Inhibition of peroxynitrite-mediated cellular toxicity, tyrosine nitration, and α1-antiproteinase inactivation by 3-mercapto-2-methylpentan-1-ol, a novel compound isolated from Alliumcepa

https://doi.org/10.1016/S0006-291X(03)00193-1Get rights and content

Abstract

Peroxynitrite formation in vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products such as thiols as “peroxynitrite scavengers”. We therefore investigated the effects of a recently identified constituent of onions, 3-mercapto-2-methylpentan-1-ol (3-MP), for its ability to inhibit peroxynitrite-mediated processes in vitro and using cultured human cells and compared its effectiveness against glutathione. 3-MP significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of α1-antiproteinase to a greater extent than glutathione at each concentration tested (15–500 μM). 3-MP also inhibited peroxynitrite-induced cytotoxicity, intracellular tyrosine nitration, and intracellular reactive oxygen species generation in human HepG2 cells in culture to a greater extent than glutathione. These data suggest that 3-MP has the potential to act as an inhibitor of ONOO-mediated processes in vivo and that the antioxidant action of 3-MP deserves further study.

Section snippets

Materials and methods

Materials. Hydrogen peroxide (30% solution), manganese dioxide, and HPLC grade methanol were purchased from BDH (Poole, Dorset, England). Tyrosine, α1-antiproteinase (A9024), porcine elastase (E0258), and N-succinyl (ala)3-p-nitroanilide (SANA), and all other reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA). Distilled water passed through a Millipore water purification system was used for all purposes. Synthesis of 3-mercapto-2-methylpentan-1-ol and hydrogen peroxide-free

Determination of oil:water partition coefficient

Using n-octanol/water, the partition coefficients [36], [37] for 3-MP and GSH were determined to be 10±0.1 and 0.01±0.001, respectively. Therefore, 3-MP is 1000-fold more lipophilic than GSH.

Inhibition of tyrosine nitration

The addition of ONOO to a solution of tyrosine led to the formation of 3-nitrotyrosine [1]. As expected, the addition of 1 mM ONOO to a buffered solution of 1 mM tyrosine led to the formation of 70–80 μM of 3-nitrotyrosine [16]. 3-MP significantly inhibited ONOO-mediated tyrosine nitration at all

Discussion

The generation of peroxynitrite in vivo is implicated in a wide range of human diseases ranging from cancer and cardiovascular diseases to chronic inflammation and influenza infection (reviewed in [14], [15]). Both free and protein bound 3-NO2 tyr are found in vivo. For example, 3-NO2 tyr in the free form is found in rheumatoid arthritis, amyotrophic lateral sclerosis, and protein bound 3-NO2 tyr is found in acute lung injury, atherosclerosis, gastrointestinal inflammatory cancers, amyotrophic

Acknowledgements

We are grateful to the National Medical Research Council of Singapore (NMRC/0474/2000 and NMRC/0481/2000) and the National University of Singapore Academic Research Fund (R183000053214) for their generous research support.

References (46)

  • P.S. Wong et al.

    Isolation and properties of oxidized α1-proteinase inhibitor from human rheumatoid synovial fluid

    Biochem. Biophys. Res. Commun.

    (1980)
  • M. Ueda et al.

    Evaluation of oxidized α-1-antitrypsin in blood as an oxidative stress marker using anti-oxidative α1-AT monoclonal antibody

    Clin. Chim. Acta

    (2002)
  • F. Tietze

    Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione: applications to mammalian blood and other samples

    Anal. Biochem.

    (1969)
  • O.W. Griffith

    Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine

    Anal. Biochem.

    (1980)
  • M. Whiteman et al.

    Thiols and disulphides can aggravate peroxynitrite-dependent inactivation of α1-antiproteinase

    FEBS Lett.

    (1997)
  • A.J. Gow et al.

    Effects of peroxynitrite-induced protein modifications on tyrosine phosphorylation and degradation

    FEBS Lett.

    (1996)
  • R.E. Huie et al.

    The reaction of NO with superoxide

    Free Radical Res. Commun.

    (1993)
  • J.S. Beckman et al.

    Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide

    Proc. Natl. Acad. Sci. USA

    (1990)
  • A. Van der Vliet et al.

    Interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion

    Biochem. J.

    (1994)
  • J.P. Spencer et al.

    Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine

    Chem. Res. Toxicol.

    (1996)
  • C. Szabo et al.

    DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite

    Proc. Natl. Acad. Sci. USA

    (1996)
  • M.R. Cookson et al.

    Peroxynitrite and hydrogen peroxide induced cell death in the NSC34 neuroblastoma x spinal cord cell line: role of poly (ADP-ribose) polymerase

    J. Neurochem.

    (1998)
  • P. Cassina et al.

    Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis

    J. Neurosci. Res.

    (2002)
  • Cited by (18)

    • An overview of sulfur-containing compounds originating from natural metabolites: Lanthionine ketimine and its analogues

      2020, Analytical Biochemistry
      Citation Excerpt :

      Besides being a ROS scavenger, AECK-DD has been shown to possess activity against reactive nitrogen species (RNS). Peroxynitrite is a typical RNS which has been reported to oxidatively modify low-density lipoproteins (LDL) [70] and inactivate α1-antiproteinase by tyrosine nitration [71]. When LDL was pre-treated with AECK-DD, peroxynitrite-mediated LDL oxidation decreased significantly and the inactivation of α1-antiproteinase, by tyrosine nitration, was also reduced [72].

    • Endogenous superoxide production and the nitrite/nitrate ratio control the concentration of bioavailable free nitric oxide in leaves

      2004, Journal of Biological Chemistry
      Citation Excerpt :

      The increasing number of novel plant-based peroxynitrite scavengers recently identified with possible therapeutic benefits (57–59) is perhaps a testament to the requirement for plants to survive in a high peroxynitrite environment, the inevitable consequence of producing NO in a photosynthetic organism.

    • Onion (allium cepa L.)

      2017, Fruit and Vegetable Phytochemicals: Chemistry and Human Health: Second Edition
    View all citing articles on Scopus

    Abbreviations: α1-AP, α1-antiproteinase; DCF-DA, dichlorofluorescein diacetate; DMSO, dimethyl sulphoxide; DNA, deoxyribonucleic acid; EBSS, Earles balanced salt solution; K2HPO4, dipotassium hydrogen phosphate; DTNB 5,5-dithio-bis(2-nitrobenzoic acid); GSH, reduced glutathione; KH2PO4, potassium dihydrogen phosphate; LDL, low density lipoprotein; MEM, minimal essential media; 3-MP, 3-mercapto-2-methylpentan-1-ol; MTT, 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide; ONOO, peroxynitrite; NO2Cl, nitryl chloride; NO, nitric oxide; PBS, phosphate-buffered saline; RNS, reactive nitrogen species; SANA, N-succinyl (ala)3p-nitroanilide.

    View full text