–101.

    Article  CAS  PubMed  Google Scholar 

  48. Huang Y, Domann FE. (1998) Redox modulation of AP-2 DNA binding activity in vitro. Biochem. Biophys. Res. Commun. 249: 307–312.

    Article  CAS  PubMed  Google Scholar 

  49. Kim W, Gates KS. (1997) Evidence for thiole-dependent production of oxygen radicals by 4-mrthyl-5-pyrazinyl-3H-1, 2-dithiole-3-thione (oltipraz) and 3H-1,2-dithiole-3-thione: Possible relevance to the anticarcinogenic properties of 1,2-dithiole-3-thiones. Chem. Res. Toxicol. 10: 296–301.

    Article  CAS  PubMed  Google Scholar 

  50. Liptay S, Schmid RM, Nabel EG, Nabel GJ. (1994) Transcriptional regulation of NF-kappa B2: Evidence for kappa B-mediated positive and negative autoregulation. Mol. Cell Biol. 14: 7695–7703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Itoh K, Wakabayashi N, Katoh Y, et al. (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13: 76–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Nguyen T, Huang HC, Pickett CB. (2000) Transcriptional regulation of the antioxidant response element: Activation by Nrf2 and repression by MafK. J. Biol. Chem. 275: 15466–15473.

    Article  CAS  PubMed  Google Scholar 

  53. Chan K, Kan YW. (1999) Nrf2 is essential for protection against acute pulmonary injury in mice. Proc. Natl. Acad. Sci. U.S.A. 96: 12731–12736.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Guyton KZ, Thompson JA, Kensler TW. (1993) Role of quinone methide in the in vitro toxicity of the skin tumor promoter butylated hydroxytoluene hydroperoxide. Chem. Res. Toxicol. 6: 731–738.

    Article  CAS  PubMed  Google Scholar 

  55. Ishii T, Itoh K, Takahashi S, et al. (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-induced genes in macrophages. J. Biol. Chem. 275: 16023–16029.

    Article  CAS  PubMed  Google Scholar 

  56. Kwong M, Kan YW, Chan JY. (1999) The CNC basic leucine zipper factor, Nrf1, is essential for cell survival in response to oxidative stress-inducing agents. J. Biol. Chem. 274: 37491–37498.

    Article  CAS  PubMed  Google Scholar 

  57. Cho H-Y, Reddy SPM, Zhang LY, et al. Quantitative trait locus analysis of hyperoxic lung injury in mice and the role of Nrf2 as a candidate susceptibility gene (Submitted).

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Acknowledgments

We thank Dr. David Eaton for providing cDNA for mouse GST Yc, Patrick Dolan for maintaining and genotyping mice, and Julie Rider, Minerva Ramos-Gomez, and Christopher Toscano for helpful discussions and assistance.

This work was supported by NIH Grants CA39416 and CA44530.

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Author notes

  1. Thomas R. Sutter

    Present address: University of Memphis, Life Science Building, Memphis, Tennessee, 38152, USA

Authors and Affiliations

  1. Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, Maryland, 21205, USA

    Mi-Kyoung Kwak, Thomas R. Sutter & Thomas W. Kensler

  2. Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tennoudai, Tsukuba, Japan

    Ken Itoh & Masayuki Yamamoto

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  1. Mi-Kyoung Kwak
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Correspondence to Thomas W. Kensler.

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Kwak, MK., Itoh, K., Yamamoto, M. et al. 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. Mol Med 7, 135–145 (2001). https://doi.org/10.1007/BF03401947

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