Claire Ducrocq

Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species

Abstract:  To face physicochemical and biological stresses, living organisms evolved endogenous chemical responses based on gas exchange with the atmosphere and on formation of nitric oxide (NO•) and oxygen derivatives. The combination of these species generates a complex network of variable extension in space and time, characterized by the nature and level of the reactive oxygen (ROS) and nitrogen species (RNS) and of their organic and inorganic scavengers. Among the latter, this review focusses on natural 3‐substituted indolic structures. Tryptophan‐derived indoles are unsensitive to NO•, oxygen and superoxide anion (O2•−), but react directly with other ROS/RNS giving various derivatives, most of which have been characterized. Though the detection of some products like kynurenine and nitroderivatives can be performed in vitro and in vivo, it is more difficult for others, e.g., 1‐nitroso‐indolic compounds. In vitro chemical studies only reveal the strong likelihood of their in vivo generation and biological effects can be a sign of their transient formation. Knowing that 1‐nitrosoindoles are NO donors and nitrosating agents indicating they can thus act both as mutagens and protectors, the necessity for a thorough evaluation of indole‐containing drugs in accordance with the level of the oxidative stress in a given pathology is highlighted.

Nitrosation of melatonin by nitric oxide and peroxynitrite

Peroxynitrite (ONOO−) is an endogenous molecule, formed by rapid coupling between NO and O2−. ONOO− is known to be a strong oxidant of thiols and metalloorganic compounds and also a nitrating agent of aromatic compounds such as tyrosine. However, its chemistry is not yet well elucidated under physiological conditions. Melatonin, which is an indole‐amine produced by the pineal gland and other organs, has antioxidant properties. We show that melatonin reacts with ONOO− in phosphate‐buffered solutions. We provide evidence of nitrosation and oxidation at the pyrrole nitrogen leading to 1‐nitrosomelatonin and 1‐hydroxymelatonin, these being the major reactions in aqueous phosphate‐buffered solutions besides other aromatic hydroxylations and nitration. 4‐Nitromelatonin is formed, but in small amounts. The kinetics of all transformations were strictly dependent on ONOO− decay, whereas yields varied with pH and the presence of CO2. The N‐oxidation became competitive with nitrosation at pH 7.4, in medium containing a sufficient amount of CO 2. A proposed mechanism involves the transient formation of melatonyl radical and ONOO radical derived from ONOO− decay.

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: Involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).

Nitration of catecholamines with nitrogen oxides in mild conditions: a hypothesis for the reactivity of NO in physiological systems

Abstract Dopamine, norepinephrine and epinephrine react at room temperature, in acetate buffer (36) with sodium nitrite or in non-deaerated phosphate buffer (pH 7.4) with NO. The corresponding 6-nitro derivatives are formed.

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: Subsequent formation of nitric oxide

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

1-Nitrosomelatonin is a spontaneous NO-releasing compound

Melatonin (N-acetyl-5-methoxytryptamin), the main hormone secreted by the pineal gland in mammals, is nitrosated by nitrite at acidic pH and by NO in the presence of oxygen under neutral conditions. Melatonin is also partly converted to 1-nitrosomelatonin by oxoperoxonitrate (ONOO-, peroxynitrite) in phosphate-buffered solutions at pH 7–10 [Blanchard, B., et al. (2000) Journal of Pineal Research 29, 184–192]. In the present report, we show that 1-nitrosomelatonin in turn behaves as an NO-donor regenerating melatonin. NO-release is evidenced by the formation of nitrite in phosphate-buffered solutions and oxidation of HbO2. No peroxynitrite was formed during that decomposition because serotonin used as a probe was converted only to 4-nitroso-serotonin as expected for a true NO-donor [Blanchard, B., et al. (2001) Free Radical Research, 34, 177–188]. The spontaneous decay of 1-nitrosomelatonin is not affected by GSH and metallic ions but its decomposition is accelerated in acidic pH or in the presence of NADH or ascorbate. Furthermore, melatonin is partially or entirely recovered in the absence or presence of ascorbate, respectively. A homolytic cleavage of 1-nitrosomelatonin is strongly suggested and discussed. Formation of 1-nitrosomelatonin from melatonin and reactive nitrogen species (RNS) followed by its decay into NO demonstrates that melatonin could reduce these RNS to NO.

Angeli's Salt (Na2N2O3) is a Precursor of HNO and NO: a Voltammetric Study of the Reactive Intermediates Released by Angeli's Salt Decomposition

Under physiological conditions, it is usually accepted that the aerobic decomposition of Angelis salt produces nitrite (NO2−) and nitroxyl (HNO), which dimerizes and leads to N2O. No consensus has yet been established on the formation of nitric oxide (NO) and/or peroxynitrite (ONOO−) by Angelis salt. Because this salt has recently been shown to have pharmacological properties for the treatment of cardiovascular diseases, identification of its follow‐up reactive intermediates is of increasing importance. In this work, we investigated the decomposition mechanism of Angelis salt by voltammetry performed at platinized carbon fiber microelectrodes. By following the decomposition process of Angelis salt, we showed that the mechanism depends on the experimental conditions. Under aerobic neutral and slightly alkaline conditions, the formation of HNO, NO2−, but also of nitric oxide NO was demonstrated. In strongly alkaline buffer (pH>10), we observed the formation of peroxynitrite ONOO− in the presence of oxygen. These electrochemical results are supported by comparison with UV spectrophotometry data.

Reactivity of Peroxynitrite with Melatonin as a Function of pH and CO2 Content

Peroxynitrite is known to be a strong oxidant and a nitrating agent of aromatic phenolic or heterocyclic rings, depending on its form in aqueous solutions: the anion ONOO−, its conjugate acid ONOOH, or its CO2 adduct ONOOCO2−. Various reactions have been observed with melatonin (1), a tryptophan derivative, in phosphate-buffered solutions. Melatonin (1) is recognized as a scavenger of several strong oxidants (HO·, H2O2, ...) accounting for its biological and pharmacological effects. Here we describe two oxidation routes that give rise to indol-2-ones 2 (probably via a 2,3-epoxyindole) and kynuramines 6 (by cleavage of the pyrrole ring), attributable to reactions of ONOOH and ONOO−, respectively, according to the effects of pH and CO2 content. At pH = 7.6 and in the presence of CO2, an important conversion is the cyclization of the lateral amide function, giving 3-substituted pyrroloindoles 4. At neutral pH, therefore, all routes coexist, with a balance between indol-2-ones 2 and pyrroloindoles 4 on the one side and kynuramines 6 on the other, depending on the CO2 content. Furthermore, under specific conditions substitutions of the hydrogen atom on the pyrrole nitrogen atom, affording the 1-nitro- (5) and the unstable 1-nitrosomelatonin (7), are among the major transformations: formation of the nitrosation product, together with that of kynuramines 6, rises sharply when the pH of the medium increases, confirming the implication of ONOO− in their synthesis; conversely, both reaction yields decrease with increasing CO2 content, to favor 1-nitromelatonin (5). Finally, nitration by aromatic substitution occurring essentially on C-4 becomes important at acidic pH, and also at pH = 7.6 over a narrow range of CO2 concentrations. Most of the reactions are typical of the indole moiety, suggesting that melatonin (1) is a model of potential use for investigation of tryptophan chemistry with peroxynitrite. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Melatonin nitrosation promoted by radical; comparison with the peroxynitrite reaction

N-nitroso species have recently been detected in animal tissues. Protein N-nitrosotryptophan is the best candidate for this N-nitroso pool. N-nitrosation of N-blocked trytophan derivatives like melatonin (MelH) by N2O3 or peroxynitrite (ONOOH/ONOO− ) has been observed under conditions of pH and reagent concentrations similar to in vivo conditions. We studied the reaction of with MelH. When was synthesized by γ-irradiation of aqueous neutral solutions of nitrate under anaerobic conditions, detected oxidation and nitration of MelH were negligible. In the presence of additional nitrite, when NO√ was also generated, formation of 1-nitrosomelatonin increased with nitrite concentration. Nitrosation is not due to N2O3 but could proceed via successive additions of and NO√. For comparison, peroxynitrite was infused into a solution of MelH under air leading to the same products as those detected in irradiated solutions but in different proportions. In the presence of additional nitrite, the formation of nitroderivatives increased significantly while N-formylkynuramine and 1-nitrosomelatonin were maintained at similar levels. Mechanistic implications are discussed.