Elsevier

Electrochimica Acta

Volume 45, Issue 21, 14 July 2000, Pages 3555-3561
Electrochimica Acta

Nitrosobenzene: electrochemical, UV-visible and EPR spectroscopic studies on the nitrosobenzene free radical generation and its interaction with glutathione

https://doi.org/10.1016/S0013-4686(00)00477-1Get rights and content

Abstract

This paper reports both the electrochemical characterization and the reactivity of the nitroso radical anion from nitrosobenzene with glutathione. The reduction of nitrosobenzene to the corresponding nitroso radical anion was kinetically characterized in acetonitrile. Free radicals exhibited a natural decay of second order, with a constant value of k2=15 555±321 M−1 s−1. Also, the radicals were characterized by UV-Visible and EPR spectroscopy. Data obtained with these two independent techniques clearly substantiated the formation of the nitrosobenzene radical under our experimental conditions. Furthermore, we have unambiguously demonstrated that glutathione (GSH) scavenged the nitroso radical anion electrochemically generated from nitrosobenzene. The scavenging effect of GSH is supported by the following experimental facts: 1. The parallel decrease of the anodic peak current in the cyclic voltammograms, corresponding to the nitroso radical anion concomitantly with the addition of GSH. 2. The significant decrease of the visible band at 560 nm corresponds to the radical after the addition of GSH. 3. The drop of the EPR signal intensity of the nitroso radical after the addition of GSH. By using a spin trapping technique, thiyl radical (GS.) was detected during the reaction between the nitroso radical anion with GSH.

Introduction

Nitroso compounds are among the most potent chemical carcinogens and are widely distributed in the environment. These compounds are also recognized as potentially reactive metabolites of a variety of toxicologically interesting compounds, which after covalent binding to proteins and DNA, become carcinogenic and/or mutagenic [1], [2].

Many nitroso compounds are reduced nonenzymatically by several endobiotic compounds, such as, NAD (P) H, glutathione, cysteine and hemoglobin with formation of free radical derivatives, as confirmed by their corresponding ESR spectra [3], [4]. On the other hand, several studies using electron spin resonance [5], [6], [7] have established the presence of radicals in chemical and electrochemical reduction of nitrosobenzene in aqueous and non-aqueous solvent systems.

Several studies [8], [9], [10] using cyclic voltammetry and EPR, have demonstrated that thiol compounds, such as glutathione significantly scavenged nitro radicals. However, the interaction between nitroso radical species and thiol compounds has not received an adequate attention up-to date.

In this paper, we examine the interaction between radical species generated from nitrosobenzene and glutathione. From these studies, the electrochemical generation of nitroso radical species from nitrosobenzene has been used. Cyclic voltammetry, UV-Visible and ESR spectroscopy were used to follow the interaction of radical species and glutathione.

Section snippets

Chemicals

Nitrosobenzene (NB), glutathione (GSH), anhydrous dimethylsulfoxide (DMSO), spectroscopic grade and anhydrous acetonitrile for UV spectroscopy were purchased from Merck. Solutions of NB were prepared in the different solvents, which were presaturated under a pure nitrogen stream. Tetrabutylammonium hexafluorophosphate (TBAHFP) was purchased from Aldrich.

Drug solutions

Stock solutions of 10 mM NB both in DMSO or acetonitrile were prepared and protected from daylight. Aliquots to obtain final concentrations

Results and discussion

The main goal of this paper was to assess the possible scavenging effect of the radical’s species from nitrosobenzene by glutathione. For this purpose electrochemical, UV-Visible and ESR spectroscopic techniques were used. Because the reduction mechanism is strongly affected by the solvent, studies were performed in two different solvents, i.e. DMSO and acetonitrile.

Concluding remarks

Our study provides experimental data that demonstrated the nitro radical anion electrochemical generated from nitrosobenzene is scavenged by GSH and could support the view that under hypoxic or anaerobic conditions, is not possible to discard the reactivity of this thiol molecule with this type of species and at least, partially could explain the significant loss of GSH under the above experimental conditions. On the other hand, the generated nitroso radical anion reacted with O2 (as was

Acknowledgements

This work was supported by Grants from FONDECYT (Project 8970023) and D.I.D. (U. de Chile). Also, the collaboration of CEPEDEQ is acknowledged.

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