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Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidantgenerating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN-, a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn2+ -thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signallingmoleculeNO·.DecreasedNO· bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smokingassociated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN- decreased active dimeric eNOS levels, and increased inactive monomer and Zn2+ release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl- -treated samples. eNOS activity was increasingly compromised byMPO/H2O2/Cl- with increasing SCN- concentrations. Exposure ofHCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl- with increasing SCN-, increased eNOS monomerization and Zn2+ release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2- /NO3- formation (products of NO· decomposition), and increased free Zn2+ . Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN- levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers. [ABSTRACT FROM AUTHOR]

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 HOSCNandOCN- significantly reduced the extent of cholesterol efflux from cholesterol-loaded macrophages when compared with unmodified apoA-I. In contrast, HOSCN did not affect the antiinflammatory 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). [ABSTRACT FROM AUTHOR]

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.

EPO (eosinophil peroxidase) and MPO (myeloperoxidase) are highly basic haem enzymes that can catalyse the production of HOBr (hypobromous acid). They are released extracellularly by activated leucocytes and their binding to the polyanionic glycosa-minoglycan components of extracellular matrix (proteoglycans and hyaluronan) may localize the production of HOBr to these materials. It is shown in the present paper that the reaction of HOBr with glycosaminoglycans (heparan sulfate, heparin, chondroitin sulfate and hyaluronan) generates polymer-derived N-bromo derivatives (bromamines, dibromamines, N-bromosulfon-amides and bromamides). Decomposition of these species, which can occur spontaneously and/or via one-electron reduction by low-valent transition metal ions (Cu+ and Fe2+), results in polymer fragmentation and modification. One-electron reduction of the N-bromo derivatives generates radicals that have been detected by EPR spin trapping. The species detected are consistent with metal ion-dependent polymer fragmentation and modification being initiated by the formation of nitrogen-centred (aminyl, N-bromoaminyl, sulfonamidyl and amidyl) radicals. Previous studies have shown that the reaction of HOBr with proteins generates N-bromo derivatives and results in fragmentation of the polypeptide backbone. The reaction of HOBr with extracellular matrix synthesized by smooth muscle cells in vitro induces the release of carbohydrate and protein components in a time-dependent manner, which is consistent with fragmentation of these materials via the formation of N-bromo derivatives. The degradation of extracellular matrix glycosaminoglycans and proteins by HOBr may contribute to tissue damage associated with inflammatory diseases such as asthma.

In gamma-irradiation, *OH is directly generated from water and causes DNA damage leading to carcinogenesis. Exposure of proteins to gamma-irradiation, in the presence of oxygen, gives high yields of hydroperoxides. To clarify whether these hydroperoxides, particularly those formed on DNA-binding histone proteins, participate in gamma-irradiation-induced carcinogenesis, experiments using 32P-labelled DNA fragments obtained from human cancer-related genes were undertaken. Histone protein-hydroperoxides induced significant DNA damage in the presence of Cu(I). Histone H1- and H3-hydroperoxides showed stronger DNA damage compared with histone H2A- and H4-hydroperoxides at 0.7 muM. Histone H1-hydroperoxides caused Cu(I)-dependent DNA damage predominantly at guanine residues, especially at 5'-GGC-3', 5'-GGA-3', 5'-GGT-3' and single G bases. In contrast, histone H3-hydroperoxides/Cu(I) induced DNA damage at 5'-G in GG sequences; this sequence specificity is identical with that generated by 2,2'-azobis (2-amidinopropane) dihydrochloride, which is known to produce peroxyl radicals (RO2*). The difference in site specificity of DNA damage induced by histone H1- and H3-hydroperoxides may arise from their amino acid composition or their mode of binding to DNA. The histone H1-hydroperoxides/Cu(I) system also induced 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in calf thymus DNA. It is concluded that histone protein-hydroperoxides can induce guanine-specific DNA damage, which may contribute to gamma-irradiation-induced carcinogenesis.