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Keywords:

  • melatonin;
  • N-acetyltryptophan;
  • nitration;
  • nitrogen dioxide;
  • nitrosation;
  • oxidation;
  • peroxynitrite

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

The synthesis of reactive oxygen (ROS) and nitrogen species (RNS) is a characteristic feature of the oxidative/nitrosative stress in the living kingdom, where stress is defined as the physiological response towards a physicochemical or biological change. These reactive species establish a particular network, variable in space, life span, and nature, depending on the presence of antioxidants. Antioxidants generally react chemically with ROS/RNS to produce inert compounds and/or other reactive species. Numerous natural substances are famous for their efficiency as oxidant scavengers in fruits and vegetables (ginseng, olive oil, garlic), Ginkgo biloba or tea leaves, and fermented beverages. These natural compounds exert reductive properties in a large range of potentials due to their variety of structures: thiol (glutathione) or phenol functions (catecholamines and catechins, tocopherol, resveratrol), flavonoids (quercetin), reduced metallic or metalloid-containing molecules, heterocyclic structures (urate, indoles). The indole nucleus is a specific class of these reducers, particularly efficient when bearing aliphatic substitutions in position 3, like that found in tryptophan (Trp) derivatives.

In this paper, we review evidence that argues that 3-substituted indole derivatives react directly with ROS/RNS and consider their biological properties related to their abilities to counteract oxidative damage. Some products of these reactions are also able to exert antioxidant properties (e.g., kynurenine and kynuramine); thus, allowing 3-substituted indoles to be highly effective antioxidants, even at low concentration, thanks to a ‘free radical scavenging cascade’ [1]. Other products have different biological properties depending on their concentration and environment. That is the case with 1-nitroso-indolic compounds which are able to release NO and indole radical cation, or nitrosate free thiols and amines. Since such compounds are mutagenic, activate enzymes and induce gene expression, their biological properties differ according to the cell, the tissue or the organism. This opens a large field of investigation to determine the weight of toxic versus therapeutic effect of 3-substituted indolic compounds under pathological conditions.

Reactive oxygen and nitrogen species involved in stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

Physiological situation

Living organisms differ in their ability to perform de novo biosynthesis of aminoacids and in the way they use nitrogen and dioxygen from their environment. However, some general mechanisms appear largely spread across the living kingdom, especially those linked with dioxygen and the stable radical nitric oxide (NO) (Fig. 1). Thus, animals use dioxygen to produce energy and NO, which is endogenously synthesized from l-arginine and dioxygen [2], to modulate many dioxygen-involving processes. NO is also provided by the reduction of nitrite and nitrate in the mouth and in the intestinal tract by symbiotic organisms or released from NO stores (nitroso- and nitrosyl-metal-compounds). Highly diffusible, NO reacts rapidly with reduced metal centers and other radicals and more slowly with dioxygen. Hence, its biological half-life is estimated to be about 1 s at micromolar concentration and 30 s in the nanomolar range in physiological aqueous compartments. In mammals, reactions with metal center molecules such as hemoglobin, guanylate cyclase in the vasculature [3, 4] and myoglobin in muscles, give pools of nitrosyl compounds (HbFe(II)NO, t1/2 < 11 min, HbFe(III)NO, t1/2 < 1 s, MbNO). When nitrosylated, guanylate cyclase is activated, inducing an elevation of the cGMP level, a biochemical signal of vasodilation or neurotransmission is produced [5]. Endogenous NO competes with dioxygen for binding to cytochrome oxidase, which inhibits the cell respiration at low dioxygen level and increases the reduction of the enzyme at elevated dioxygen concentrations [6]. Besides the reactions with hemoproteins, NO combines rapidly with radicals, the most widespread of which is the superoxide anion (O2•−). O2•− is provided by several dioxygen-reducing enzymatic systems (NADPH oxidases, xanthine oxidase, monoamine oxidases, prostaglandin synthases). O2•− has a short life-span due to its dismutation, either spontaneous or catalyzed by superoxide dismutase (SOD), that yields hydrogen peroxide and dioxygen, and due to its reaction with NO, it generates peroxynitrite (ONOO). In particular in the mitochondria, the formation of ONOO is the result of the concomitant production of NO by NO synthase and of O2•− from incomplete reduction of dioxygen by the respiratory chain [7].

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Figure 1.  Major ROS/RNS interaction pathways.

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In aqueous solution at physiological pH, ONOO is in equilibrium with its protonated form ONOOH (pKa = 6.8) and gives ONOOCO2 in the presence of CO2, which is in equilibrium with bicarbonate anion HCO3 (Fig. 2). ONOO has a biological half-life of 10–20 ms. All these species together (ONOOH/ONOO/ONOOCO2) account for ONOO reactivity towards organic and inorganic molecules [8, 9]. Under physiological conditions, ONOO oxidizes redox sensitive molecules: the first targets are the ascorbate and tocopherol vitamins, reduced hemoproteins and thiols.

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Figure 2.  Peroxynitrite-related species.

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In physiological medium, the reaction of NO and dioxygen yields nitrite (NO2) and nitrosation products (nitrosophenols, nitrosothiols in the presence of cystein-containing molecules or nitrosoamines in the presence of secondary amines), via the formation of ONOO and then N2O3 (Fig. 3). In the absence of oxygen, other electron acceptors such as NO2 or reduced transition metals could allow the reaction of nitrosation to occur. In mammals, NO2 is either carried by the inhaled polluted air in the lungs or formed in cells. Inside the cells, NO2 could arise either from the reduction of nitrite by peroxidases, or from ONOO decay. NO autoxidation, NO2 release and nitrosation are more likely in lipid compartments [10].

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Figure 3.  Nitrosation of biomolecules.

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S-nitrosation, which prevents critical protein thiols from undergoing a further oxidative modification, has emerged as an important post-translational protein modification. A review by Sun [11] lists the S-nitrosated proteins. This process is governed by a consensus acid–base motif in proteins [12]. Little is known regarding the potential role of other nitrosative protein modifications like the formation of N-nitrosoaminoacids. The latter were distinguished from nitrosylheme and S-nitrosocompounds as constituants of NO pools in tissues [13].

Stress responses

Stress is a general word including both the causes and the physiological or pathological reaction of the organism towards a physicochemical or biological change. The latter can be either external such as a change in the environment (diet, atmosphere, irradiation) or internal due to contact with infectious agents, sustained exercise, or psychological events. The adaptation response to the initial aggression is characterized by an imbalance between ROS/RNS production and the cell defenses in living organisms, including bacteria, plants, and mammals. Slight or acute variations of the concentrations of ROS/RNS modulate the specific network of their interactions with reducing equivalents in a limited area or in the whole organism [9, 14]. In this context, bioorganic molecules such as aminoacids, proteins, hormones, and nucleic acids can undergo modifications by direct reactions with ROS/RNS [15]. However, stress responses differ according to the nature and the proportion of the ROS/RNS [16] and partner fluxes [17].

An illustrative instance of stress is the mechanical pressure of the blood flux on the vessel wall due to movement, which provokes a shear stress. Vessel walls are lined with endothelial cells. The pressure on the endothelial cells leads to a movement of calcium cations through the membrane and an activation of the endothelial NO synthase and NADPH oxidase inserted therein. Both oxidases use oxygen and NADPH but generate NO and O2•−, respectively. NO and ONOO activate guanylate cyclase, leading to the dilation of the vessel, and regulate the cellular respiration in the mitochondria and the consumption of oxygen; thus the energy supply [6, 18]. It has been demonstrated that increases of fluid shear stress at physiological levels have beneficial effects on vascular homeostasis [19], which could explain the beneficial actions of physical exercise in preventing atherosclerosis and diabetes [20].

When the amounts of oxidants such as ONOO or NO2 increase and the reducer pool is deprived, some molecular targets appear especially sensitive to oxidation and/or nitration. Thus, exposed guanine, tyrosine, methionine and tryptophan residues yield 5-nitroguanine, 3-nitrotyrosine, methionine sulfoxide, and 6-nitrotryptophan as relatively stable products, respectively [21–23]. Hence, the function or the addressing (signaling, catabolism, molecular trafficking) of sensitive targets can be modified, as exemplified by calmodulin and GAPDH [11, 24]. In the case of endotoxemia, infection, Alzheimer's [25] and Parkinson’s diseases, levels of nitrite, nitrate and/or nitrotyrosine in proteins and nitroguanine in nucleic acids are modified suggesting the involvement of ROS/RNS. An excessive burst of ONOO or its sustained synthesis may be detrimental and is suspected to cause pathological situations related to inflammation such as diabetes [26], heart failure [27], cancer, atherosclerosis, arthritis, and most degenerative disorders [28]. However, the formation of transient ONOO has been demonstrated beneficial in terms of host defense against invading microorganisms and in stress responses [29, 30]. This may sometimes be explained at molecular level because ONOO could induce NO release from nitrosyl stores such as HbFe(II)NO and MbFe(II)NO [31] or form labile nitrosated compounds. In cancer, the synthesis of ROS/RNS inside or outside the tumor appears to depend on the nature and evolution of the illness. Ridnour et al. [32] described the dual or biphasic responses of cancer to NO depending on the level and duration of the NO flux together and on the chemical redox environment.

Natural 3-substituted indoles

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

Since most present indolic compounds in living organisms derive from l-tryptophan (Trp), these derivatives deserve consideration (Fig. 4). Higher vertebrates are not able to synthesize aromatic aminoacids while most species of bacteria and plants do. Synthesized from indoles by tryptophan-dependent or -independent pathways, indole-3-acetic acid is the source of numerous 3-substituted indoles in plants and microorganisms. Indole-3-acetic acid is a key phytohormone in most plants and belongs to the auxin family, which is involved in plant growth regulation [33]. The effect of auxins on a growing plant depends on their structure and their concentration. At low concentration, they activate or repress genes controlling structure and function in the plant [34].

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Figure 4.  Important 3-substituted indoles.

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In animals, food and drugs provide Trp and it enters cells by active transport. Its first role is its part as a building block in protein synthesis; however, Trp is also the precursor of biomolecules with specialized functions such as serotonin (5-hydroxytryptamine, a neurotransmitter), melatonin (N-acetyl-5-methoxytryptamine, a hormone) and their derivatives.

Serotonin is synthesized in neurons, platelets, and intestinal cells by a specific l-tryptophan hydroxylase and is mainly sequestered in vesicles. Thus, concentrated in vesicles at several millimolar concentration, its exocytosis is thoroughly controlled and biological functions are mediated by specific receptors in the cardiovascular, digestive, and neurosystems. When present in the extracellular medium, serotonin is retrieved by an efficient uptake mechanism.

Melatonin is mainly synthesized at night in the pineal gland and retina following circadian and seasonal rhythms. Highly diffusible, it spreads through the organism and regulates circadian rhythms through specific receptors and signal transduction processes. Thus, melatonin is often recommended as a drug to prevent the ‘jet lag’. Melatonin synthesis occurs at many other sites including skin, bone marrow, brain, thymus, and lymphocytes and the level of synthesis can reach high levels, for example in keratinocytes where the concentration is unexpectedly high [1]. Interestingly, the amount of melatonin is substantial in tissues that are exposed to external aggressions (such as the gut or the skin) or in places of high oxygen metabolism (brain) in accordance with its protective role against ROS/RNS. Its synthesis is controlled by the activities of serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase. Due to its lipophilicity, melatonin concentrates in membranes including those of mitochondria and in the nucleus in cells.

In physiological fluids, serotonin and melatonin (1–100 nm in plasma) are in lower concentrations than Trp (10–100 μm) provided by diet and hydrolysis of proteins. A rich Trp diet increases the central serotonin concentration, its metabolism and functions leading to oxidative stress. Indeed, serotonin is able to promote oxidative stress in acellular systems or tissues [35] and an increased serotonin synthesis corroborates the markers of oxidative stress and the generation of ROS/RNS [36]. On the contrary, Trp depletion lowers central serotonin metabolism affecting mood and brain performance [37].

Serotonin and melatonin can act through specific membrane receptors involved in numerous physiological functions. Serotonin regulates sleep, mood, and appetite and is implicated in pathological situations as mental disorders. It is particularly well known for its crucial role in emotional behavior [38]. Melatonin has three membrane melatonin receptors that have been cloned. At picomolar concentrations, melatonin acts through receptors MT1 and MT2 involved in the chronobiotic properties and in the vasoconstrictor activities in vessels [39]. This hormone modulates a myriad of physiological functions including vision and the cerebrovascular, reproductive, neuroendocrine, and neuroimmunological systems [40].

The endogenous level of the indolic compounds is also regulated by a more or less specific catabolism. Serotonin is specifically degraded by monoamine oxidases. Two heme-containing dioxygenases catalyze the oxidative degradation of Trp to N-formylkynurenine: tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO), using molecular dioxygen, superoxide anion or H2O2 [41, 42]. Though they catalyze the same reaction, the two enzymes engage in different physiological functions and tryptophan 2,3-dioxygenase has a higher substrate specificity than IDO, which also converts melatonin to kynuramine. Myeloperoxidase too oxidizes Trp to kynurenine and melatonin to kynuramine (using H2O2 and O2•−) [43]. In the liver, Trp and melatonin are mainly eliminated by aromatic hydroxylation and sulfate conjugation. The availability of substrates and cofactors regulates most of these enzymes.

IDO is the rate-limiting step of the kynurenine pathway in extrahepatic tissues [44]. Moreover, it is both inhibited by NO and its derivatives [45] and induced by inflammatory cytokines. Thus, it is involved in the immunoregulating system and numerous disorders [46], among which are asthma and allergy [47]. IDO-induced immunological tolerance participates in successful allogeneic pregnancy and application of this enzyme to improve the outcome of allogenic transplantation is currently under study [48]. Since Trp is essential for lymphocyte activation and proliferation, its depletion in IDO-expressing tumors establishes a local immunosuppressive microenvironment and plays a role in cancer immune escape. A clinical trial treating cancer patients with 1-methyltryptophan, an inhibitor of IDO, has been started [48].

In turn, kynurenine can be metabolized in the brain by two separate pathways (Fig. 5), either to kynurenic acid or to 3-hydroxykynurenine and quinolinic acid, the precursors of nicotinamide adenine dinucleotide (NAD). Kynurenic acid is neuroprotective through its excitatory amino acid receptor blocking activity and downregulates the initial neuroinflammation, whereas an excess of quinolinic acid has the opposite effect, being a strong agonist of the glutamatergic N-methyl-d-aspartate receptor and a potent neurotoxin with a free radical producing activity. An alteration of the critical balance between these two metabolic branches of the kynurenine pathway in the brain is observed in many neurodegenerative disorders (Parkinson’s, Alzheimer’s and Huntington’s diseases, etc.) [49].

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Figure 5.  Kynurenine pathway (simplified overview): (a) indoleamine 2,3-dioxygenase or tryptophan 2,3-dioxygenase; (b) kynurenine formamidase; (c) kynurenine aminotransferase; (d) kynurenine 3-hydroxylase, kynureninase, 3-hydroxyanthranilic acid oxidase and non-enzymic cyclisation; (e) quinolinic acid phosphoribosyltransferase.

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Numerous studies evaluated the ability of 3-substituted indole derivatives to scavenge radicals or inhibit oxidation reactions in in vitro models. A few examples are presented in Table 1.

Table 1.   Comparison of the antioxidant properties of indolic compounds with those of ascorbate, thiols, and dopamine
Antioxidant assayIC50 (μm)Reference
  1. ABTS•+ (2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) is a stable radical cation with a characteristic absorption at 734 nm. The Trolox-equivalent antioxidant capacity (TEAC) assay is based on the suppression of the absorbance of ABTS•+ by antioxidants in the test sample when ABTS incubates with a peroxidase and H2O2. The TEAC is defined as the concentration of Trolox with equivalent inhibition potency to 1 mm Trp derivatives. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable radical absorbing at 517 nm; TBARS (thiobarbituric acid reactive substances) are measured in tissue extract incubations in the presence of ferric cation, thiobarbituric acid, and 2,6-ditert-butyl-4-methylphenol.

Melatonin  
 Inhibition of OH hydroxylation11[50]
 Inhibition of 3-NO2-Tyr by ONOO290[51]
 DPPH scavenging50[52]
 H2O2200[53]
 ABTS+ scavenging690[54]
 TEAC = 480[55]
 Inhibition of TBARS formation600[54]
 Inhibition of lipid peroxidation300[56]
Serotonin  
 Inhibition of lipid peroxidation30[56]
 Inhibition of 3-NO2-Tyr by ONOO80[51]
Other substituted indoles  
 ABTS scavenging20–300[54]
 TEAC = 1.3–4 [55]
 DPPH scavenging12–200[53]
Pinoline  
 Inhibition of OH hydroxylation60[50]
Ascorbate  
 DPPH scavenging65[52]
 11[53]
 H2O252[53]
Thiols  
 H2O2255[53]
 Inhibition of 3-NO2-Tyr by ONOO40[51]
Dopamine  
 Inhibition of 3-NO2-Tyr by ONOO40[57]

Direct interactions between tryptophan derivatives and ROS/RNS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

Trp derivatives are recognized as efficient hydroxyl (HO) [58, 59], peroxyl (ROO), and oxoferryl [60] scavengers. Melatonin and tryptophan react with hydroxyl radical with similar rate constants = 1010 m/s [61]. They trap tert-butoxyl radical (CH3)3CO as efficiently as flavonoids with a rate constant of 2.8 × 109 m/s [61].

The initial monoelectronic oxidation product is the indolyl radical cation (Fig. 6) [62, 63], which reacts with oxygen to form a peroxyl radical. Decay of such a radical depends on the structure and the presence of reducers like thiols or ascorbate [64].

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Figure 6.  Oxidation products of 3-substituted indoles.

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Trp and melatonin are fundamentally inert for H2O2, NO, or O2•− under physiological conditions in aqueous solutions deprived of metal cations [65, 66]. Chemical reactions with NO and O2•− require a preliminary oxidation, either of the tryptophan derivative or of NO. Nitrosation of primary amine results in diazotation and further degradation, so that only N-blocked indole derivatives are converted to 1-nitrosoindoles by NO in aerated solutions (i.e. via ONOO or N2O3), under thiol-, amine-, or phenol-free conditions. The kinetic rate is limited by the autoxidation of NO.

The other nitrosation pathway is the reaction of NO with one-electron oxidized indoles. Indeed, the electron-rich aromatic ring allows the indolamine to act as an electron donor leading to a radical cation. The kinetic rates of the oxidation by the strong oxidants HO and CO3•2− are identical for melatonin and N-blocked tryptophan (k = 1.3 × 1010 and 7 × 108 m/s, respectively). The radical cation reacts very rapidly with radicals like NO, with electron-rich molecules or with dioxygen [67], rather than undergoing disproportionation.

  • image([68])
  • image
  • image

It was of interest to understand the reactivity of the weak oxidant NO2 towards Trp derivatives. Our study performed with melatonin argued for an addition of the NO2 radical on the indole ring rather than for a monoelectronic oxidation. When both NO2 and NO were generated by radiolysis of aqueous solutions of nitrite and/or nitrate, the unique conversion of melatonin was the formation of 1-nitrosomelatonin suggesting the following mechanism if extrapolation to indole ring is allowed [71]:

  • image

For melatonin: k = 3.7 × 106 m/s [61]

  • image

Trp-derivative conversions by ONOO are diverse and in part not easy to detect. The first step is the formation of the indolyl radical [72]. Suzuki [73] and we [74, 75] reported that a large part of the products obtained from N-acetyltryptophan and melatonin with ONOO are not identified by classical chromatographic analyses probably due to the formation of highly hydrophilic oxidized products. The products that are recovered are identified (Fig. 7) and led us to study the reaction with ONOO in aqueous buffered solutions under various conditions of ratio of the reactants, pH, CO2 content, and additional agents.

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Figure 7.  Major tryptophan derivatives produced by peroxynitrite.

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Analyses performed with other indolic structures showed that ONOO reacts preferentially with 3-substituted indoles such as Trp derivatives rather than with unsubstituted indole. However, a methoxy group in position 5 of the indole nucleus discourages further nucleophilic substitutions as seen in the case of melatonin. The most important products observed at physiological pH with N-acetylTrp and melatonin are 1-nitrosotryptophan derivatives, kynurenines/kynuramines obtained by opening of the pyrrole ring, 2,3-epoxides on the indole ring and nitroindoles (6-nitroTrp and 4-nitromelatonin). When the level of CO2 is low enough and the ONOO anion is not entirely converted to ONOOCO2, kynuramines or kynurenines and 1-nitrosoderivatives are the major products. The nitrosation by ONOO is discussed in the literature [73, 74, 76]. The oxidant OH or CO3•− induce the formation of an indolyl radical cation. NO and NO2 could be produced either by homolytic rupture of ONOOH or through the reaction between ONOO and oxidant radicals. Subsequent combinations depending on the flux of NO and NO2 result in the nitrosation and nitration processes, respectively. At high CO2 concentration, ONOO yields nitromelatonin compounds and indol-2-one at the expense of the nitrosated product and of kynuramine. Similar patterns with other proportions of the class of products were described for peroxidases/H2O2/NO2 [77–79].

In proteins, Trp residues are surrounded by aminoacids that are generally more reactive than Trp itself (tyrosine, cysteine, etc.) and offer a relative protection to Trp residues preventing their oxidation and conversion. Only very few proteins incubated with ONOO have Trp residues modified by oxidation and/or nitration [73]. A single Trp residue is modified by ONOO in human recombinant Cu,Zn-SOD that contains no tyrosine residue. This conversion, corresponding to 30% nitration besides various oxidations, partially inhibits the enzyme [76]. Recently, the same authors detected proteins modified on their Trp residues in PC12 cells [80], but the substitution on N1 by NO or NO2 group was not detected for all proteins reacted with RNS, probably due to the very labile N–N bond that does not withstand the treatment for analysis. However, using several natural biologically active peptides, we showed that nitrosation occurred with the N-blocked terminal peptide of endothelin treated with ONOO [75].

Not only can modification of Trp residues induce changes in the structure and function of the protein they belong to, but also circulating ROS/RNS-modified Trp derivatives are likely to have specific roles in the organism. This aspect still requires further investigation. For instance, nitroindole derivatives display affinity and selectivity for melatoninergic-binding sites MT3 [81]. Moreover, kynurenines and kynuramine (N1-acetyl-N2-formyl-5-methoxykynuramine) are able to donate an electron and protect macromolecules against oxidative modifications in vivo and in vitro [82]. As substrates of kynurenine aminotransferases, they intervene in the transamination of 2-oxoglutamic acid to glutamine and thus play a role in glutaminergic neurotransmission [83].

N-nitrosoindoles as NO donors: mutagens and protectors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

Here we focus on N-nitrosoindoles, an important class of products formed by the reactions discussed above. Their generation through these routes is restricted to the close vicinity of sources of ROS/RNS production. However, larger amounts are produced in the digestive tract when nitrate is ingested. Around 20–30% of the nitrate from the diet is reduced to nitrite in the mouth by the salivary flora, where its protective role is recognized [84]. It then yields nitrous acid in the stomach that stimulates mucus secretion and performs nitrosation of a large variety of food components (phenols, polyphenols, and peptides), despite the presence of nitrosation inhibitors and competitors such as ascorbic acid, flavonoids, anthocyanins, melatonin, free thiol, and free amine-containing compounds in the human diet. Phenols yield C-nitrosated products, while secondary amines like proline, arginine, cysteine, and tryptophan-containing peptides yield N-nitrosoamines [85]. N-nitroso compounds have been known to be present in the intestinal tract of mammals for over 40 years [86–88]. The derivatives of the N-nitrosoindole formed (from melatonin, N-blocked tryptophan derivatives, peptides, or auxine) are largely unknown.

The mutagenic properties of C- and N-nitrosocompounds [85] and of nitrocompounds [89, 90] are thorougly discussed in the literature. It is advisable to distinguish between C-nitroso-aromatic, N-nitroso-aliphatic and -aromatic compounds, and N-nitroso-indoles. The endogenous formation of N-nitroso aliphatic compounds or their presence in the diet has been associated with an elevated cancer risk in the population [91]. Some aromatic nitrosourea and aromatic N-nitrosamines are known to be carcinogens and mutagens. In fact, the mechanisms involved in the mutagenicity depend on the structure, its chemical properties and sometimes involve the products released by the degradation rather than the parent molecule itself. For example, N-nitrosourea was first used as an antitumor agent on the basis of the observation that methylnitrosoguanidine and methylnitrosourea exhibited modest antitumor activity in experimental animal tumor models. Careful structure–function studies showed that several nitrosourea derivatives demonstrated a significant activity against brain tumors which is highly correlated to their biodegradation [92].

Regarding C-nitroso aromatic compounds, their facile addition on thiol groups of GSH or of lateral chains in proteins [93] provokes a depletion of the thiol content and cellular damage. Such an addition of 4-nitrososerotonin (which we have shown to be formed with serotonin and NO under physiological conditions) on GSH was characterized in our laboratory (C. Ducrocq, unpublished results). This example showed clearly the importance of understanding the chemical properties of nitrosocompounds to evaluate their implication in living organisms.

Mutagenic activity tests were developed to gain insight into the role of metabolites by comparing the mutagenic activity of the drug against a specific strain of bacteria with or without prior incubation with liver homogenates [94]. Relying on such tests, the International Agency of Research on Cancer (IARC) classified nitro- and nitrosocompounds as mutagens and potential carcinogens. However, the hypothesis that exposure to any nitroso and nitrocompounds might lead to cancer in vivo has not been scientifically ascertained for all of them and necessitates active investigations.

Aromatic nitrosourea and aromatic N-nitrosamines can act as NO donors [95]. N-nitrosomelatonin and N-nitrosotryptophan decompose spontaneously, following the same rate in physiological aqueous solutions, without effect of free thiol and metal cations. The decay is accelerated by nucleophiles such as ascorbate [96–98]. The release of NO is likely to induce an ambivalent behavior for these compounds. On the one hand, there is some genetic and mechanistic evidence that NO can suppress tumorigenesis [99]. The part of N-nitrosoindoles that is absorbed by digestion could deliver NO through NO release or nitrosation of thiols or phenols, which might be protective by trapping deleterious oxidants. On the other hand, NO elevated production associated with chronic inflammation has been postulated to play an important role in DNA damage [100]. Several results indicate that the mutagenicity of endogenous NO and its derivatives may contribute to spontaneous mutagenesis [101, 102] and characteristic ROS/RNS-induced mutations were observed when macrophages were stimulated [103]. Moreover, characterized oxidative stress with cellular redox imbalance has been found in various cancer cells compared with normal ones [104]. N-acetyl-1-nitrosotryptophan is described to be mutagenic in a series of Escherischia coli and Salmonella typhimurium strains without requirement for metabolic activation [105] and this activity is related to the presence of the N–NO function. Yet, actual NO donors have not been proven carcinogenic in vivo [106]. In the case of 1-nitrosomelatonin, we initiated independent evaluations of its mutagenic properties and a comparison with NO donors, such as DEA and MAMAH NONOates or SIN-1, the molsidomine metabolite. Assays were performed with the E. coli strains developed by Blanco and coworkers according to the protocol previously described for NO donors [107, 108]. Under these conditions, a qualitative evaluation of mutagenesis can be obtained together with a quantitative one and it is possible to differentiate between oxidative (via peroxynitrite, HO, NO2, and CO3•−) and nitrosative (via N2O3) mutagenesis induced by NO [109]. Conclusions are: (i) 1-nitrosomelatonin showed similar mutagenicity to DEA and MAMAH NONOates, but 20-fold less than N-ethyl-N-nitrosourea under the same conditions (results obtained by Manuel Blanco and collaborators and not yet published); (ii) the mutagenicity observed with 1-nitrosomelatonin would be the result of nitrosative deamination of DNA bases such as cytosine and is inhibited by ascorbate; (iii) taking into account that 1-nitrosoindoles can perform transnitrosation to nucleophilic sites on the purine bases, mutageneicity could occur by depurination as described [110].

Biological perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References

Endogenous l-tryptophan and melatonin should trap ROS/RNS in excess in tissues or cells. Interestingly, melatonin synthesis is stimulated, as a protective adaptation response, when organisms are subjected to a mild stress such as diet restriction or moderate exercise in animals or humans, while plasma Trp and melatonin levels drop rapidly (even though high synthesis in maintained) in rats forced to swim or exposed to a toxic environmental contaminant [1, 111]. At the same time, the usual catabolism by liver into 6-hydroxymelatonin sulfate is not affected. In humans, circulating melatonin is significantly decreased in many disease states such as cancer, diabetes [17], coronary heart and Alzheimer’s diseases [112]. In the case of rheumatoid arthritis, a deficiency of tryptophan in the serum of patients associated with an increased degradation into kynurenine was recently measured [113].

Following these observations, 3-substituted indolic compounds have been recognized as promising drugs in several diseases such as neurodegenerative or renal disorders where oxidative stress is well demonstrated [114]. High doses of l-tryptophan and serotonin are not recommended in food supplements due to their effect on mood and functional changes in the activity of the brain [115]. Other indolic derivatives including melatonin, indole-3-carbinol, and their metabolites could be recommended in numerous acute pathological situations. The more abundant results concern melatonin but the therapeutic window in humans is not clearly established [1], even though melatonin is generally recognized safe in short-term treatment. Positive effects of melatonin are numerous, provided that the dose was sufficient to increase the circulating concentration, from attenuation of the effects of age, inhibition of some features characteristic of atherosclerosis, and reducing cancer prevalence and cancer growth. Biochemical markers of its action are decreased oxidized organic compounds (malondialdehyde, 4-hydroxynonenal, etc.) and increased antioxidant enzymes (SOD and glutathione peroxidase) [116]. These effects are not mediated by melatonin receptors. Also, in acute pathological models, beneficial pleiotropic actions of melatonin (3–10 mg/kg) are described following administration of lipopolysaccharide [117], metallic ions (copper, aluminium, mercury, iron, etc.), and chemical toxins, ischemia, ischemia–reperfusion, UV exposure [118] and models of Parkinson’s disease [119]. Therapeutic clinical trials with melatonin gave beneficial effects in Alzheimer’s patients [120]. In chemotherapeutic treatment of cancers [121, 122], melatonin and indole-3-carbinol, alone or in combination with traditional therapy, have been shown to have protective effects in experimental animals and humans [123–126]. However, some adverse effects have been reported in people with immune-system disorders and severe mental illness [127].

Based on our results, the effects of indolic compounds in mammals have to be considered in strong relation with the nitrergic system. It would be of interest to consider the effects of melatonin, indole-3-carbinol and relative drugs with the other components in the diet-like nitrite and nitrate in parallel to the activities of the NO and the O2•− generating enzymatic systems (NADPH oxidases, xanthine oxidase). For example, it is noteworthy that melatonin slows down the l-arginine influx [128] and the NO production [129]. This indoleamine inhibits the expression of inducible NO synthase [130] and counteracts inducible mitochondrial NO synthase [131]. Furthermore, serotonin also interferes with NO-synthase. Taking into account that serotonin and neuronal NO synthase are co-localized in some areas of the central nervous system, in platelets and probably also in the intestinal tract, we demonstrated that serotonin modulated NOS activity to generate reactive oxygen species (O2•− and H2O2) without preventing the production of NO [132]. However, the biological significance of this effect is not known yet.

Only a more global knowledge of these interactions in tissues or organisms will clarify some contradictions found in the literature. Furthermore, research of new indole-derived protective drugs is a valuable concept in the prevention and in the therapy of oxidative stress-related diseases [52, 53, 133].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Reactive oxygen and nitrogen species involved in stress
  5. Natural 3-substituted indoles
  6. Direct interactions between tryptophan derivatives and ROS/RNS
  7. N-nitrosoindoles as NO donors: mutagens and protectors
  8. Biological perspectives
  9. References