Molecular and Cellular Biochemistry

, Volume 307, Issue 1–2, pp 185–191 | Cite as

Generation of superoxide from reaction of 3H-1,2-dithiole-3-thione with thiols: implications for dithiolethione chemoprotection

  • Zhenquan Jia
  • Hong Zhu
  • Michael A. Trush
  • Hara P. Misra
  • Yunbo Li


3H-1,2-Dithiole-3-thione (D3T), a potent member of dithiolethiones, induces phase 2 enzymes by activating an Nrf2/Keap1-dependent signaling pathway. It was proposed that interaction between D3T and two adjacent sulfhydryl groups of Keap1 might cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation. This study was undertaken to investigate the reactions between D3T and thiols, including the dithiol compound, dithiothreitol (DTT), and the monothiol, glutathione (GSH). We reported here that under physiologically relevant conditions incubation of D3T with DTT caused remarkable oxygen consumption, indicating a redox reaction between D3T and the dithiol molecule. Incubation of D3T with GSH also led to oxygen consumption, but to a less extent. Electron paramagnetic resonance (EPR) studies showed that the redox reaction between D3T and DTT generated superoxide. Superoxide was also formed from the redox reaction of D3T with GSH. These findings demonstrate that D3T reacts with thiols, particularly a dithiol, generating superoxide, which may provide a mechanistic explanation for induction of Nrf2-dependent phase 2 enzymes by D3T.


EPR Superoxide D3T Oxygen consumption Thiols 



Antioxidant response element




5-(Diethoxyphosphoryl)-5-methylpyrroline N-oxide


Diethylenetriaminepentaacetic acid




Electron paramagnetic resonance




Mitogen-activated protein kinases


Nitroblue tetrazolium


Phosphate-buffered saline


Reactive oxygen species


Superoxide dismutase



This work was supported in part by NIH grant HL71190 (Y. L.). M. A. T. was supported by NIH grants ES03760, ES03819 and ES08078.


  1. 1.
    Bailar JC 3rd, Gornik HL (1997) Cancer undefeated. N Engl J Med 336:1569–1574PubMedCrossRefGoogle Scholar
  2. 2.
    Jacobson LP, Zhang BC, Zhu YR, Wang JB, Wu Y, Zhang QN, Yu LY, Qian GS, Kuang SY, Li YF, Fang X, Zarba A, Chen B, Enger C, Davidson NE, Gorman MB, Gordon GB, Prochaska HJ, Egner PA, Groopman JD, Munoz A, Helzlsouer KJ, Kensler TW (1997) Oltipraz chemoprevention trial in Qidong, People’s Republic of China: study design and clinical outcomes. Cancer Epidemiol Biomarkers Prev 6:257–265PubMedGoogle Scholar
  3. 3.
    Kwak MK, Egner PA, Dolan PM, Ramos-Gomez M, Groopman JD, Itoh K, Yamamoto M, Kensler TW (2001) Role of phase 2 enzyme induction in chemoprotection by dithiolethiones. Mutat Res 480–481:305–315PubMedGoogle Scholar
  4. 4.
    Nguyen T, Sherratt PJ, Pickett CB (2003) Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu Rev Pharmacol Toxicol 43:233–260PubMedCrossRefGoogle Scholar
  5. 5.
    Motohashi H, Yamamoto M (2004) Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med 10:549–557PubMedCrossRefGoogle Scholar
  6. 6.
    Li Y, Cao Z, Trush MA (2004) Chemical carcinogenesis and mutagenesis. In: F Barile (ed) Clinical toxicology: principles and mechanisms. CRC Press, Florida, pp 359–376Google Scholar
  7. 7.
    Kim W, Gates KS (1997) Evidence for thiol-dependent production of oxygen radicals by 4-methyl-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 10:296–301PubMedCrossRefGoogle Scholar
  8. 8.
    Velayutham M, Villamena FA, Fishbein JC, Zweier JL (2005) Cancer chemopreventive oltipraz generates superoxide anion radical. Arch Biochem Biophys 435:83–88PubMedCrossRefGoogle Scholar
  9. 9.
    Velayutham M, Villamena FA, Navamal M, Fishbein JC, Zweier JL (2005) Glutathione-mediated formation of oxygen free radicals by the major metabolite of oltipraz. Chem Res Toxicol 18:970–975PubMedCrossRefGoogle Scholar
  10. 10.
    Li Y, Kuppusamy P, Zweir JL, Trush MA (1996) Role of Cu/Zn-superoxide dismutase in xenobiotic activation. II. Biological effects resulting from the Cu/Zn-superoxide dismutase-accelerated oxidation of the benzene metabolite 1,4-hydroquinone. Mol Pharmacol 49:412–421PubMedGoogle Scholar
  11. 11.
    Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier JL, Trush MA (1998) Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 273:2015–2023PubMedCrossRefGoogle Scholar
  12. 12.
    Cao Z, Tsang M, Zhao H, Li Y (2003) Induction of endogenous antioxidants and phase 2 enzymes by alpha-lipoic acid in rat cardiac H9C2 cells: protection against oxidative injury. Biochem Biophys Res Commun 310:979–985PubMedCrossRefGoogle Scholar
  13. 13.
    Frejaville C, Karoui H, Tuccio B, Le Moigne F, Culcasi M, Pietri S, Lauricella R, Tordo P (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals. J Med Chem 38:258–265PubMedCrossRefGoogle Scholar
  14. 14.
    Shaked Z, Szajewski RP, Whitesides GM (1980) Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values. Biochemistry 19:4156–4166PubMedCrossRefGoogle Scholar
  15. 15.
    Carey KA, Kensler TW, Fishbein JC (2001) Kinetic constraints for the thiolysis of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) and related dithiole-3-thiones in aqueous solution. Chem Res Toxicol 14:939–945PubMedCrossRefGoogle Scholar
  16. 16.
    Delmas-Beauvieux MC, Peuchant E, Thomas MJ, Dubourg L, Pinto AP, Clerc M, Gin H (1998) The place of electron spin resonance methods in the detection of oxidative stress in type 2 diabetes with poor glycemic control. Clin Biochem 31:221–228PubMedCrossRefGoogle Scholar
  17. 17.
    Timmins GS, Liu KJ, Bechara EJ, Kotake Y, Swartz HM (1999) Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO· and SO4 ·-. Free Radic Biol Med 27:329–333PubMedCrossRefGoogle Scholar
  18. 18.
    Stolze K, Udilova N, Nohl H (2000) Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase. Free Radic Biol Med 29:1005–1014PubMedCrossRefGoogle Scholar
  19. 19.
    Ross D, Cotgreave I, Moldeus P (1985) The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine. Biochim Biophys Acta 841:278–282PubMedGoogle Scholar
  20. 20.
    Armstead WM (2001) Vasopressin induced cyclooxygenase dependent superoxide generation contributes to K+ channel function impairment after brain injury. Brain Res 910:19–28PubMedCrossRefGoogle Scholar
  21. 21.
    Choi HS, Kim JW, Cha YN, Kim C (2006) A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem 27:31–44PubMedCrossRefGoogle Scholar
  22. 22.
    Garrett IR, Boyce BF, Oreffo RO, Bonewald L, Poser J, Mundy GR (1990) Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest 85:632–639PubMedCrossRefGoogle Scholar
  23. 23.
    Nguyen T, Huang HC, Pickett CB (2000) Transcriptional regulation of the antioxidant response element: activation by Nrf2 and repression by MafK. J Biol Chem 275:15466–15473PubMedCrossRefGoogle Scholar
  24. 24.
    Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, Yamamoto M (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 275:16023–16029PubMedCrossRefGoogle Scholar
  25. 25.
    Huang HC, Nguyen T, Pickett CB (2000) Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci USA 97:12475–12480PubMedCrossRefGoogle Scholar
  26. 26.
    Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 13:76–86PubMedCrossRefGoogle Scholar
  27. 27.
    Rosenberger J, Petrovics G, Buzas B (2001) Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-kappaB. J Neurochem 79:35–44PubMedCrossRefGoogle Scholar
  28. 28.
    Schreck R, Albermann K, Baeuerle PA (1992) Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Radic Res Commun 17:221–237PubMedCrossRefGoogle Scholar
  29. 29.
    Zhou LZ, Johnson AP, Rando TA (2001) NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells. Free Radic Biol Med 31:1405–1416PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic MedicineVirginia Tech Corporate Research CenterBlacksburgUSA
  2. 2.Division of Toxicology, Department of Environmental Health SciencesThe Johns Hopkins University Bloomberg School of Public HealthBaltimoreUSA

Personalised recommendations