Quercetin Attenuates Vascular Endothelial Dysfunction in Atherosclerotic Mice by Inhibiting Myeloperoxidase and NADPH Oxidase Function.

Myeloperoxidase (MPO) exhibits a unique property to use H2O2 to oxidize chloride and lead to the generation of a strong oxidant, hypochlorous acid (HOCl), which plays important roles in atherosclerosis. A lot of evidence indicates that quercetin, a natural polyphenol derived from human diet, effectively contributes to cardiovascular health. Herein, we found that dietary quercetin significantly inhibited vascular endothelial dysfunction and atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Mechanistic studies revealed that dietary quercetin effectively suppressed the MPO level and activity in the vessels of ApoE-/- animals, and p47phox expression and NADPH oxidase activity were simultaneously attenuated after quercetin treatment. In vascular endothelial cells, NADPH oxidase was demonstrated to be the major source of H2O2 formation. Moreover, quercetin effectively attenuated MPO/H2O2-mediated HOCl production and toxicity to human vascular endothelial cells, and this compound was not toxic. The inhibitory effect on MPO activity was likely attributed to that quercetin significantly inhibited NADPH oxidase-derived H2O2 formation in human endothelial cells and could act as an effective mediator for MPO intermediates, subsequently preventing HOCl production by the MPO/H2O2 system. Collectively, it was suggested that quercetin effectively suppressed endothelial dysfunction in atherosclerotic vasculature through the reduction of MPO/NADPH oxidase-mediated HOCl production.

Serum albumin acted as an effective carrier to improve the stability of bioactive flavonoid.

The health-improving functions of bioactive flavonoids in vitro and in vivo are often limited by their low stability, which could be counteracted by the application of proteins as carriers of flavonoids. Clarification of the mechanism of protein-ligand interaction is crucial for the encapsulation of bioactive components. Herein, common plasma proteins [i.e., bovine serum albumin (BSA), human serum albumin (HSA), human immunoglobulin G (IgG) and fibrinogen (FG)] were compared for their binding characteristics to quercetin, the main component of flavonoids in human diet, in the absence and presence of free Cu2+ (an accelerator for flavonoids' instability) using multi-spectroscopic and computational methods. As a flexible open structure of proteins, both BSA and HSA were found to be the most promising carriers for quercetin and Cu2+ with an affinity on the order of 104 M-1. HSA-diligand complex (i.e., HSA-quercetin-Cu2+) was successfully generated when both quercetin and Cu2+ were added to the HSA solution. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the HSA-diligand complex relative to quercetin-Cu2+ complex. Quercetin/Cu2+ system could induce the formation of reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxide radical (·OH), which were significantly suppressed upon HSA binding. Consistently, the cytotoxicity of the quercetin/Cu2+ system to endothelial cells was reduced in the HSA-diligand complex. These results demonstrate the possibility of developing serum albumin-based carriers for the protection of bioactive flavonoids in their nutritional application.

Bovine Serum Albumin as a Potential Carrier for the Protection of Bioactive Quercetin and Inhibition of Cu(II) Toxicity.

Considering the protective ability of proteins and the potential toxicity of free Cu(II), it was proposed herein that the co-presence of protein could play an important role in suppressing the toxicity of free Cu(II) to the stability of bioactive quercetin if a flavonoid-protein-Cu(II) complex could be formed. In this study, the interaction between quercetin (a major flavonoid in the human diet) and bovine serum albumin (BSA) was investigated in the absence and presence of free Cu(II). The results demonstrated that both quercetin and free Cu(II) had a strong ability to quench the intrinsic fluorescence of BSA through a static procedure (i.e., formation of a BSA-monoligand complex). Site marker competitive experiments illustrated that the binding of both quercetin and Cu(II) to BSA mainly took place in subdomain IIA. The quenching process of free Cu(II) with BSA was easily affected by quercetin, and the increased binding capacity possibly resulted from the generation of a ternary quercetin-BSA-Cu(II) complex. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the BSA-diligand complex relative to a quercetin-Cu(II) complex. A quercetin-Cu(II) system could generate reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), which were significantly inhibited upon BSA binding. Consistently, the cytotoxicity of the quercetin-Cu(II) system to endothelial cells was decreased in the BSA-diligand complex, where the co-presence of BSA played an important role. These results suggest the possibility and advantage of developing albumin-based carriers for the protection of bioactive components and suppression of Cu(II) toxicity in their biomedical and nutritional applications.

Myeloperoxidase Targets Apolipoprotein A-I for Site-Specific Tyrosine Chlorination in Atherosclerotic Lesions and Generates Dysfunctional High-Density Lipoprotein.

We previously demonstrated that apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), is an important target for myeloperoxidase (MPO)-catalyzed tyrosine chlorination in the circulation of subjects with cardiovascular diseases. Oxidation of apoA-I by MPO has been reported to deprive HDL of its protective properties. However, the potential effects of MPO-mediated site-specific tyrosine chlorination of apoA-I on dysfunctional HDL formation and atherosclerosis was unclear. Herein, Tyr192 in apoA-I was found to be the major chlorination site in both lesion and plasma HDL from humans with atherosclerosis, while MPO binding to apoA-I was demonstrated by immunoprecipitation studies in vivo. In vitro, MPO-mediated damage of lipid-free apoA-I impaired its ability to promote cellular cholesterol efflux by the ABCA1 pathway, whereas oxidation to lipid-associated apoA-I inhibited lecithin:cholesterol acyltransferase activation, two key steps in reverse cholesterol transport. Compared with native apoA-I, apoA-I containing a Tyr192 → Phe mutation was moderately resistant to oxidative inactivation by MPO. In high-fat-diet-fed apolipoprotein E-deficient mice, compared with native apoA-I, subcutaneous injection with oxidized apoA-I (MPO treated) failed to mediate the lipid content in aortic plaques while mutant apoA-I (Tyr192 → Phe) showed a slightly stronger ability to reduce the lipid content in vivo. Our observations suggest that oxidative damage of apoA-I and HDL involves MPO-dependent site-specific tyrosine chlorination, raising the feasibility of producing MPO-resistant forms of apoA-I that have stronger antiatherosclerotic activity in vivo.

Generation of a Bovine Serum Albumin-Diligand Complex for the Protection of Bioactive Quercetin and Suppression of Heme Toxicity.

As an abundant protein in milk and blood serum, bovine serum albumin (BSA) contains various sites to bind a lot of bioactive components, generating BSA-monoligand complex. Demonstration of the interaction between BSA and bioactive components (such as heme, flavonoids) is important to develop effective carrier for the protection of bioactive ligands and to reduce cytotoxicity of heme. Herein, the bindings of BSA to quercetin and/or heme were investigated by multispectroscopic and molecular docking methods. The fluorescence of protein was significantly quenched by both quercetin and heme in a static mode (i.e., generation of BSA-ligand complex). Although quercetin had lower affinity to protein than heme, the interactions of both compounds with protein did locate in site I (i.e., subdomain IIA). BSA-diligand complex was successfully generated after the coaddition of quercetin and heme. The cytotoxicity of free heme to endothelial cells was reduced in the BSA-diligand complex relative to that of heme or BSA-monoligand complex, while the stability of bioactive quercetin was promoted in the complex relative to free flavonoid. The complex provided a better inhibition on the cytotoxicity of heme than BSA-monoligand complex, in which the copresence of quercetin played a vital role.

Dietary nitrate attenuated endothelial dysfunction and atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: A critical role for NADPH oxidase.

Nitric oxide (NO) deficiency and NADPH oxidase plays key roles in endothelial dysfunction and atherosclerotic plaque formation. Recent evidence demonstrates that nitrate-nitrite-NO pathway in vivo exerts beneficial effects upon the cardiovascular system. We aimed to investigate the effects of dietary nitrate on endothelial function and atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet. It was shown that dietary nitrate significantly attenuated aortic endothelial dysfunction and atherosclerosis in ApoE-/- mice. Mechanistic studies revealed that dietary nitrate significantly improved plasma nitrate/nitrite, inhibited vascular NADPH oxidase activity and oxidative stress in ApoE-/- mice, while xanthine oxidoreductase (XOR) expression and activity was enhanced in ApoE-/- mice in comparison with wide type animals. These beneficial effects of nitrate in ApoE-/- mice were abolished by PTIO (NO scavenger) and significantly prevented by febuxostat (XOR inhibitor). In the presence of nitrate, no further effect of apocynin (NADPH oxidase inhibitor) was observed, suggesting NADPH oxidase as a possible target. In vitro, NO donor significantly inhibited NADPH oxidase activity in vascular endothelial cells via the induction of heme oxygenase-1. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of vascular NADPH oxidase-derived oxidative stress and endothelial dysfunction, and consequently protected ApoE-/- mice against atherosclerosis. These findings may have novel nutritional implications for the preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.

Supplementation of dietary nitrate attenuated oxidative stress and endothelial dysfunction in diabetic vasculature through inhibition of NADPH oxidase.

The metabolic disorders in diabetes, which are usually accompanied by oxidative stress and impaired nitric oxide (NO) bioavailability, increase the risk of detrimental cardiovascular complications. Herein, we investigated the therapeutic potential of dietary nitrate, which is found in high content in green leafy vegetables, on vascular oxidative stress and endothelial dysfunction in diabetic mice induced by high-fat diet and streptozotocin injection. Dietary nitrate in drinking water fuelled a nitrate-nitrite-NO pathway, which inhibited vascular oxidative stress, endothelial dysfunction and many features of metabolic syndrome in diabetic mice. These beneficial effects of nitrate on diabetic mice were abolished by PTIO (NO scavenger) treatment and significantly prevented by febuxostat (xanthine oxidoreductase inhibitor), demonstrating the central importance of NO in bioactivation of nitrate. The favorable effects of nitrate were not further influenced by apocynin (NADPH oxidase inhibitor), suggesting NADPH oxidase as a possible target. In high glucose-incubated vascular endothelial cells, NO donor attenuated oxidative stress and endothelial dysfunction via the inhibition of NADPH oxidase, where a heme oxygenase-1 (HO-1)-dependent mechanism was demonstrated for the antioxidant abilities of NO. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of NADPH oxidase-derived oxidative stress, endothelial dysfunction and metabolic disorders in diabetic vasculature. These findings may have novel implications for the preventive strategy against diabetes-induced vascular dysfunction and associated complications.

Quercetin, but not rutin, attenuated hydrogen peroxide-induced cell damage via heme oxygenase-1 induction in endothelial cells.

Oxidative stress plays an important role in the pathogenesis of cardiovascular disease. Quercetin, a naturally occurring flavonoid presents in plants and human diet, has been reported to exert antioxidant properties in vivo and in vitro. The upregulation of antioxidant enzyme heme oxygenase-1 (HMOX1) in endothelial cells is considered to be beneficial in cardiovascular disease. In this work, we tested whether quercetin might suppress hydrogen peroxide (H2O2)-induced cell damage in endothelial cells by augmenting this cellular antioxidant defense. It was found that quercetin upregulated HMOX1 expression to protect endothelial cells against oxidative stress, and the protective effects of quercetin on H2O2-induced endothelial cell damage (such as loss of cell viability and reduction of nitric oxide) could be abolished by the specific small-interfering RNA against HMOX1 expression or HMOX1 activity inhibitor. In addition, the activation of ERK/Nrf2 signaling pathway was critical to the upregulation of HMOX1 induced by quercetin. Consistent with its non-effective ability to induce HMOX1, rutin (the glycoside of quercetin) showed less protective effects on H2O2-induced cell damage than quercetin. Therefore, quercetin could attenuate oxidative stress-induced endothelial cell damage at least partly through ERK/Nrf2/HMOX1 pathway. Our results also suggested a novel mechanism for the anti-oxidant property of quercetin and might explain in part the protective cardiovascular effects of diets rich in these compounds.

Quercetin suppressed NADPH oxidase-derived oxidative stress via heme oxygenase-1 induction in macrophages.

NADPH oxidase-derived superoxide (O2.-) generation and oxidative stress is usually considered as an important factor to the pathogenesis of inflammatory diseases. Quercetin, widely known for their anti-oxidant and anti-inflammatory properties in vitro and in vivo, is recently identified to induce expression of antioxidant enzyme heme oxygenase-1 (HO-1). Previous studies suggest that HO-1 induction and/or subsequent HO-1 end product generation in vitro and in vivo may suppress NADPH oxidase-derived oxidative stress. In this study, we tested whether quercetin might modulate NADPH oxidase activity in macrophages via induction of HO-1. In RAW264.7 macrophages, quercetin significantly attenuated NADPH oxidase-derived O2.- generation via a HO-1-dependent mechanism. Mechanistically, the protective effects of quercetin were (1) linked to increased expression of HO-1 in the presence or absence of lipopolysaccharide (LPS), (2) similar to that observed with the NADPH oxidase inhibitor apocynin, and (3) could be abolished by the specific small-interfering RNA against HO-1 expression or HO-1 activity inhibitor tin protoporphyrin. The induction of HO-1 by quercetin was associated with the nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. In addition, this flavonoid also inhibited the overproduction of nitric oxide and inflammatory cytokines in LPS-stimulated macrophages via simultaneous induction of HO-1 expression. In agreement with the observations in macrophages, pretreatment with quercetin significantly alleviated LPS-induced inflammation in mice which was concomitant with decreased NADPH oxidase activity and increased HO-1 expression. Our results suggested that quercein could modulate NADPH oxidase-derived O2.- production in macrophages at least partly through HO-1 induction. Suppression of NADPH oxidase-dependent oxidative stress may represent a novel mechanism underlying the anti-oxidant and anti-inflammatory properties of quercetin/HO-1 pathway.

NADPH oxidase is a primary target for antioxidant effects by inorganic nitrite in lipopolysaccharide-induced oxidative stress in mice and in macrophage cells.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Inorganic nitrite, previously viewed as a harmful substance in our diet or inert metabolites of endogenous NO, is recently identified as an important biological NO reservoir in vasculature and tissues. Stimulation of a nitrite-NO pathway shows organ-protective effects on oxidative stress and inflammation, but the mechanisms or target are not clear. In this study, the hypothesis that inorganic nitrite attenuated lipopolysaccharide (LPS)-induced oxidative stress in mice and in macrophage cells by modulating NADPH oxidase activity and NO bioavailability were investigated. We showed that nitrite treatment, in sharp contrast with the worsening effect of NO synthases inhibition, significantly attenuated aortic oxidative stress, endothelial dysfunction and mortality in LPS-induced shock in mice. Mechanistically, protective effects of nitrite were abolished by NO scavenger and xanthine oxidase inhibitor, and inhibition of NADPH oxidase with apocynin attenuated LPS-induced oxidative stress similar to that of nitrite. In the presence of nitrite, no further effect of apocynin was observed, suggesting NADPH oxidase as a possible target. In LPS-activated macrophage cells, nitrite reduced NADPH oxidase activity and oxidative stress and these effects of nitrite were also abolished by NO scavenger and xanthine oxidase inhibitor, where xanthine oxidase-mediated reduction of nitrite attenuated NADPH oxidase activity in activated macrophages via a NO-dependent mechanism. In conclusion, these novel findings position NADPH oxidase in the inflammatory vasculature as a prime target for the antioxidant effects of inorganic nitrite, and open a new direction to modulate the inflammatory response.

Adsorption of Plasma Proteins on Single-Walled Carbon Nanotubes Reduced Cytotoxicity and Modulated Neutrophil Activation.

Proteins in the bloodstream bind to carbon nanotubes (CNTs) through noncovalent interactions to form a protein corona, thereby effectively influencing the biological properties and blood biocompatibility of the CNTs. Here, we investigated the binding of common plasma proteins (i.e., human immunoglobulin G (IgG), human serum albumin (HSA), and fibrinogen (FG)) to carboxylated single-walled CNTs (SWCNTs), and evaluated the effects of these different protein coronas on cytotoxicity to endothelial cells and immune response to neutrophils in the bloodstream. Measurements of adsorption parameters revealed tight binding of proteins to SWCNTs, and the SWCNTs adsorption capacities followed the order FG > HSA > IgG. In addition, the basic residues (Arg, Lys, His) were found to play an important role in the formation of protein-SWCNTs corona complexes and determine their adsorption capacity. Consistent with the higher protein adsorption capacity, FG more significantly reduced the cytotoxicity of CNTs to human umbilical vein endothelial cells than the other two proteins. However, only treatment of SWCNTs with IgG resulted in the enhancement of CNT-induced myeloperoxidase (MPO) release (i.e., neutrophil activation) in neutrophils, while MPO-dependent degradation of CNTs induced less cytotoxicity than initial nanomaterials. Consistent with these effects of protein coronas, the presence of serum attenuated the cytotoxicity of CNTs and CNTs could induce neutrophil activation in human blood plasma. Our study demonstrates the ability of adsorbed plasma proteins to influence cytotoxicity and neutrophil response caused by CNTs in the bloodstream.

Fibrinogen binding-dependent cytotoxicity and degradation of single-walled carbon nanotubes.

Carbon nanotubes are widely used in the area of biomedicine, and the binding of protein to carbon nanotubes are believed to play an important role in the potential cytotoxicity of nanomaterials. In this work, we investigated the effects of human fibrinogen-surface coatings on the biodegradation and cytotoxicity of carboxylated single-walled carbon nanotubes (SWCNTs). It was found that the electrostatic and π-π stacking interactions might be the crucial factors in stabilizing the binding of fibrinogen with SWCNTs by both theoretical and experimental approaches. Although naked SWCNTs could induce significant toxicity to macrophages, coating these nanomaterials with fibrinogen could greatly attenuate their toxicity. On the other hand, although SWCNTs and fibrinogen-preincubated SWCNTs were resistant to biodegradation in resting macrophages, both naked and fibrinogen-coated SWCNTs could be effectively and similarly degraded through myeloperoxidase (MPO) and peroxynitrite (ONOO-)-dependent pathways in activated macrophages, where NADPH oxidase played a determinant role in the biodegradation process. Importantly, degraded SWCNTs by ONOO- pathway in vitro induced less cytotoxicity than non-degraded nanotubes. These findings demonstrated that the binding of fibrinogen to SWCNTs could reduce cytotoxicity without affecting the biodegradation of nanotubes in activated inflammatory cells, providing a new route to design the safer nanotubes for future biomedical applications.

Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H2O2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H2O2-MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases.

NADPH oxidase-dependent degradation of single-walled carbon nanotubes in macrophages.

Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) could be oxidatively biodegraded by neutrophil myeloperoxidase (MPO) and peroxynitrite (ONOO-). However, the biodegradation mechanism of nanotubes in macrophages has not been explored enough. Here, we showed that both MPO and ONOO- could effectively oxidize SWCNTs to generate shorter and oxidative nanotubes in vitro. SWCNTs were significantly degraded in zymosan-stimulated macrophages, and the degradation mechanism was dependent on MPO and ONOO--driven oxidative pathways of activated macrophages, where NADPH oxidase was found to be a major determinant of the biodegradation process. Moreover, the functionalization of IgG to SWCNTs could stimulate MPO release and ONOO- formation in macrophages, thereby creating the conditions favorable for degradation of nanotubes and subsequently contributing to the higher degradation degree of IgG-coated SWCNTs. Therefore, our discovery of NADPH oxidase-dependent SWCNTs degradation in activated macrophages will open new opportunities for the regulation of SWCNTs fate in vivo.

Key roles of Tyr 10 in Cu bound Aß complexes and its relevance to Alzheimer's disease.

Recent studies show that the accumulation of redox-active Cu mediates the aggregation of amyloid ß-peptide (Aß) and conspicuous oxidative damage to the brain in Alzheimer's disease (AD). However, the key roles for Tyr 10 in Aß-Cu(II) complex and its potential biological relevance to AD etiology under oxidative stress, were not stressed enough. Interestingly, our results indicated that Aß40 (not Aß16)-Cu(II) complex showed obviously enhanced peroxidase activity than free Cu(II). Although Tyr 10 was not the residue binding Cu(II), the mutation of Tyr 10 residue in Aß40 decreased the peroxidase activity of Aß40-Cu(II) complex, and the mutation of Tyr 10 could inhibit Aß40 aggregation. Under oxidative and nitrative stress conditions, the Aß-Cu(II) complex caused oxidation and nitration of the Aß Tyr 10 residue through peroxidase-like reactions, where the formation of Cu(I) and hydroxyl radical (OH) was proposed as a chemical mechanism. We also showed that, when Aß40 aggregates were bound to Cu(II), they retained peroxidase-like activity. Therefore, Tyr 10 residue is pivotal in Aß-Cu(II) complex and shows important relevance to oxidative stress, implicating the novel significance of Tyr 10 residue as well as Aß-Cu(II) complex in the pathology of AD.

Key roles for tyrosine 10 in aß-heme complexes and its relevance to oxidative stress.

Amyloid ß-peptide (Aß) aggregation in the brain, known as amyloid plaques, is a pathological feature of Alzheimer's disease (AD). Recent studies show that heme binds to the His residue of Aß with the iron center and subsequently forms an Aß-heme complex, which can inhibit Aß aggregation. Although Tyr-10 was not the residue binding heme, the key roles for Tyr-10 in Aß-heme complexes and its potential biological relevance to AD etiology under oxidative stress were not sufficiently evaluated. Here, we used wild-type and Tyr-10-mutated human Aß peptides and studied the impact of the mutation on Aß-heme peroxidase activity, heme-bound Aß aggregation, and oxidation and nitration under oxidative and nitrative stresses. Our results indicate that the mutation of Tyr-10 in Aß16 and Aß40 decreased the peroxidase activity of Aß-heme complexes and that the mutation of Tyr-10 could inhibit Aß40 self-assembly aggregation. Under oxidative (H2O2) and nitrative (H2O2/NaNO2) stress conditions, the Aß40-heme complexes caused oxidation and nitration of the Aß Tyr-10 residue through promoting peroxidase-like reactions, which were different from the classic inhibitive effect of heme on Aß aggregation. To our knowledge, this is the first time that the formation of a heme-to-protein cross-linked Aß40-heme complex under oxidative stress has been reported; in addition, the mutation of Tyr-10 could inhibit the cross-link formation. Therefore, Tyr-10 is pivotal in Aß-heme complexes and plays key roles in Aß aggregation under oxidative and nitrative stresses, demonstrating a novel significance of Tyr-10 as well as Aß-heme complexes in the pathology of AD.

Key roles of Arg(5), Tyr(10) and his residues in Aß-heme peroxidase: relevance to Alzheimer's disease.

Recent reports show that heme binds to amyloid ß-peptide (Aß) in the brain of Alzheimer's disease (AD) patients and forms Aß-heme complexes, thus leading a pathological feature of AD. However, the important biological relevance to AD etiology, resulting from human Aß-heme peroxidase formation, was not well characterized. In this study, we used wild-type and mutated human Aß1-16 peptides and investigated their Aß-heme peroxidase activities. Our results indicated that both histidine residues (His(13), His(14)) in Aß1-16 and free histidine enhanced the peroxidase activity of heme, hence His residues were essential in peroxidase activity of Aß-heme complexes. Moreover, Arg(5) was found to be the key residue in making the Aß1-16-heme complex as a peroxidase. Under oxidative and nitrative stress conditions, the Aß1-16-heme complexes caused oxidation and nitration of the Aß Tyr(10) residue through promoting peroxidase-like reactions. Therefore, these residues (Arg(5), Tyr(10) and His) were pivotal in human Aß-heme peroxidase activity. However, three of these residues (Arg(5), Tyr(10) and His(13)) identified in this study are all absent in rodents, where rodent Aß-heme complex lacks peroxidase activity and it does not show AD, implicating the novel significance of these residues as well as human Aß-heme peroxidase in the pathology of AD.

Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein-SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π-π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl- (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA-SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl- generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

The dual effects of nitrite on hemoglobin-dependent redox reactions.

Evidence to support the role of heme proteins-dependent reactions as major inducers of oxidative damage is increasingly present. Nitrite (NO2(-)) is one of the major end products of NO metabolism, and from the daily consumption. Although the biological significance of heme proteins/NO2(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO2(-) on heme proteins-dependent redox reactions have been greatly underestimated. In this study, we investigated the influence of NO2(-) on met-hemoglobin (Hb)-dependent oxidative and nitrative stress. It was found that NO2(-) effectively reduced cytotoxic ferryl intermediate back to ferric Hb in a biphasic kinetic reaction. However, the presence of NO2(-) surprisingly exerted pro-oxidant effect on Hb-H2O2-induced protein (bovine serum albumin, enolase) oxidation at low concentrations and enhanced the loss of HepG2 cell viability. In the reduction of ferryl Hb to ferric state, NO2(-) was decreased and oxidized to a nitrating agent NO2, Tyr12 and Tyr191 in enolase were subsequently nitrated. In contrast to the frequently inhibitive effect of nitrotyrosine, NO2(-)-triggered tyrosine nitration might play an important role in enolase activation. These data provided novel evidence that the dietary intake and potential therapeutic application of NO2(-) would possess anti- and pro-oxidant activities through interfering in hemoglobin-dependent redox reactions. Besides the classic role in protein tyrosine nitration, the dual effects on hemoglobin-triggered oxidative stress may provide new insights into the physiological and toxicological implications of NO2(-) with heme proteins.

The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology.

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.