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Archives of Toxicology

, Volume 86, Issue 10, pp 1613–1625 | Cite as

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide

  • Eric V. PatridgeEmail author
  • Emma S. E. Eriksson
  • Philip G. Penketh
  • Raymond P. Baumann
  • Rui Zhu
  • Krishnamurthy Shyam
  • Leif A. Eriksson
  • Alan C. Sartorelli
Biologicals

Abstract

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 μM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

Keywords

Benzofurazan Reactive oxygen species Oxidative stress Electrochemistry Electrophilic stress 

Abbreviations

BFZ

Benzofurazan

NBF

7-Nitrobenzofurazan

ROS

Reactive oxygen species

NBF-SPh

7-Nitro-4-(phenylthio)benzofurazan

GST

Glutathione S-transferases

SBF-SPh

7-Sulfo-4-(phenylthio)benzofurazan

DMEM

Dulbecco’s modified Eagle’s medium

G6P

Glucose-6-phosphate

SOD

Superoxide dismutase

G6PDH

Glucose-6-phosphate dehydrogenase

P450Red

NADPH:cytochrome P450 reductase

TBARS

2-Thiobarbituric acid-reactive substances

DP

Differential pulse

CV

Cyclic voltammetry

NBDHEX

7-Nitro-4-(hexylthio)benzofurazan

Notes

Acknowledgments

The authors are grateful to James Blakemore for his assistance with electrochemical studies and to Dr. Tukiet Lam and Edward Voss for their services and help in mass spectroscopy analysis. This work was supported in part by U.S. Public Health Service Grants CA-090671, CA-122112, and CA-129186 from the National Cancer Institute and a Grant from the National Foundation for Cancer Research.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Eric V. Patridge
    • 1
    Email author
  • Emma S. E. Eriksson
    • 2
    • 3
  • Philip G. Penketh
    • 1
  • Raymond P. Baumann
    • 1
  • Rui Zhu
    • 1
  • Krishnamurthy Shyam
    • 1
  • Leif A. Eriksson
    • 2
    • 3
  • Alan C. Sartorelli
    • 1
  1. 1.Department of PharmacologyYale University School of MedicineNew HavenUSA
  2. 2.School of ChemistryNational University of Ireland-GalwayGalwayIreland
  3. 3.Department of Chemistry and Molecular BiologyUniversity of GothenburgGothenburgSweden

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