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

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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.

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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

References

  • Andrews J, Ghosh P, Ternai B, Whitehouse M (1982) Ammonium 4-chloro-7-sulfobenzofurazan: a new fluorigenic thiol-specific reagent. Arch Biochem Biophys 214(1):386–396

    Article  PubMed  CAS  Google Scholar 

  • Baez S, Segura-Aguilar J (1995) Effects of superoxide dismutase and catalase during reduction of adrenochrome by DT-diaphorase and NADPH-cytochrome P450 reductase. Biochem Mol Med 56(1):37–44

    Article  PubMed  CAS  Google Scholar 

  • Bard A, Faulkner L (2001) Electrochemical methods: fundamentals and applications, 2nd edn. Wiley, Hoboken

    Google Scholar 

  • Barone V, Cossi M (1998) Quantum calculation of molecular energies and energy gradients in solution by a conductor solvent model. J Phys Chem 102(11):1995–2001

    Article  CAS  Google Scholar 

  • Baumann RP, Seow HA, Shyam K, Penketh PG, Sartorelli AC (2005) The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation. Oncol Res 15(6):313–325

    PubMed  CAS  Google Scholar 

  • Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J Chem Phys 98(7):5648–5652

    Article  CAS  Google Scholar 

  • Belton JG (1974) A Novel N → S oxygen migration in 2,1,3-benzoxadiazole systems. Proc R Ir Acad B 74:185–192

    CAS  Google Scholar 

  • Bindoli A, Scutari G, Rigobello MP (1999) The role of adrenochrome in stimulating the oxidation of catecholamines. Neurotox Res 1(2):71–80

    Article  PubMed  CAS  Google Scholar 

  • Birkett DJ, Price NC, Radda GK, Salmon AG (1970) The reactivity of SH groups with a fluorogenic reagent. FEBS Lett 6(4):346–348

    Article  PubMed  CAS  Google Scholar 

  • Caccuri AM, Ricci G (2006) Italy Patent No. EP1615638B1. EP Office

  • Castro F, Mariani D, Panek AD, Eleutherio EC, Pereira MD (2008) Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cerevisiae. PLoS ONE 3(12):e3999

    Article  PubMed  Google Scholar 

  • Cenas N, Nemeikaite A, Dickancaite E, Anusevicius Z, Nivinskas H, Bironaite D (1995) The toxicity of aromatic nitrocompounds to bovine leukemia virus-transformed fibroblasts: the role of single-electron reduction. Biochim Biophys Acta 1268(2):159–164

    Article  PubMed  Google Scholar 

  • Cossi M (2003) Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model. J Comp Chem 24(6):669–681

    Article  CAS  Google Scholar 

  • Federici L, Lo Sterzo C, Pezzola S, Di Matteo A, Scaloni F, Federici G, Caccuri AM (2009) Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione s-transferases. Cancer Res 69(20):8025–8034

    Article  PubMed  CAS  Google Scholar 

  • Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA et al (2009) Gaussian09. Gaussian, Inc., Wallingford

    Google Scholar 

  • Ghosh PB (1968) Preparation and study of some 5- and 7-substituted 4-nitrobenzofurazans and their N-oxides; a retro-Boulton–Katritzky rearrangement. J Chem Soc B 1:334–338

    Article  Google Scholar 

  • Ghosh PB, Whitehouse MW (1968) Potential antileukemic and immunosuppressive drugs. Preparation and in vitro pharmacological activity of some benzo-2,1,3-oxadiazoles (benzofurazans) and their N-oxides (benzofuroxans). J Med Chem 11(2):305–311

    Article  PubMed  CAS  Google Scholar 

  • Ghosh PB, Whitehouse MW (1969) Potential antileukemic and immunosuppressive drugs. II. Further studies with benzo-2,1,3-oxadiazoles (benzofurazans) and their N-oxides (benzofuroxans). J Med Chem 12(3):505–507

    Article  PubMed  CAS  Google Scholar 

  • Ghosh PB, Ternai B, Whitehouse MW (1972) Potential antileukemic and immunosuppressive drugs. 3. Effects of homocyclic ring substitution on the in vitro drug activity of 4-nitrobenzo-2,1,3-oxadiazoles (4-nitrobenzofurazans) and their N-oxides (4-nitrobenzofuroxans). J Med Chem 15(3):255–260

    Article  PubMed  CAS  Google Scholar 

  • Ghosh PB, Ternai B, Whitehouse MW (1981) Benzofurazans and benzofuroxans: biochemical and pharmacological properties. Med Res Rev 1(2):159–187

    Article  PubMed  CAS  Google Scholar 

  • Giulivi C, Cadenas E (1994) One- and two-electron reduction of 2-methyl-1,4-naphthoquinone bioreductive alkylating agents: kinetic studies, free-radical production, thiol oxidation and DNA-strand-break formation. Biochem J 301(Pt 1):21–30

    PubMed  CAS  Google Scholar 

  • Heimbrook DC, Sartorelli AC (1986) Biochemistry of misonidazole reduction by NADPH-cytochrome c (P-450) reductase. Mol Pharmacol 29(2):168–172

    PubMed  CAS  Google Scholar 

  • Heyne B (2007) Synthesis and characterization of a new fluorescent probe for reactive oxygen species. Org Biomol Chem 5(9):1454–1458

    Article  PubMed  CAS  Google Scholar 

  • Heyne B, Ahmed S, Scaiano JC (2008) Mechanistic studies of fluorescent sensors for the detection of reactive oxygen species. Org Biomol Chem 6(2):354–358

    Article  PubMed  CAS  Google Scholar 

  • Imai K, Fukushima T, Uzu S (1993) Sensitive determination of enantiomers of amino acids derivatized with the fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole, separated on a Pirkle-type column, Sumichiral OA 2500(S). Biomed Chromatogr 7(3):177–178

    Article  PubMed  CAS  Google Scholar 

  • Inbaraj JJ, Chignell CF (2004) Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 17(1):55–62

    Article  PubMed  CAS  Google Scholar 

  • Johnson SA, Dalton AE, Pardini RS (1998) Time-course of hypericin phototoxicity and effect on mitochondrial energies in EMT6 mouse mammary carcinoma cells. Free Radic Biol Med 25(2):144–152

    Article  PubMed  CAS  Google Scholar 

  • Juchau MR, Fantel AG, Harris C, Beyer BK (1986) The potential role of redox cycling as a mechanism for chemical teratogenesis. Environ Health Perspect 70:131–136

    Article  PubMed  CAS  Google Scholar 

  • Kappus H, Sies H (1981) Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia 37(12):1233–1241

    Article  PubMed  CAS  Google Scholar 

  • Kennedy KA, Teicher BA, Rockwell S, Sartorelli AC (1980) The hypoxic tumor cell: a target for selective cancer chemotherapy. Biochem Pharmacol 29(1):1–8

    Article  PubMed  CAS  Google Scholar 

  • Klamt A (1998) Refinement and parametrization of COSMO-RS. J Phys Chem A 102(26):5074–5085

    Article  CAS  Google Scholar 

  • Klamt A, Schuurmann G (1993) COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J Chem Soc Perkin Trans 2(5):799–805

    Google Scholar 

  • Knox RJ, Knight RC, Edwards DI (1983) Studies on the action of nitroimidazole drugs. The products of nitroimidazole reduction. Biochem Pharmacol 32(14):2149–2156

    Article  PubMed  CAS  Google Scholar 

  • Moreno SN, Docampo R (1985) Mechanism of toxicity of nitro compounds used in the chemotherapy of trichomoniasis. Environ Health Perspect 64:199–208

    Article  PubMed  CAS  Google Scholar 

  • Onoda M, Uchiyama S, Endo A, Tokuyama H, Santa T, Imai K (2003) First fluorescent photoinduced electron transfer (PET) reagent for hydroperoxides. Org Lett 5(9):1459–1461

    Article  PubMed  CAS  Google Scholar 

  • Ricci G, De Maria F, Antonini G, Turella P, Bullo A, Stella L, Filomeni G, Federici G, Caccuri AM (2005) 7-Nitro-2,1,3-benzoxadiazole derivatives, a new class of suicide inhibitors for glutathione S-transferases. Mechanism of action of potential anticancer drugs. J Biol Chem 280(28):26397–26405

    Article  PubMed  CAS  Google Scholar 

  • Santa T, Okamoto T, Uchiyama S, Mitsuhashi K, Imai K (1999) A new fluorogenic reagent for carboxylic acids, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH), derived from an empirical method for predicting fluorescence characteristics. Analyst 124(11):1689–1693

    Article  CAS  Google Scholar 

  • Stradyn YP, Kadysh VP, Giller SA (1974) Polarography of heterocyclic compounds. Chem Heterocycl Comp 10(2):129–141

    Article  Google Scholar 

  • Sun M, Zigman S (1978) An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation. Anal Biochem 90(1):81–89

    Article  PubMed  CAS  Google Scholar 

  • Takabatake T, Hasegawa M, Nagano T, Hirobe M (1990) Toxicities of dicyanobenzofurazans with formation of superoxide in Escherichia coli. Chem Pharm Bull (Tokyo) 38(1):128–132

    Article  CAS  Google Scholar 

  • Takabatake T, Hasegawa M, Nagano T, Hirobe M (1991) Formation of superoxide by benzofurazans in Escherichia coli under aerobic incubation. Chem Pharm Bull (Tokyo) 39(5):1352–1354

    Article  CAS  Google Scholar 

  • Takabatake T, Hasegawa M, Nagano T, Hirobe M (1992a) Bacteriostatic effect of 4,7-dicyanobenzofurazan due to inactivation of 2,3-dihydroxyisovalerate dehydratase. Chem Pharm Bull (Tokyo) 40(6):1644–1646

    Article  CAS  Google Scholar 

  • Takabatake T, Hasegawa M, Nagano T, Hirobe M (1992b) Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat. J Biol Chem 267(7):4613–4618

    PubMed  CAS  Google Scholar 

  • Toyo’oka T, Imai K (1983) High-performance liquid chromatography and fluorometric detection of biologically important thiols, derivatized with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). J Chromatogr 282:495–500

    Article  PubMed  Google Scholar 

  • Toyo’oka T, Ishibashi M, Takeda Y, Nakashima K, Akiyama S, Uzu S, Imai K (1991) Precolumn fluorescence tagging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7-aminoalkylamino-2,1,3-benzoxadiazoles. J Chromatogr 588(1–2):61–71

    PubMed  Google Scholar 

  • Tsveniashvili V, Zhdanov SI, Todres ZV (1966) Polarography of piazothiol and piazoselenol in aqueous solutions. Fresen J Anal Chem 224(1):389–406

    Article  Google Scholar 

  • Turella P, Cerella C, Filomeni G, Bullo A, De Maria F, Ghibelli L, Ciriolo MR, Cianfriglia M, Mattei M, Federici G et al (2005) Proapoptotic activity of new glutathione S-transferase inhibitors. Cancer Res 65(9):3751–3761

    Article  PubMed  CAS  Google Scholar 

  • Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86(1):271–278

    Article  PubMed  CAS  Google Scholar 

  • Uchiyama S, Santa T, Okiyama N, Fukushima T, Imai K (2001) Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton. Biomed Chromatogr 15(5):295–318

    Article  PubMed  CAS  Google Scholar 

  • Watanabe Y, Imai K (1981) High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole. Anal Biochem 116(2):471–472

    Article  PubMed  CAS  Google Scholar 

  • Weiss RF (1970) The solubility of nitrogen, oxygen and argon on water and seawater. Deep Sea Res 17:721–735

    CAS  Google Scholar 

  • Whitehouse MW, Ghosh PB (1968) 4-nitrobenzofurazans and 4-nitrobenzofuroxans: a new class of thiol-neutralising agents and potent inhibitors of nucleic acid synthesis in leucocytes. Biochem Pharmacol 17(1):158–161

    Article  PubMed  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Pharmacology, Yale University School of Medicine, New Haven, CT, 06520, USA

    Eric V. Patridge, Philip G. Penketh, Raymond P. Baumann, Rui Zhu, Krishnamurthy Shyam & Alan C. Sartorelli

  2. School of Chemistry, National University of Ireland-Galway, Galway, Ireland

    Emma S. E. Eriksson & Leif A. Eriksson

  3. Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

    Emma S. E. Eriksson & Leif A. Eriksson

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  1. Eric V. Patridge
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  2. Emma S. E. Eriksson
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Correspondence to Eric V. Patridge.

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Patridge, E.V., Eriksson, E.S.E., Penketh, P.G. et al. 7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide. Arch Toxicol 86, 1613–1625 (2012). https://doi.org/10.1007/s00204-012-0872-9

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  • DOI: https://doi.org/10.1007/s00204-012-0872-9

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