Bilirubin: an endogenous scavenger of nitric oxide and reactive nitrogen species

Bilirubin is the final product of heme metabolism. Until recently, bilirubin was considered as a mere by-product of heme degradation but, in the last 20 years, many papers have appeared in the literature demonstrating that this bile pigment is endowed with a strong antioxidant activity, being able to counteract the cellular damage elicited by reactive oxygen species in many in vitro and in vivo experimental systems. Interestingly, compelling evidence has shown that BR can serve as an endogenous scavenger of both nitric oxide and reactive nitrogen species, thus widening the protective role of bilirubin to other reactive species originating within the cellular milieu. The aim of this paper is to give an overview of the interaction between bilirubin and nitric oxide/reactive nitrogen species; furthermore, the possible pathophysiological and clinical relevance of this interaction will be discussed.     

Nitric Oxide Production by Necrotrophic Pathogen Macrophomina phaseolina and the Host Plant in Charcoal Rot Disease of Jute: Complexity of the Interplay between Necrotroph–Host Plant Interactions

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.     

The interdependence of the reactive species of oxygen, nitrogen, and carbon

This mini-review tries to summarize the main interdependences between the free radicals of oxygen, nitrogen, and carbon. Also, the main metabolic pathways for these radical species are described, as well as how these affect their interaction and functional implications. Emphasis is made on the metabolic disturbances induced by stressing aggressions that produce radical species. In this way, cellular oxidative imbalances created by the superiority of reactive oxygen species over the antioxidant systems produce both activation of nitroxide synthases and the oxidation of terminal nitrogen from l-arginine, as well as the metabolization of heme until carbon monoxide by nitric oxide-activated hemoxygenase. Also, multiple cellular protein and nucleoprotein alterations determined by these three kinds of radical species are completed by the involvement of hydrogen sulfide, which results from the degradation of l-cysteine by cistationine-γ-lyase. In this way, sufficient experimental data tend to demonstrate the involvement of hydrogen sulfide and other thiol derivatives in the interrelations between oxygen, nitrogen, and carbon, which results in a true radical cascade. Thus, oxidative stress, together with nitrosative and carbonilic stress, may constitute a central point where other factors of vulnerability meet, and their interactions could have an important impact in many modern diseases. Considering that the actions of reactive species can be most of the time corrected, future studies need to establish the therapeutical importance of various agents which modulate oxidative, nitrosative, or carbonilic stress.     

Structural and functional diversity of transient heme binding to bacterial proteins

BackgroundHeme is an important nutritional iron source for almost all bacteria. Elevated heme concentrations, in contrast, are toxic e.g. due to the generation of reactive oxygen species. The cellular heme concentration thus requires tight regulation. The observation of heme acting as an effector molecule in heme-uptake and -utilization processes is rather new and many of these processes are unknown or rarely understood on the molecular level.Scope of reviewWe describe processes involving transient heme-protein interaction in bacteria and highlight the regulatory function of heme at key steps during heme uptake and utilization. We furthermore focus on essential structural aspects of heme binding to respective proteins.Major conclusionsThe structural and functional basis for heme-regulated processes in bacteria is diverse and ranges from increased degradation to extended half-life and from inhibition to activation of the respective heme-regulated protein. The large variety of effects is attributed to the versatile ability of heme to interact with proteins in different ways.General significanceKnowledge of the molecular mechanism of transient heme-protein interaction is central to understand the heme-regulated processes in bacteria. The heme-binding proteins involved in these processes represent potential targets for the development of novel antibacterial drugs. New antibacterial strategies are urgently needed to combat antibiotic resistance.     

Silibinin induces the generation of nitric oxide in human breast cancer MCF-7 cells

Abstract

Increasing research has concentrated on the anti-tumour efficacy of silibinin, a flavonolignan that is clinically used as an hepatoprotectant. However, previous work has found that silibinin-induced apoptosis is accompanied by protective superoxide (O₂??) generation in MCF-7 cells. This study further reports the formation of reactive nitrogen species (RNS) in the same system. It finds that silibinin induces nitric oxide (?NO) generation in a time- and concentration-dependent manner. Moreover, the results support that there exists an inter-regulation pattern between RNS and reactive oxygen species (ROS) generation. In addition, silibinin is also found to induce RNS and ROS generation in the isolated populations of mouse peripheral blood mononuclear cells (PBMCs) and a simple in vivo model of Caenorhabditis elegans.     

Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin

Eukaryotic assimilatory nitrate reductase (NR) is a multi-domain protein that catalyzes the rate-limiting step in nitrate assimilation. This protein is highly conserved and has been extensively characterized in plants and algae. Here, we report hybrid NRs (NR2-2/2HbN) identified in two microalgal species, Heterosigma akashiwo and Chattonella subsalsa, with a 2/2 hemoglobin (2/2Hb) inserted into the hinge 2 region of a prototypical NR. 2/2Hbs are a class of single-domain heme proteins found in bacteria, ciliates, algae and plants. Sequence analysis indicates that the C-terminal FAD/NADH reductase domain of NR2-2/2HbN retains identity with eukaryotic NR, suggesting that the 2/2Hb domain was inserted interior to the existing NR domain architecture. Phylogenetic analysis supports the placement of the 2/2Hb domain of NR2-2/2HbN within group I (N-type) 2/2Hbs with high similarity to mycobacterial 2/2HbNs, known to convert nitric oxide to nitrate. Experimental data confirms that H. akashiwo is capable of metabolizing nitric oxide and shows that HaNR2-2/2HbN expression increases in response to nitric oxide addition. Here, we propose a mechanism for the dual function of NR2-2/2HbN in which nitrate reduction and nitric oxide dioxygenase reactions are cooperative, such that conversion of nitric oxide to nitrate is followed by reduction of nitrate for assimilation as cellular nitrogen.     

The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells

Background: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2).Aims: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state).Methods: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay.Results: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells.Conclusions: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.     

The mechanisms for nitration and nitrotyrosine formation in vitro and in vivo: Impact of diet

The detection of 3-nitro-L-tyrosine residues associated with many disease states, including gastric cancer, has implicated a role for peroxynitrite in vivo, and thus endogenously produced nitric oxide and superoxide. Additionally, dietary nitrate has been suggested to be involved in the pathogenesis of gastric cancer through a mechanism involving reduction to nitrite and subsequent formation of potentially mutagenic nitrosocompounds. Studies have now demonstrated that a multitude of reactive nitrogen species other than peroxynitrite are capable of producing nitrotyrosine. Thus, we have reviewed the evidence that dietary nitrate, amongst other reactive nitrogen species, may contribute to the body burden of nitrotyrosine.     

Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b₅, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b₅ reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.     

Similarity and dissimilarity of thiols as anti-nitrosative agents in the nitric oxide-superoxide system

Concomitant production of nitric oxide and superoxide in biological systems has been proposed to generate numerous reactive oxygen and nitrogen species that cause oxidative and nitrosative stress. Thiols, especially glutathione, play an important role in cellular defense against radical species. In the present study, we investigated and compared the anti-nitrosative activity of a wide range of thiols in a simplified chemical system of co-generated nitric oxide and superoxide. Of the 13 thiols studied, three groups of thiols are distinguishable: (i) Group I includes cysteine and its four congeners (cysteine methyl ester, cysteine ethyl ester, homocysteine, cysteamine); they are subject to rapid oxidative decomposition and have the least anti-nitrosative activity. (ii) Group II consists of glutathione, penicillamine, tiopronin and mesna; they have the greatest effect on delaying the nitrosation reaction. (iii) Group III comprises N-acetylcysteine, N-acetylpenicillamine, captopril, and thioglycolate; they all have high pK_a for the mercapto group and show the strongest inhibitory effect on the rate and extent of nitrosation in the system studied.     

Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants

Effects of water stress on plants have been well-documented. However, the combined responses to drought and rewatering and their underlying mechanisms are relatively unknown. The present study attempts to describe spatiotemporal alterations in the physiology and cellular status of Medicago truncatula tissues that result from and subsequently follow a period of moderate water deficit. Physiological processes and cellular damage levels were monitored in roots and leaves by determining lipid peroxidation levels, as well as nitric oxide and hydrogen peroxide content, further supported by stomatal conductance and chlorophyll fluorescence measurements in leaves. During water stress, cells in both organs displayed increased damage levels and reactive oxygen and nitrogen species content, while leaves showed reduced stomatal conductance. Furthermore, both tissues demonstrated increased proline content. Upon rewatering, plants recovered displaying readings similar to pre-stress control conditions. Furthermore, molecular analysis of antioxidant gene expression by quantitative real-time RT-PCR revealed differential spatiotemporal regulation in a number of genes examined (including catalase, cytosolic ascorbate peroxidase, copper/zinc and iron superoxide dismutase and alternative oxidase). Overall, M. truncatula plants demonstrated increased sensitivity to drought-induced oxidative damage; however, this was reversed following rewatering indicating a great elasticity in the plant''s capacity to cope with free oxygen and nitrogen radicals.Key words: drought stress, antioxidants, cellular damage, medicago, proline, hydrogen peroxide, nitric oxide, reactive oxygen species, reactive nitrogen species     

Nitric oxide generation from heme/copper assembly mediated nitrite reductase activity

Nitric oxide (NO) as a cellular signaling molecule and vasodilator regulates a range of physiological and pathological processes. Nitrite (NO₂ ^?) is recycled in vivo to generate nitric oxide, particularly in physiologic hypoxia and ischemia. The cytochrome c oxidase binuclear heme a ₃/Cu_B active site is one entity known to be responsible for conversion of cellular nitrite to nitric oxide. We recently reported that a partially reduced heme/copper assembly reduces nitrite ion, producing nitric oxide; the heme serves as the reductant and the cupric ion provides a Lewis acid interaction with nitrite, facilitating nitrite (N–O) bond cleavage (Hematian et al., J. Am. Chem. Soc. 134:18912–18915, 2012). To further investigate this nitrite reductase chemistry, copper(II)–nitrito complexes with tridentate and tetradentate ligands were used in this study, where either O,O′-bidentate or O-unidentate modes of nitrite binding to the cupric center are present. To study the role of the reducing ability of the ferrous heme center, two different tetraarylporphyrinate–iron(II) complexes, one with electron-donating para-methoxy peripheral substituents and the other with electron-withdrawing 2,6-difluorophenyl substituents, were used. The results show that differing modes of nitrite coordination to the copper(II) ion lead to differing kinetic behavior. Here, also, the ferrous heme is in all cases the source of the reducing equivalent required to convert nitrite to nitric oxide, but the reduction ability of the heme center does not play a key role in the observed overall reaction rate. On the basis of our observations, reaction mechanisms are proposed and discussed in terms of heme/copper heterobinuclear structures.     

Attenuation of oxidative stress by Allylpyrocatechol in synovial cellular infiltrate of patients with Rheumatoid Arthritis

Abstract

Free radicals are involved in the pathogenesis of Rheumatoid arthritis, a systemic autoimmune disorder characterized by unchecked synovial inflammation. Allylpyrocatechol, a phytoconstituent of Piper betle leaves, has potent anti-inflammatory activity and this study evaluated its anti-oxidant effect on the synovial infiltrate of patients with Rheumatoid arthritis. The ex vivo effect of allylpyrocatechol upon generation of reactive oxygen species in neutrophils, macrophages and lymphocytes was measured by flow cytometry using dichlorodihydrofluorescein diacetate, wherein it significantly decreased basal levels as also scavenged phorbol myristate acetate generated reactive oxygen species. Furthermore, its effect on generation of superoxide and hydroxyl radicals produced within infiltrated neutrophils was measured by cytochrome c and deoxyribose assay, respectively. Allylpyrocatechol significantly scavenged superoxide and hydroxyl radicals in infiltrated neutrophils. The effect of allylpyrocatechol on nitric oxide was measured in macrophages using 4,5-diaminofluorescein diacetate by flow cytometry wherein it decreased production of nitric oxide in infiltrated macrophages, which correlated with its in vitro nitric oxide scavenging activity. Taken together, this ex vivo study has established that allylpyrocatechol has potent scavenging activity and could be considered as an add-on therapy in the treatment of inflammation-associated disorders like Rheumatoid Arthritis.     

Cellular uptake mechanisms and responses to NO transferred from mono- and poly-S-nitrosated human serum albumin

Abstract

Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects.     

An apoptotic model for nitrosative stress

Nitric oxide overproduction has been implicated in the pathogenesis of many disorders, including artherosclerosis, neurodegenerative diseases, inflammatory and autoimmune diseases, and cancer. The common view holds that nitric oxide-induced cellular injury is caused by oxidative stress. This theory predicts that interactions between reactive nitrogen species and reactive oxygen species produce powerful oxidants that initiate cell death programs. Cytokine-treated murine macrophages are the prototype of this form of cellular injury. Here we report that generation of reactive nitrogen species upon lipopolysacharide/interferon-gamma stimulation of RAW 264.7 cells is largely divorced from production of reactive oxygen species, and that oxidative stress is not principally responsible for cell death (in this model). Rather, the death program is induced mainly by a nitrosative challenge, characterized by the accrual of nitrosylated proteins without a major alteration in cellular redox state. Moreover, interactions between reactive oxygen and nitrogen species may alter the balance between pathways that yield nitrite and nitrate, without impacting the level of S-nitrosylation or extent of cell death. Our results thus (1) provide new insights into NO-related metabolic pathways, (2) demonstrate that apoptotic injury can be caused by nitrosative mechanisms, and (3) establish a model for nitrosative stress in mammalian cells.     

Nitrosation-modulating effect of ascorbate in a model dynamic system of coexisting nitric oxide and superoxide

Abstract

The coexistence of nitric oxide and superoxide leads to complex oxidative and nitrosative chemistry, which has been implicated in many pathophysiological conditions. The present study investigated the role of ascorbate in affecting the kinetics of nitrosative chemistry in a model dynamic snystem of coexisting nitric oxide and superoxide. SIN-1 (3-morpholinosydnonimine) was used to elicit various degrees of nitroxidative stress in a reaction buffer and DAN (2,3-diaminonaphthalene) was used as a probe for N-nitrosation reaction. The nitrosation kinetics in the absence and presence of ascorbate was followed by measuring the formation of the fluorescent product over time. Computational modelling was used to provide quantitative or semi-quantitative insights into the studied system. The results show that ascorbate effectively quenches N-nitrosation reaction, which could be partially attributed to the free radical scavenging and repairing effect of ascorbate. Computational modelling reveals an interesting temporal distribution of superoxide, nitric oxide and peroxynitrite. The model predicts that peroxynitrite is the most predominant species in the SIN-1 system. Furthermore, ascorbate might alter the system dynamics by removing superoxide and, thereby, increasing the availability of nitric oxide.     

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction

Abstract

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.