Luke Carroll

Reaction of low-molecular-mass organoselenium compounds (and their sulphur analogues) with inflammation-associated oxidants

Abstract Selenium is an essential trace element in mammals, with the majority specifically encoded as seleno-L-cysteine into a range of selenoproteins. Many of these proteins play a key role in modulating oxidative stress, via either direct detoxification of biological oxidants, or repair of oxidised residues. Both selenium- and sulphur-containing residues react readily with the wide range of oxidants (including hydrogen peroxide, radicals, singlet oxygen and hypochlorous, hypobromous, hypothiocyanous and peroxynitrous acids) that are produced during inflammation and have been implicated in the development of a range of inflammatory diseases. Whilst selenium has similar properties to sulphur, it typically exhibits greater reactivity with most oxidants, and there are considerable differences in the subsequent reactivity and ease of repair of the oxidised species that are formed. This review discusses the chemistry of low-molecular-mass organoselenium compounds (e.g. selenoethers, diselenides and selenols) with inflammatory oxidants, with a particular focus on the reaction kinetics and product studies, with the differences in reactivity between selenium and sulphur analogues described in the selected examples. These data provide insight into the therapeutic potential of low-molecular-mass selenium-containing compounds to modulate the activity of both radical and molecular oxidants and provide protection against inflammation-induced damage. Progress in their therapeutic development (including modulation of potential selenium toxicity by strategic design) is demonstrated by a brief summary of some recent studies where novel organoselenium compounds have been used as wound healing or radioprotection agents and in the prevention of cardiovascular disease.

Reactivity of selenium-containing compounds with myeloperoxidase-derived chlorinating oxidants: Second-order rate constants and implications for biological damage.

Hypochlorous acid (HOCl) and N-chloramines are produced by myeloperoxidase (MPO) as part of the immune response to destroy invading pathogens. However, MPO also plays a detrimental role in inflammatory pathologies, including atherosclerosis, as inappropriate production of oxidants, including HOCl and N-chloramines, causes damage to host tissue. Low molecular mass thiol compounds, including glutathione (GSH) and methionine (Met), have demonstrated efficacy in scavenging MPO-derived oxidants, which prevents oxidative damage in vitro and ex vivo. Selenium species typically have greater reactivity toward oxidants compared to the analogous sulfur compounds, and are known to be efficient scavengers of HOCl and other hypohalous acids produced by MPO. In this study, we examined the efficacy of a number of sulfur and selenium compounds to scavenge a range of biologically relevant N-chloramines and oxidants produced by both isolated MPO and activated neutrophils and characterized the resulting selenium-derived oxidation products in each case. A dose-dependent decrease in the concentration of each N-chloramine was observed on addition of the sulfur compounds (cysteine, methionine) and selenium compounds (selenomethionine, methylselenocysteine, 1,4-anhydro-4-seleno-L-talitol, 1,5-anhydro-5-selenogulitol) studied. In general, selenomethionine was the most reactive with N-chloramines (k2 0.8-3.4×10(3)M(-1) s(-1)) with 1,5-anhydro-5-selenogulitol and 1,4-anhydro-4-seleno-L-talitol (k2 1.1-6.8×10(2)M(-1) s(-1)) showing lower reactivity. This resulted in the formation of the respective selenoxides as the primary oxidation products. The selenium compounds demonstrated greater ability to remove protein N-chloramines compared to the analogous sulfur compounds. These reactions may have implications for preventing cellular damage in vivo, particularly under chronic inflammatory conditions.

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

Abstract Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side‐chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second‐order rate constants were determined for Trp• dimerization in Trp‐containing peptides to be 2–6 × 108 M−1 s−1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp‐containing peptides to steady‐state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC‐MS of up to 4 different isomeric Trp‐Trp cross‐links. Similar species were detected with other oxidants, including CO3•‐ (from the HCO3‐ ‐dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3‐. Trp‐Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp‐Trp cross‐linked species. These products may serve as markers of radical‐mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease. Graphical abstract No caption available. HighlightsTryptophan‐derived indolyl radicals (Trp•) have been implicated in protein dimerization.Trp• formed on Trp‐containing peptides dimerize with high rate constants: k2 2–6 × 108 M−1 s−1.Multiple carbon‐carbon and carbon‐nitrogen Trp‐Trp dimers detected by LC‐MS analyses.Trp‐Trp dimers formed by multiple oxidants, including N3•, ONOOH + bicarbonate, and CO3•‐.Trp‐Trp dimers detected after alkaline hydrolysis of oxidized lysozyme.

Selenium-containing indolyl compounds: Kinetics of reaction with inflammation-associated oxidants and protective effect against oxidation of extracellular matrix proteins

Abstract Activated white blood cells generate multiple oxidants in response to invading pathogens. Thus, hypochlorous acid (HOCl) is generated via the reaction of myeloperoxidase (from neutrophils and monocytes) with hydrogen peroxide, and peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent is formed from superoxide radicals and nitric oxide, generated by stimulated macrophages. Excessive or misplaced production of these oxidants has been linked to multiple human pathologies, including cardiovascular disease. Atherosclerosis is characterized by chronic inflammation and the presence of oxidized materials, including extracellular matrix (ECM) proteins, within the artery wall. Here we investigated the potential of selenium‐containing indoles to afford protection against these oxidants, by determining rate constants (k) for their reaction, and quantifying the extent of damage on isolated ECM proteins and ECM generated by human coronary artery endothelial cells (HCAECs). The novel selenocompounds examined react with HOCl with k 0.2–1.0 × 108 M−1 s−1, and ONOOH with k 4.5–8.6 ‐ × 105 M−1 s−1. Reaction with H2O2 is considerably slower (k < 0.25 M−1 s−1). The selenocompound 2‐phenyl‐3‐(phenylselanyl)imidazo[1,2‐a]pyridine provided protection to human serum albumin (HSA) against HOCl‐mediated damage (as assessed by SDS‐PAGE) and damage to isolated matrix proteins induced by ONOOH, with a concomitant decrease in the levels of the biomarker 3‐nitrotyrosine. Structural damage and generation of 3‐nitroTyr on HCAEC‐ECM were also reduced. These data demonstrate that the novel selenium‐containing compounds show high reactivity with oxidants and may modulate oxidative and nitrosative damage at sites of inflammation, contributing to a reduction in tissue dysfunction and atherogenesis. Graphical abstract No caption available. HighlightsAtherosclerosis is characterized by chronic inflammation and oxidative damage.Rate constants for reaction of novel selenocompounds with oxidants were determined.3‐Selanylindoles and imidazopyridines protect HSA against HOCl‐induced oxidation.3‐Selanyl‐imidazopyridine protects matrix proteins against ONOOH‐induced damage.3‐Selanyl‐imidazopyridine reduced 3‐nitroTyr formation on matrix proteins.

Catalytic oxidant scavenging by selenium-containing compounds: Reduction of selenoxides and N-chloramines by thiols and redox enzymes

Myeloperoxidase produces strong oxidants during the immune response to destroy invading pathogens. However, these oxidants can also cause tissue damage, which contributes to the development of numerous inflammatory diseases. Selenium containing compounds, including selenomethionine (SeMet) and 1,4-anhydro-5-seleno-D-talitol (SeTal), react rapidly with different MPO-derived oxidants to form the respective selenoxides (SeMetO and SeTalO). This study investigates the susceptibility of these selenoxides to undergo reduction back to the parent compounds by intracellular reducing systems, including glutathione (GSH) and the glutathione reductase and thioredoxin reductase systems. GSH is shown to reduce SeMetO and SeTalO, with consequent formation of GSSG with apparent second order rate constants, k2, in the range 103–104 M−1 s−1. Glutathione reductase reduces both SeMetO and SeTalO at the expense of NADPH via formation of GSSG, whereas thioredoxin reductase acts only on SeMetO. The presence of SeMet and SeTal also increased the rate at which NADPH was consumed by the glutathione reductase system in the presence of N-chloramines. In contrast, the presence of SeMet and SeTal reduced the rate of NADPH consumption by the thioredoxin reductase system after addition of N-chloramines, consistent with the rapid formation of selenoxides, but only slow reduction by thioredoxin reductase. These results support a potential role of seleno compounds to act as catalytic scavengers of MPO-derived oxidants, particularly in the presence of glutathione reductase and NADPH, assuming that sufficient plasma levels of the parent selenoether can be achieved in vivo following supplementation.

Superoxide radicals react with peptide-derived tryptophan radicals with very high rate constants to give hydroperoxides as major products.

ABSTRACT Oxidative damage is a common process in many biological systems and proteins are major targets for damage due to their high abundance and very high rate constants for reaction with many oxidants (both radicals and two‐electron species). Tryptophan (Trp) residues on peptides and proteins are a major sink for a large range of biological oxidants as these side‐chains have low radical reduction potentials. The resulting Trp‐derived indolyl radicals (Trp•) have long lifetimes in some circumstances due to their delocalized structures, and undergo only slow reaction with molecular oxygen, unlike most other biological radicals. In contrast, we have shown previously that Trp• undergo rapid dimerization. In the current study, we show that Trp• also undergo very fast reaction with superoxide radicals, O2•−, with k 1–2 × 109 M−1 s−1. These values do not alter dramatically with peptide structure, but the values of k correlate with overall peptide positive charge, consistent with positive electrostatic interactions. These reactions compete favourably with Trp• dimerization and O2 addition, indicating that this may be a major fate in some circumstances. The Trp• + O2•− reactions occur primarily by addition, rather than electron transfer, with this resulting in high yields of Trp‐derived hydroperoxides. Subsequent degradation of these species, both stimulated and native decay, gives rise to N‐formylkynurenine, kynurenine, alcohols and diols. These data indicate that reaction of O2•− with Trp• should be considered as a major pathway to Trp degradation on peptides and proteins subjected to oxidative damage. Graphical abstract Figure. No caption available. HighlightsTryptophan (Trp) residues are a major sink for oxidants on peptides and proteins.Trp radicals in peptides undergo fast reactions with O2•− (k ˜ 109 M−1 s−1).Trp hydroperoxides are major products of Trp• + O2•− reactions in peptides.Hydroperoxide degradation gives N‐formylkynurenine, kynurenine and alcohols.Hydroperoxide formation competes favourably with dimerization and O2 addition.

Aggregation of α- and β- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation

Abstract The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO•). We hypothesized that the selectivity of ROO• and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical‐radical reactions, and di‐tyrosine (di‐Tyr) and di‐tryptophan (di‐Trp) formation. Solutions of &agr;‐ and &bgr;‐caseins were exposed to ROO• from thermolysis and photolysis of AAPH (2,2′‐azobis(2‐methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross‐linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di‐Tyr and di‐Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di‐Tyr in both model peptides and &agr;‐ and &bgr;‐ caseins; di‐Trp was only detected in peptides and &agr;‐casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical‐radical reactions, the low flux of ROO• generated during AAPH thermolysis disfavours the formation of dimeric radical‐radical cross‐links such as di‐Tyr and di‐Trp, instead favoring other O2‐dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products. Graphical abstract Figure. No Caption available. Highlights&agr;‐ and &bgr;‐caseins are extensively oxidized by peroxyl radicals.Protein crosslinking and aggregation are the most significant pathways.Extensive oxidation of Tyr and Trp was evidenced.Low fluxes of peroxyl radicals favor Schiff bases rather than di‐Tyr and di‐Trp.

Chapter 9. Reaction of Selenium Compounds with Reactive Oxygen Species and the Control of Oxidative Stress

Selenium is an essential trace element in mammals, as a result of the requirement for the encoded amino acid seleno-l-cysteine (Sec) that is present in a range of selenoproteins. Selenium is also present in other forms in biological systems, including selenomethionine and inorganic species. While the role of some Sec-containing proteins has yet to be fully resolved, some of these, in particular isoforms of the thioredoxin reductase, glutathione peroxidase and methionine sulfoxide reductases, play key roles in modulating oxidative stress. Consequently, there is considerable interest in the reactions of selenium species with oxidants, the reasons for the biological use of selenium versus sulfur and the potential use of low-molecular-mass selenium species as protective agents against oxidative damage. Here we review the kinetics and mechanisms of the reactions of oxidants produced during inflammation, with low-molecular-mass organoselenium compounds (e.g. selenoethers, diselenides and selenols). While selenium has some similar properties to sulfur, it typically exhibits greater reactivity with oxidants (e.g. hydrogen peroxide, radicals, singlet oxygen and hypochlorous, hypobromous, hypothiocyanous and peroxynitrous acids), and there are considerable differences in the subsequent reactivity and ease of repair of the resulting oxidation products. These data provide important insights for the development of therapeutic compounds.