Detrimental Actions of Chlorinated Nucleosides on the Function and Viability of Insulin-Producing Cells.

Neutrophils are innate immune cells that play a key role in pathogen clearance. They contribute to inflammatory diseases, including diabetes, by releasing pro-inflammatory cytokines, reactive oxygen species, and extracellular traps (NETs). NETs contain a DNA backbone and catalytically active myeloperoxidase (MPO), which produces hypochlorous acid (HOCl). Chlorination of the DNA nucleoside 8-chloro-deoxyguanosine has been reported as an early marker of inflammation in diabetes. In this study, we examined the reactivity of different chlorinated nucleosides, including 5-chloro-(deoxy)cytidine (5ClC, 5CldC), 8-chloro-(deoxy)adenosine (8ClA, 8CldA) and 8-chloro-(deoxy)guanosine (8ClG, 8CldG), with the INS-1E ß-cell line. Exposure of INS-1E cells to 5CldC, 8CldA, 8ClA, and 8CldG decreased metabolic activity and intracellular ATP, and, together with 8ClG, induced apoptotic cell death. Exposure to 8ClA, but not the other nucleosides, resulted in sustained endoplasmic reticulum stress, activation of the unfolded protein response, and increased expression of thioredoxin-interacting protein (TXNIP) and heme oxygenase 1 (HO-1). Exposure of INS-1E cells to 5CldC also increased TXNIP and NAD(P)H dehydrogenase quinone 1 (NQO1) expression. In addition, a significant increase in the mRNA expression of NQO1 and GPx4 was seen in INS-1E cells exposed to 8ClG and 8CldA, respectively. However, a significant decrease in intracellular thiols was only observed in INS-1E cells exposed to 8ClG and 8CldG. Finally, a significant decrease in the insulin stimulation index was observed in experiments with all the chlorinated nucleosides, except for 8ClA and 8ClG. Together, these results suggest that increased formation of chlorinated nucleosides during inflammation in diabetes could influence ß-cell function and may contribute to disease progression.

Role of macrophage extracellular traps in innate immunity and inflammatory disease.

Macrophages play an integral role in initiating innate immune defences and regulating inflammation. They are also involved in maintaining homeostasis and the resolution of inflammation, by promoting tissue repair and wound healing. There is evidence that like neutrophils, macrophages can release extracellular traps following exposure to a range of pathogenic and pro-inflammatory stimuli. Extracellular traps are released by a specialised cell death pathway termed 'ETosis', and consist of a backbone of DNA and histones decorated with a range of other proteins. The composition of extracellular trap proteins can be influenced by both the cell type and the local environment in which the traps are released. In many cases, these proteins have an antimicrobial role and assist with pathogen killing. Therefore, the release of extracellular traps serves as a means to both immobilise and destroy invading pathogens. In addition to their protective role, extracellular traps are also implicated in disease pathology. The release of neutrophil extracellular traps (NETs) is causally linked to the development of wide range of human diseases. However, whether macrophage extracellular traps (METs) play a similar role in disease pathology is less well established. Moreover, macrophages are also involved in the clearance of extracellular traps, which could assist in the resolution of tissue damage associated with the presence of extracellular traps. In this review, we will provide an overview of the pathways responsible for macrophage extracellular trap release, and discuss the role of these structures in innate immunity and disease pathology and possible therapeutic strategies.

Detection, identification, and quantification of oxidative protein modifications.

Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.

8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages.

The exposure of RNA and DNA nucleobases to the oxidant hypochlorous acid (HOCl) results in the generation of different stable chlorinated products. These chlorinated nucleobases are formed in vivo, particularly in chronic inflammatory pathologies, which are characterized by the overproduction of HOCl by myeloperoxidase. As such, chlorinated nucleosides are used as biomarkers of inflammation. However, these compounds have also attracted attention as potential chemotherapeutic agents with 8-chloro-adenosine (8ClA), for example, currently in clinical trials for the treatment of hematological cancers, including chronic lymphocytic leukemia. 8ClA has mainly RNA-directed effects in malignant cells, with exposure resulting in ATP depletion and apoptotic cell death. Whether 8ClA has significant reactivity with nonmalignant cells has not been widely studied. Here we show that prolonged incubation of J774A.1 macrophage-like cells with 8ClA results in the perturbation of cellular metabolism and apoptotic cell death. These effects are associated with an accumulation of 8-chloroadenosine triphosphate (8Cl-ATP), an effect not seen in experiments utilizing other chlorinated nucleosides. Exposure of the macrophages to 8ClA did not significantly change basal mitochondrial respiration or glycolysis but resulted in an increase in maximal mitochondrial respiration as well as spare respiratory capacity within these cells. Additionally, 8ClA exposure also altered the mRNA expression of a range of antioxidant and DNA damage repair genes in the macrophages in a manner consistent with a reduction in the capacity of the cells to cope with oxidative stress and repair DNA damage. Taken together, these results provide new insight into pathways by which the production of HOCl during chronic inflammation could perturb immune cell function and may also have implications for the use of 8ClA as a chemotherapeutic drug.

Human Indoleamine 2,3-Dioxygenase 1 Is an Efficient Mammalian Nitrite Reductase.

The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.

Characterization of the cellular effects of myeloperoxidase-derived oxidants on H9c2 cardiac myoblasts.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury, which is in part due to the release of the enzyme myeloperoxidase (MPO) from activated infiltrating leukocytes, and the subsequent production of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Although exposure of various cell types to either oxidant is known to cause cellular dysfunction within a variety of pathological settings, the precise role of HOCl and HOSCN in the initiation of tissue damage evident following cardiac I/R injury remains unclear. In this study, we have employed the use of the cardiac myoblast cell line H9c2 as a model for cardiac myocytes and demonstrate that exposure to either oxidant elicits a dose-dependent increase in cytosolic calcium accumulation, depletion of the cellular thiol pool, reduction of glutathione (GSH) levels and loss of mitochondrial inner trans-membrane potential, concomitant with increased necrotic cell death. H9c2 cell recovery from the initial oxidant exposure involves the initiation of cell survival signalling pathways centred around Nrf2-antioxidant response element (ARE) and activator protein 1 (AP-1) activation, with cell survival accompanied by restoration of mitochondrial function following exposure to HOSCN, but not HOCl. These data highlight the cellular responses elicited by HOCl and HOSCN in cardiac myocytes furthering our understanding of the pathogenesis of oxidant injury following cardiac I/R injury.

A Role for Chlorinated Nucleosides in the Perturbation of Macrophage Function and Promotion of Inflammation.

During inflammation, myeloperoxidase released from activated phagocytes generates the highly reactive oxidant hypochlorous acid (HOCl). This oxidant plays an important role in the immune response but can also promote tissue damage and has been strongly linked with the development of numerous inflammatory diseases. HOCl reacts with cellular DNA forming chlorinated nucleobases, which induce strand breaks, mutations, and cross-links. Although it has been shown that chlorinated nucleosides are present within inflammatory pathologies and diseased tissue, whether or not these species are biomarkers formed as a byproduct of chronic inflammation or play a role in the disease progression has not been ascertained. In this study, we show that exposure of J774A.1 macrophage-like cells to chlorinated ribose and deoxyribose nucleosides results in the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA), and 8-chloro-guanosine (8ClG) into the cellular RNA and 5-chloro-deoxycytidine (5CldC) but not 8-chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA) into cellular DNA. Evidence was obtained for the clearance of 5ClC from the RNA, with a loss of 8ClA and 8ClG observed to a lesser extent, whereas an increase in the level of 5CldC in DNA was seen on further incubation of treated cells in the absence of chlorinated nucleosides. Importantly, exposure of the macrophages to chlorinated nucleosides, particularly 8ClG and 5ClC, resulted in the increased expression of interleukin-1ß, and other pro-inflammatory cytokines and chemokines. With 5ClC, this inflammatory response was associated with the increased nuclear translocation of the NF-κB subunit, p65, rather than inflammasome activation. This alteration in gene expression appeared to be unrelated to the extent of incorporation of the chlorinated nucleosides into RNA or DNA and was not associated with any significant changes in cell viability or proliferation. Taken together, these results highlight a potential biological role for chlorinated nucleosides to promote inflammatory disease, in addition to their utility as biomarkers.

Protein carbamylation: a key driver of vascular calcification during chronic kidney disease.

Vascular calcification is a frequent complication of advanced chronic kidney disease. Protein carbamylation is implicated in the acceleration of vascular disease in chronic kidney disease, but the mechanisms are not clear. Mori et al. report that protein carbamylation exacerbates vascular calcification by decreasing ectonucleotide pyrophosphate/phosphodiesterase 1 expression, owing to carbamylation of mitochondrial proteins and oxidative stress. This provides new insight into the pathways responsible for calcification in chronic kidney disease.

Molecular Alterations in a Mouse Cardiac Model of Friedreich Ataxia: An Impaired Nrf2 Response Mediated via Upregulation of Keap1 and Activation of the Gsk3ß Axis.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a master regulator of the antioxidant response. However, studies in models of Friedreich ataxia, a neurodegenerative and cardiodegenerative disease associated with oxidative stress, reported decreased Nrf2 expression attributable to unknown mechanisms. Using a mouse conditional frataxin knockout (KO) model in the heart and skeletal muscle, we examined the Nrf2 pathway in these tissues. Frataxin KO results in fatal cardiomyopathy, whereas skeletal muscle was asymptomatic. In the KO heart, protein oxidation and a decreased glutathione/oxidized glutathione ratio were observed, but the opposite was found in skeletal muscle. Decreased total and nuclear Nrf2 and increased levels of its inhibitor, Kelch-like ECH-associated protein 1, were evident in the KO heart, but not in skeletal muscle. Moreover, a mechanism involving activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3ß (Gsk3ß) signaling was demonstrated in the KO heart. This process involved the following: i) increased Gsk3ß activation, ii) ß-transducin repeat containing E3 ubiquitin protein ligase nuclear accumulation, and iii) Fyn phosphorylation. A corresponding decrease in Nrf2-DNA-binding activity and a general decrease in Nrf2-target mRNA were observed in KO hearts. Paradoxically, protein levels of some Nrf2 antioxidant targets were significantly increased in KO mice. Collectively, cardiac frataxin deficiency reduces Nrf2 levels via two potential mechanisms: increased levels of cytosolic Kelch-like ECH-associated protein 1 and activation of Gsk3ß signaling, which decreases nuclear Nrf2. These findings are in contrast to the frataxin-deficient skeletal muscle, where Nrf2 was not decreased.

A pivotal role for NF-κB in the macrophage inflammatory response to the myeloperoxidase oxidant hypothiocyanous acid.

Atherosclerosis is characterised by the infiltration of macrophages at sites of inflammation within the vessel wall and the release of myeloperoxidase (MPO), which forms hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). HOCl is a damaging oxidant implicated in the development of atherosclerosis. Preferential formation of HOSCN occurs under conditions where thiocyanate ions are elevated, as is the case in smokers. HOSCN reacts selectively with thiols, which can result in more enzyme inactivation and damage than HOCl at susceptible sites, which may contribute to atherosclerosis in smokers. In this study, we show that exposure of macrophages to HOSCN results in a time- and dose-dependent increase in the mRNA expression and release of pro-inflammatory cytokines and chemokines, including monocyte chemotactic protein 1, tumour necrosis factor alpha, and interleukins 6, 8 and 1ß. At high oxidant concentrations (>200 µM), a significant loss of cellular thiols and increased cell death is observed. HOSCN-induced cytokine/chemokine expression and cell death were decreased on pharmacological inhibition of nuclear factor kappa B. These data highlight a pathway by which HOSCN could promote inflammation and the development of atherosclerosis, in the presence of supra-physiological levels of the precursor thiocyanate, which are achievable by cigarette smoking.

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity.

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity.

Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function.

Mitochondria are a major source of intracellular energy and reactive oxygen species in cells, but are also increasingly being recognized as a controller of cell death. Here, we review evidence of signal transduction control by mitochondrial superoxide generation via the nuclear factor-κB (NF-κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA signal transductions by mitochondrial superoxide. Oxidative phosphorylation via the electron transfer chain, glycolysis, and generation of superoxide from mitochondria could be important factors in regulating signal transduction, cellular homeostasis, and cell death.

Chasing great paths of Helmut Sies "Oxidative Stress".

Prof. Dr. Helmut Sies is a pioneer of "Oxidative Stress", and has published over 18 papers with the name of "Oxidative Stress" in the title. He has been Editor-in-Chief of the journal "Archives of Biochemistry and Biophysics" for many years, and is a former Editor-in-Chief of the journal "Free Radical Research". He has clarified our understanding of the causes of chronic developing diseases, and has studied antioxidant factors. In this article, importance of "Oxidative Stress" and our mitochondrial oxidative stress studies; roles of mitochondrial ROS, effects of vitamin E and its homologues in oxidative stress-related diseases, effects of antioxidants in vivo and in vitro, and a mitochondrial superoxide theory for oxidative stress diseases and aging are introduced, and some of our interactions with Helmut are described, congratulating and appreciating his great path.

Detection and characterisation of radicals in biological materials using EPR methodology.

BACKGROUND: Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the "gold standard" for the detection and characterisation of radicals in biological systems. SCOPE OF REVIEW: The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment. MAJOR CONCLUSIONS: When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors. GENERAL SIGNIFICANCE: EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Comparative reactivity of the myeloperoxidase-derived oxidants HOCl and HOSCN with low-density lipoprotein (LDL): Implications for foam cell formation in atherosclerosis.

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo, and has implications for the progression of atherosclerosis.

Manganese superoxide dismutase promotes interaction of actin, S100A4 and Talin, and enhances rat gastric tumor cell invasion.

It has been demonstrated that cancer cells are under high levels of oxidative stress and express high levels of Manganese superoxide dismutase (MnSOD) to protect themselves and support the anabolic metabolism needed for growth and cell motility. The aim of this study was to identify proteins that may have a correlation with invasion and redox regulation by mitochondrial reactive oxygen species (ROS). MnSOD scavenges superoxide anions generated from mitochondria and is an important regulator of cellular redox status. Oxidative posttranslational modification of cysteine residues is a key mechanism that regulates protein structure and function. We hypothesized that MnSOD regulates intracellular reduced thiol status and promotes cancer invasion. A proteomic thiol-labeling approach with 5-iodoacetamidofluorescein was used to identify changes in intracellular reduced thiol-containing proteins. Our results demonstrate that overexpression of MnSOD maintained the major structural protein, actin, in a reduced state, and enhanced the invasion ability in gastric mucosal cancer cells, RGK1. We also found that the expression of Talin and S100A4 were increased in MnSOD-overexpressed RGK1 cells. Moreover, Talin bound not only with actin but also with S100A4, suggesting that the interaction of these proteins may, in part, contribute to the invasive ability of rat gastric cancer.

Role of cyanate in the induction of vascular dysfunction during uremia: more than protein carbamylation?

Cyanate is a uremic toxin responsible for the carbamylation of proteins, which has been implicated as playing a key role in accelerating the progression of atherosclerosis in patients with chronic kidney disease. El-Gamal et al. report that while cyanate promotes protein carbamylation in vivo, the resulting endothelial dysfunction observed is consistent with reactions mediated by cyanate itself, rather than by carbamylated proteins. This provides new insight into the relationship between uremia and cardiovascular disease.

Tryptophan oxidation in proteins exposed to thiocyanate-derived oxidants.

Human defensive peroxidases, including lactoperoxidase (LPO) and myeloperoxidase (MPO), are capable of catalyzing the oxidation of halides (X(-)) by H2O2 to give hypohalous acids (HOX) for the purpose of cellular defense. Substrate selectivity depends upon the relative abundance of the halides, but the pseudo-halide thiocyanate (SCN(-)) is a major substrate, and sometimes the exclusive substrate, of all defensive peroxidases in most physiologic fluids. The resulting hypothiocyanous acid (HOSCN) has been implicated in cellular damage via thiol oxidation. While thiols are believed to be the primary target of HOSCN in vivo, Trp residues have also been implicated as targets for HOSCN. However, the mechanism involved in HOSCN-mediated Trp oxidation was not established. Trp residues in proteins appeared to be susceptible to oxidation by HOSCN, whereas free Trp and Trp residues in small peptides were found to be unreactive. We show that HOSCN-induced Trp oxidation is dependent on pH, with oxidation of free Trp, and Trp-containing peptides observed when the pH is below 2. These conditions mimic those employed previously to precipitate proteins after treatment with HOSCN, which accounts for the discrepancy in the results reported for proteins versus free Trp and small peptides. The reactant in these cases may be thiocyanogen ((SCN)2), which is produced by comproportionation of HOSCN and SCN(-) at low pH. Reaction of thiocyanate-derived oxidants with protein Trp residues at low pH results in the formation of a number of oxidation products, including mono- and di-oxygenated derivatives, which are also formed with other hypohalous acids. Our data suggest that significant modification of Trp by HOSCN in vivo is likely to have limited biological relevance.

Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctional high-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorous acid (HOCl).

Oxidative modification of HDLs (high-density lipoproteins) by MPO (myeloperoxidase) compromises its anti-atherogenic properties, which may contribute to the development of atherosclerosis. Although it has been established that HOCl (hypochlorous acid) produced by MPO targets apoA-I (apolipoprotein A-I), the major apolipoprotein of HDLs, the role of the other major oxidant generated by MPO, HOSCN (hypothiocyanous acid), in the generation of dysfunctional HDLs has not been examined. In the present study, we characterize the structural and functional modifications of lipid-free apoA-I and rHDL (reconstituted discoidal HDL) containing apoA-I complexed with phospholipid, induced by HOSCN and its decomposition product, OCN- (cyanate). Treatment of apoA-I with HOSCN resulted in the oxidation of tryptophan residues, whereas OCN- induced carbamylation of lysine residues to yield homocitrulline. Tryptophan residues were more readily oxidized on apoA-I contained in rHDLs. Exposure of lipid-free apoA-I to HOSCN and OCN- significantly reduced the extent of cholesterol efflux from cholesterol-loaded macrophages when compared with unmodified apoA-I. In contrast, HOSCN did not affect the anti-inflammatory properties of rHDL. The ability of HOSCN to impair apoA-I-mediated cholesterol efflux may contribute to the development of atherosclerosis, particularly in smokers who have high plasma levels of SCN- (thiocyanate).

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes.

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.