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

Abstract

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.     

Destructive effect of anticancer oxali-palladium on heme degradation through the generation of endogenous hydrogen peroxide

The interaction between human hemoglobin (Hb) and oxali-palladium was studied using different spectroscopic methods of UV–vis, fluorescence, circular dichroism (CD), and chemiluminescence at two temperatures of 25 and 37°C. The experimental results showed that both dynamic and static quenching is occurred simultaneously when oxali-palladium quenches the fluorescence of Hb. According to the fluorescence quenching method, the binding site number, apparent binding constant, and corresponding thermodynamic parameters were measured at two temperatures. The values of ΔH°, ΔS°, and ΔG° indicate that process of the formation of oxali-palladium–Hb complex is a spontaneous interaction procedure in which electrostatic interaction plays a major role. In addition, UV–vis and CD results showed that the addition of oxali-palladium changes the conformation of Hb. To evaluate the functional changes of Hb via destruction of the heme structure, fluorescence studies were performed. The results demonstrated that two fluorescent heme degradation products are found during the interaction of oxali-palladium with Hb. Also, the amount of hydrogen peroxide produced in the solution of Hb due to the interaction of oxali-palladium with Hb using chemiluminescence method indicated heme degradation in the protein is occurred. Structural and functional changes induced in Hb via heme degradation are considered as side effects of this synthesized anticancer drug.     

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.     

ROS-mediated heme degradation and cytotoxicity induced by iron nanoparticles: hemoglobin and lymphocyte cells as targets

Nanoparticles (NPs) due to their small size and high surface area induce remarkable adverse effects on the biological systems. However, the exact mechanism by which NPs interacted with biological system and induce their adverse effects is still an enigma. Herein, the interaction of zero valent iron NPs (ZVFe NPs) with human hemoglobin (Hb) was evaluated using a variety of techniques including circular dichroism, fluorescence, and UV–visible (UV–vis) spectroscopy methods. Also, the cytotoxicity of ZVFe NPs on the human lymphocyte cell line as a model of blood system cell line was investigated by reactive oxygen species (ROS), caspase-9, and caspase-3 activities assays. It was revealed that ZVFe NP interaction resulted in heme displacement and degradation and induction of protein cabonylation. It was also shown that ZVFe NPs impaired the complexity of lymphocyte cells through ROS generation and apoptotic pathway. Together, these data suggest that NPs influence the biological system and induce adverse effects through ROS generation.     

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K,PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L ‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O, H₂O₂, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009. © 2009 Wiley‐Liss, Inc.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Effects of Diabetes and Obesity on Vascular Reactivity,Inflammatory Cytokines,and Growth Factors

We examined the influences of obesity and diabetes on endothelium‐dependent and ‐independent vasodilation, inflammatory cytokines, and growth factors. We included 258 subjects, age 21–80 years in four groups matched for age and gender: 40 healthy nonobese (BMI <30 kg·m^?2) nondiabetic subjects, 76 nonobese diabetic patients, 37 obese (BMI >30) nondiabetic subjects, and 105 obese (BMI >30) diabetic patients. The flow‐mediated dilation (FMD, endothelium‐dependent) and nitroglycerin‐induced dilation (NID, endothelium‐independent) in the brachial artery, the vascular reactivity at the forearm skin and serum growth factors and inflammatory cytokines were measured. FMD was reduced in the nonobese diabetic patients, obese nondiabetic controls, and obese diabetic patients (P < 0.0001). NID was different among all four groups, being highest in the obese nondiabetic subjects and lowest in the obese diabetic patients (P < 0.0001). The resting skin forearm blood flow was reduced in the obese nondiabetic subjects (P < 0.01). Vascular endothelial growth factor (VEGF) was higher in the obese nondiabetic subjects (P < 0.05), tumor necrosis factor–α was higher in the obese diabetic patients (P < 0.0001) and C‐reactive protein was higher in both the obese nondiabetic and diabetic subjects (P < 0.0001). Soluble intercellular adhesion molecule‐1 was elevated in the two diabetic groups and the obese nondiabetic subjects (P < 0.05). We conclude that diabetes and obesity affect equally the endothelial cell function but the smooth muscle cell function is affected only by diabetes. In addition, the above findings may be related to differences that were observed in the growth factors and inflammatory cytokines.     

Effect of antioxidant <Emphasis Type="Italic">N</Emphasis>-acetylcysteine on diabetic retinopathy and expression of VEGF and ICAM-1 from retinal blood vessels of diabetic rats

Diabetic retinopathy (DR) is a leading cause of blindness globally and its pathogenesis has still not been completely elucidated. Some studies show a close relation between oxidative stress and DR. This study was aimed to investigate the effects of anti-oxidant in DR and expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) from retinal blood vessels in diabetic rats. Diabetic rat models were established by intraperitoneal injection of streptozotocin (60 mg/kg) and confirmation of high serum glucose levels in the animals. Antioxidant N-acetylcysteine was given to diabetic rats to elicit antioxidative responses, and rats were sacrificed at 3 and 5 months. Ultrastructures of retinal vascular tissues were observed under transmission electron microscope, and pathology of retinal capillaries was examined using retinal vascular digest preparations. Changes in the expression of VEGF and ICAM-1 were examined by immunofluorescence; and reactive oxygen species contents in the retinas were detected using dichlorofluorescein assay. Compared with normal rats, diabetic rats displayed significant retinopathy both under electronic and light microscopy, accompanied by elevated reactive oxygen species contents in the retinas; N-acetylcysteine treatment alleviated the pathological changes and also decreased reactive oxygen species, most significantly at 5 months. VEGF and ICAM-1 expressions were significantly up-regulated in retinal blood vessels from diabetic rats, and such up-regulation was attenuated by N-acetylcysteine treatment. The expression of both factors returned to basal levels after 5-month treatment with N-acetylcysteine. Long-term N-acetylcysteine treatment exerts protective effects on the diabetic retinas, possibly through its down-regulation of the expression of VEGF and ICAM-1, and reduction of reactive oxygen species content in retinal vascular tissues in diabetic rats.     

Hemoglobin Warsaw (Phe beta 42(CD1)----Val), an unstable variant with decreased oxygen affinity. Characterization of its synthesis, functional properties, and structure

In Hb Warsaw Val replaces the Phe normally present at the heme contact position beta 42 (CD1). This variant is unstable, and it readily undergoes methemoglobin formation. In DEAE-cellulose chromatography, the variant hemoglobin co-eluted with Hb A; a partially heme-depleted fraction of the variant, representing 5-6% of the total hemoglobin, eluted separately and in pure form. The heme replete form of Hb Warsaw exhibited decreased oxygen affinity with a normal Bohr effect and normal cooperativity and interaction with 2,3-diphosphoglycerate (DPG). The heme-depleted Hb Warsaw had a higher oxygen affinity than that of Hb A, decreased cooperativity and 2,3-DPG interaction, and a very low alkaline Bohr effect. Gel filtration of the heme-depleted form showed it to exist entirely as alpha beta dimers. Globin chain synthesis by Hb Warsaw-containing reticulocytes followed a balanced alpha/beta ratio. In short-term synthesis experiments, a major portion of incorporated radiolabeled L-leucine was recovered from the dimeric, heme-depleted Hb Warsaw fraction, suggesting that subunit association precedes the incorporation of heme into the beta subunits in the post-synthetic assembly of this hemoglobin. Structural analysis of deoxyhemoglobin containing roughly equal proportions of normal and variant beta chains showed that the replacement leaves a cavity next to the heme that is large enough to hold a water molecule, which may account for the instability of Hb Warsaw. The heme and the pyrrol nearest to ValCD1 tilt into the cavity. The resulting increase in the tilt of the proximal histidine relative to the heme plane, coupled with a possible stretching of the Fe-N epsilon bond may account for the low oxygen affinity.     

Maize Sep15‐like functions in endoplasmic reticulum and reactive oxygen species homeostasis to promote salt and osmotic stress resistance

In animals, the Sep15 protein participates in disease resistance, growth, and development, but the function of its plant homologues remains unclear. Here, the function of maize Sep15 was analysed by characterization of two independent Sep15‐like loss‐of‐function mutants. In the absence of ZmSep15‐like, seedling tolerance to both water and salinity stress was compromised. The mutants experienced a heightened level of endoplasmic reticulum stress, and over‐accumulated reactive oxygen species, resulting in leaf necrosis. Characterization of Arabidopsis thaliana atsep15 mutant as well as like with ectopic expression of ZmSep15‐like indicated that ZmSep15‐like contributed to tolerance of both osmotic and salinity stress. ZmSep15‐like interacted physically with UDP‐glucose: glycoprotein glucosyltransferase1 (UGGT1). When the interaction was disrupted, the response to both osmotic and salinity stresses was impaired in maize or Arabidopsis. Co‐expressing ZmUGGT1 and ZmUGGT2 enhanced the tolerance of A. thaliana to both stressors, indicating a functional interaction between them. Together, the data indicated that plants Sep15‐like proteins promote osmotic and salinity stress resistance by influencing endoplasmic reticulum stress response and reactive oxygen species level.     

Human hemoglobin adsorption onto colloidal cerium oxide nanoparticles: a new model based on zeta potential and spectroscopy measurements

The nanoparticle (NP)-induced conformational changes of protein and NP agglomeration have gained a remarkable interest in medical and biotechnological fields. Herein, the effect of human hemoglobin (Hb) on the colloidal stability of cerium oxide NP (CNP) was investigated by dynamic light scattering (DLS), zeta potential, and TEM analysis. In addition, the effect of CNP on the heme degradation and structural changes of Hb was studied using fluorescence, circular dichroism (CD), and UV–visible (UV–vis) spectroscopic methods. DLS and TEM analysis showed that the presence of Hb can increase the mean diameter of CNP. Zeta potential measurements revealed that CNP demonstrated a higher charge distribution relative to CNP/Hb complex. Besides, fluorescence studies indicated that two fluorescent heme degradation products are revealed during the interaction of CNP with Hb. Near UV-CD spectroscopy also showed that the microenvironmental changes of heme groups occur after interaction of Hb with CNP. The result of thermal behavior of Hb confirmed the structural changes of protein, which referred to decrease in the Hb stability in the presence of CNP. Indeed, the finding related to structural and functional changes of Hb induced by CNP may be crucial to obtain information regarding the side effects of NPs. Finally, this data reveal much insight into the effects of the interaction on protein structural changes and NP agglomeration, and can correlate the zeta potential of NP-protein complexes with the nature of the principle NP-protein interaction.     

Structural basis for binding and transfer of heme in bacterial heme‐acquisition systems

Periplasmic heme‐binding proteins (PBPs) in Gram‐negative bacteria are components of the heme acquisition system. These proteins shuttle heme across the periplasmic space from outer membrane receptors to ATP‐binding cassette (ABC) heme importers located in the inner‐membrane. In the present study, we characterized the structures of PBPs found in the pathogen Burkholderia cenocepacia (BhuT) and in the thermophile Roseiflexus sp. RS‐1 (RhuT) in the heme‐free and heme‐bound forms. The conserved motif, in which a well‐conserved Tyr interacts with the nearby Arg coordinates on heme iron, was observed in both PBPs. The heme was recognized by its surroundings in a variety of manners including hydrophobic interactions and hydrogen bonds, which was confirmed by isothermal titration calorimetry. Furthermore, this study of 3 forms of BhuT allowed the first structural comparison and showed that the heme‐binding cleft of BhuT adopts an “open” state in the heme‐free and 2‐heme‐bound forms, and a “closed” state in the one‐heme‐bound form with unique conformational changes. Such a conformational change might adjust the interaction of the heme(s) with the residues in PBP and facilitate the transfer of the heme into the translocation channel of the importer.     

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl⁻) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl⁻ was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.     

The concentration dependence of the heme heme interaction of hemoglobin S

Data for oxygen equilibrium curves for Hb SS erythrocytes, both before and after separation into fractions of varying density by ultracentrifugation technique, were fitted to Hill plots and the ‘n’ values, which is a measure of the heme heme interaction of the Hb molecule, were analyzed. The heme heme interaction for the bottom fractions, which consist mainly of dense deformed cells with a very high MCHC, was found to be smaller than that for the top (undeformed cells) and middle fractions or unfractioned erythrocytes. This finding indicates that the high concentration of Hb S in the dense deformed cells is associated not only with a reduced affinity for oxygen but also a reduced heme heme interaction.     

Sampangine inhibits heme biosynthesis in both yeast and human

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.     

Altered ligand rebinding kinetics due to distal-side effects in hemoglobin chico (Lysbeta66(E10) --> thr)

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues.     

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.     

Roles of distal Asp in heme oxygenase from Corynebacterium diphtheriae, HmuO: A water-driven oxygen activation mechanism

Heme oxygenases found in mammals, plants, and bacteria catalyze degradation of heme using the same mechanism. Roles of distal Asp (Asp-136) residue in HmuO, a heme oxygenase of Corynebacterium diphtheriae, have been investigated by site-directed mutagenesis, enzyme kinetics, resonance Raman spectroscopy, and x-ray crystallography. Replacements of the Asp-136 by Ala and Phe resulted in reduced heme degradation activity due to the formation of ferryl heme, showing that the distal Asp is critical in HmuO heme oxygenase activity. D136N HmuO catalyzed heme degradation at a similar efficiency to wild type and D136E HmuO, implying that the carboxylate moiety is not required for the heme catabolism by HmuO. Resonance Raman results suggest that the inactive ferryl heme formation in the HmuO mutants is induced by disruption of the interaction between a reactive Fe-OOH species and an adjacent distal pocket water molecule. Crystal structural analysis of the HmuO mutants confirms partial disappearance of this nearby water in D136A HmuO. Our results provide the first experimental evidence for the catalytic importance of the nearby water molecule that can be universally critical in heme oxygenase catalysis and propose that the distal Asp helps in positioning the key water molecule at a position suitable for efficient activation of the Fe-OOH species.