Hypothiocyanous acid is a more potent inducer of apoptosis and protein thiol depletion in murine macrophage cells than hypochlorous acid or hypobromous acid (original) (raw)
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Redox Biology, 2020
A host of chronic inflammatory diseases are accelerated by the formation of the powerful oxidant hypochlorous acid (HOCl) by myeloperoxidase (MPO). In the presence of thiocyanate (SCN-), the production of HOCl by MPO is decreased in favour of the formation of a milder oxidant, hypothiocyanous acid (HOSCN). The role of HOSCN in disease has not been fully elucidated, though there is increasing interest in using SCNtherapeutically in different disease settings. Unlike HOCl, HOSCN can be detoxified by thioredoxin reductase, and reacts selectively with thiols to result in reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. In this study, we show that exposure of macrophages, a key inflammatory cell type, to HOSCN results in the reversible modification of multiple mitochondrial proteins, leading to increased mitochondrial membrane permeability, decreased oxidative phosphorylation and reduced formation of ATP. The increased permeability and reduction in ATP could be reversed by pre-treatment of the macrophages with cyclosporine A, implicating a role for the mitochondrial permeability transition pore. HOSCN also drives cells to utilise fatty acids as an energetic substrate after the inhibition of oxidative phosphorylation. Raman imaging studies highlighted the ability of HOSCN to perturb the electron transport chain of mitochondria and redistribute these organelles within the cell. Taken together, these data provide new insight into the pathways by which HOSCN can induce cytotoxicity and cellular damage, which may have relevance for the development of inflammatory disease, and therapeutic strategies to reduce HOCl-induced damage by supplementation with SCN- .
Free Radical Biology and Medicine, 2012
Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.
Journal of Biological Chemistry, 2013
Background: Secreted hypothiocyanous acid (HOSCN) kills pathogens but paradoxically is tolerated by many mammalian cells. Results: Mammalian thioredoxin reductase (H-TrxR) reduces HOSCN, whereas bacterial L-TrxR is inhibited by it, corresponding to differential cytotoxicity. Conclusion: Mammalian H-TrxR confers resistance against HOSCN, enabling its use as a selective biocide. Significance: Findings directly link mammalian H-TrxR to innate immunity and inflammatory lung disease. The endogenously produced oxidant hypothiocyanous acid (HOSCN) inhibits and kills pathogens but paradoxically is well tolerated by mammalian host tissue. Mammalian high molecular weight thioredoxin reductase (H-TrxR) is evolutionarily divergent from bacterial low molecular weight thioredoxin reductase (L-TrxR). Notably, mammalian H-TrxR contains a selenocysteine (Sec) and has wider substrate reactivity than L-TrxR. Recombinant rat cytosolic H-TrxR1, mouse mitochondrial H-TrxR2, and a purified mixture of both from rat selectively turned over HOSCN (k cat ؍ 357 ؎ 16 min ؊1 ; K m ؍ 31.9 ؎ 10.3 M) but were inactive against the related oxidant hypochlorous acid. Replacing Sec with Cys or deleting the final eight C-terminal peptides decreased affinity and turnover of HOSCN by H-TrxR. Similarly, glutathione reductase (an H-TrxR homologue lacking Sec) was less effective at HOSCN turnover. In contrast to H-TrxR and glutathione reductase, recombinant Escherichia coli L-TrxR was potently inhibited by HOSCN (IC 50 ؍ 2.75 M). Similarly, human bronchial epithelial cell (16HBE) lysates metabolized HOSCN, but E. coli and Pseudomonas aeruginosa lysates had little or no activity. HOSCN selectively produced toxicity in bacteria, whereas hypochlorous acid was nonselectively toxic to both bacteria and 16HBE. Treatment with the H-TrxR inhibitor auranofin inhibited HOSCN metabolism in 16HBE lysates and significantly increased HOSCN-mediated cytotoxicity. These findings demonstrate both the metabolism of HOSCN by mammalian H-TrxR resulting in resistance to HOSCN in mammalian cells and the potent inhibition of bacterial L-TrxR resulting in cytotoxicity in bacteria. These data support a novel selective mechanism of host defense in mammals wherein HOSCN formation simultaneously inhibits pathogens while sparing host tissue.
International Journal of Molecular Sciences
This review discusses the formation of hypochlorous acid HOCl and the role of reactive chlorinated species (RCS), which are catalysed by the enzyme myeloperoxidase MPO, mainly located in leukocytes and which in turn contribute to cellular oxidative stress. The reactions of RCS with various organic molecules such as amines, amino acids, proteins, lipids, carbohydrates, nucleic acids, and DNA are described, and an attempt is made to explain the chemical mechanisms of the formation of the various chlorinated derivatives and the data available so far on the effects of MPO, RCS and halogenative stress. Their presence in numerous pathologies such as atherosclerosis, arthritis, neurological and renal diseases, diabetes, and obesity is reviewed and were found to be a feature of debilitating diseases.
International Journal of Molecular Medicine, 2002
We examined whether generation of H 2 0 2 is a critical event for the apoptotic pathway upstream of mitochondrial involvement and caspase-3 protease activation. Perylquinone photosensitizers such as Hypocrellin A (HA), Hypocrellin B (HB) and Hypericin (HY) induced activation of caspase-3 and apoptosis upon photoactivation. Generation of H 2 0 2 was commonly detected after photoactivation within an hour, and scavenging of H 2 0 2 caused cells to fail to undergo apoptosis. Flow cytometry demonstrated that H 2 0 2 production preceded loss of mitochondrial membrane potential (A17m) in photoactivated cells treated with HA, HB and HY. Then caspase-3 activity was activated, followed by DNA fragmentation. These findings suggest that HA, HB and HY upon photoactivation induce H 2 0 2 generation, which causes (AWm) and subsequently caspase-3 activation, resulting in apoptosis. These findings suggest that generation of H 2 0 2 by photoactivation of HA, HB and HY causes activation of caspase-3. Therefore, H 2 0 2 may function as a common mediator for apoptosis induced by HA, HB and HY. The present study also demonstrated that upon photoactivation HA, HB and HY induced a decrease in intracellular acidification, glutathione (GSH) depletion and an array of mitochondrial damage together with apoptotic morphological changes in the irradiated cells.
Tryptophan residues are targets in hypothiocyanous acid-mediated protein oxidation
Biochemical Journal, 2008
Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H2O2. These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the reactions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of 14C from HOS14CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable 14C-containing products and oxidation of tryptophan residu...
European Journal of Pharmacology, 1997
Nitric oxide (NO) plays an important role in the cytotoxic activity of macrophages towards tumour cells and microbial pathogens. We investigated whether alteration of intracellular thiol levels modulates the cytotoxic effects of different NO donors and lipopolysaccharideinduced NO in the murine macrophage cell line J774A.1. The NO-releasing compound S-nitroso-N-acetylpenicillamine caused a significant concentration-dependent loss of viability of the macrophages only under glucose-limiting conditions. The cytotoxic effect of S-nitroso-N-acetylpenicillamine was prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (carboxy-PTIO). Depletion of total glutathione before exposure to S-nitroso-N-acetylpenicillamine further decreased cell viability while pretreatment with N-acetylcysteine was protective. Comparing equimolar concentrations of various NO donors including S-nitrosoglutathione, S-nitrosocysteine and 3-morpholino-sydnonimine hydrochloride, cytotoxicity appeared to be related to the relative stability of the test compound. Both the order of stability and the order of potency for cell killing was S-nitrosoglutathione > S-nitroso-Nacetylpenicillamine > S-nitrosocysteine = 3-morpholino-sydnonimine hydrochloride. Stimulation of the macrophages with lipopolysaccharide and interferon-~ resulted in dose-dependent cell injury and NO production. Glutathione depletion prior to stimulation considerably decreased macrophage viability as well as the NO production. In contrast to the protective effect on S-nitroso-N-acetylpenicillamine-mediated injury, pretreatment with N-acetylcysteine did not influence the lipopolysaccharide-mediated cytotoxicity. These results demonstrate that (a) reduction in the availability of glucose and intracellular glutathione renders the cells more vulnerable to the cytotoxic effects of NO donors, (b) in this model of cytotoxicity, long-lived NO donors were more cytotoxic than short-lived NO donors, (c) the differential effects of N-acetylcysteine on S-nitroso-N-acetylpenicillamine-induced and bacterial lipopolysaccharide-mediated cytotoxicity support the existence of other toxic species different from NO or NO-related compounds with a potent cytotoxic activity in immunostimutated macrophages, and (d) other non-protein thiols like N-acetylcysteine may substitute for glutathione as a major component of the cellular antioxidant defense system.
Free Radical Biology and Medicine, 2013
Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. 34 Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger 35 receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that 36 are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), 37 results in extensive lipid accumulation by macrophages. However, the reactivity of the other major 38 MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given 39 that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the 40 reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage 41 observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl 42 esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) 43 and F 2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though 44 generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of 45 HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature 46 of LDL oxidation in vivo, and has implications for the progression of atherosclerosis. 47 65 pattern of reactivity of oxLDL is dependent on the type of oxidising 66 system and hence the specific fingerprint of the resulting oxidative 67 modifications [3]. 68 Human lesions are enriched in myeloperoxidase (MPO) [4], an 69 enzyme released by activated neutrophils and monocytes that 70 produces a number of potent oxidants, including hypochlorous 71 acid (HOCl) [5]. High circulating MPO levels are also a significant 72 independent risk factor for the development of coronary artery 73 disease [6], and a prognostic agent for patients with cardiac symp-74 toms (e.g. [7]). MPO interacts with LDL in plasma, with complexes 75 containing LDL and MPO reported in the plasma of a subset of 76 patients with atherosclerosis and high plasma MPO levels [8].
Biology of the Cell, 2002
Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose-and time-dependent. At concentrations below 5 µM, AA was not toxic; at 10-400 µM, AA induced apoptosis and at concentrations beyond 400 µM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6-24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative-reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E 2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB 4 . AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis.