Jihan Talib

High plasma thiocyanate levels in smokers are a key determinant of thiol oxidation induced by myeloperoxidase.

Smokers have an elevated risk of atherosclerosis but the origins of this elevated risk are incompletely defined, though evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall. We hypothesized that smokers would have a high level of thiocyanate (SCN(-)), a preferred substrate for MPO, which in turn would predispose to thiol oxidation, an established independent risk factor for atherosclerosis. In this study it is shown that on exposure to MPO/H(2)O(2), thiols on plasma proteins from nonsmokers were increasingly oxidized with increasing added SCN(-) concentrations. Plasma from smokers contained significantly higher endogenous levels of SCN(-) than that from nonsmokers (131±31 vs 40±24 μM, P<0.0001). When plasma from smokers and nonsmokers was exposed to MPO/H(2)O(2)-stimulated oxidation, a strong positive correlation (r=0.8139, P<0.0001) between the extent of thiol oxidation and the plasma SCN(-) concentrations was observed. Computational calculations indicate a changeover from HOCl to HOSCN as the major MPO-generated oxidant in plasma, with increasing SCN(-) levels. These data indicate that plasma SCN(-) levels are a key determinant of the extent of thiol oxidation on plasma proteins induced by MPO, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage to proteins in smokers.

Low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation

BACKGROUND Oxidative stress with dysregulated inflammation are hallmarks of sepsis. Zinc and selenium have important antioxidant functions, such that they could be important in patients with sepsis. We used an in vitro approach to assess the effect of zinc and selenium on oxidative stress, mitochondrial function, and inflammatory responses in conditions mimicking sepsis and related the findings to plasma concentrations and biomarkers in patients with and without sepsis. METHODS Human endothelial cells were exposed to a range of zinc and selenium concentrations in conditions mimicking sepsis. Zinc, selenium, and a series of biomarkers of oxidative stress and inflammation were measured in plasma from critically ill patients with and without sepsis. RESULTS Culturing cells with different concentrations of zinc caused altered zinc transporter protein expression and cellular zinc content, and selenium affected glutathione peroxidase 3 activity. Although zinc or selenium at physiological concentrations had no effect on interleukin-6 release in vitro, higher concentrations of the trace elements were associated with improved mitochondrial function. Plasma zinc and selenium concentrations were low in patients [zinc: median (range) 4.6 (2.1-6.5) μM in control patients without sepsis and 3.1 (1.5-5.4) μM in patients with sepsis, P=0.002; and selenium: 0.78 (0.19-1.32) μM in control patients and 0.42 (0.22-0.91) μM in sepsis patients, P=0.0009]. Plasma concentrations of interleukin-6, other biomarkers of inflammation, and markers of oxidative damage to proteins and lipids were elevated, particularly in patients with sepsis, and were inversely related to plasma zinc and selenium concentrations. CONCLUSIONS Zinc and selenium concentrations were reduced in critically ill patients, with increased oxidative stress and inflammatory biomarkers, particularly in patients with sepsis. Oxidative stress as a result of suboptimal selenium and zinc concentrations might contribute to damage of key proteins. CLINICAL TRIAL REGISTRATION ClinicalTrials.gov: registration number NCT01328509.

High plasma thiocyanate levels modulate protein damage induced by myeloperoxidase and perturb measurement of 3-chlorotyrosine.

Smokers have an elevated risk of atherosclerosis but the origin of this elevated risk is incompletely defined, though increasing evidence supports a role for the oxidant-generating enzyme myeloperoxidase (MPO). In previous studies we have demonstrated that smokers have elevated levels of thiocyanate ions (SCN(-)), relative to nonsmokers, and increased thiol oxidation, as SCN(-) is a favored substrate for MPO, and the resulting hypothiocyanous acid (HOSCN) targets thiol groups rapidly and selectively. In this study we show that increased HOSCN formation by MPO diminishes damage to nonthiol targets on both model proteins and human plasma proteins. Thus high SCN(-) levels protect against HOCl- and MPO-mediated damage to methionine, tryptophan, lysine, histidine, and tyrosine residues on proteins. Furthermore, levels of the HOCl-mediated marker compound 3-chlorotyrosine and the cross-linked product dityrosine are decreased. Plasma protein 3-chlorotyrosine levels induced by HOCl exposure in nonsmokers are elevated over the levels detected in smokers when exposed to identical oxidative insult (P<0.05), and a strong inverse correlation exists between plasma SCN(-) levels and 3-chlorotyrosine concentrations (r=0.6182; P<0.0001). These correlations were also significant for smokers (r=0.2724; P<0.05) and nonsmokers (r=0.4141; P<0.01) when analyzed as individual groups. These data indicate that plasma SCN(-) levels are a key determinant of the extent and type of protein oxidation induced by MPO on isolated and plasma proteins and that smoking status and resulting high SCN(-) levels can markedly modulate the levels of the widely used biomarker compound 3-chlorotyrosine.

The smoking-associated oxidant hypothiocyanous acid induces endothelial nitric oxide synthase dysfunction

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 oxidant-generating 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 Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NO•. Decreased NO• bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated 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 Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) 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 Zn(2+). 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.

Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN−, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]4 cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]4 cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]4 cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.

Disruption of the iron-sulfur cluster of aconitase by myeloperoxidase-derived oxidants.

Oxidative damage catalysed by the heme enzyme myeloperoxidase (MPO) has been linked with multiple inflammatory pathologies. The major oxidant species generated by MPO are hypochlorous (HOCl) and hypothiocyanous acids (HOSCN). The damage induced by HOCl is well characterized and has been linked to multiple diseases, however the role of HOSCN is less well understood. It is known that HOSCN can cause selective damage, as this oxidant selectively targets thiol (e.g. Cys) residues and selenium-containing species. The aim of the current study was to assess whether HOCl and HOSCN can disrupt the [4Fe-4S] cluster of aconitase causing iron release and loss of activity. It is shown that HOSCN induces rapid and efficient release of iron from aconitase, with 80% removed at an oxidant concentration of 3 micromoles/mg protein; this is markedly more efficient than HOCl. In contrast the extent of loss of enzymatic activity was comparable between the two oxidants at the same concentration. Blocking the [4Fe-4S] cluster inhibited HOSCN-mediated inactivation, but did not have dramatic effects on HOCl-mediated damage, consistent with HOSCN, but not HOCl, interacting with the cluster. This data is supported by peptide mass mapping studies that indicate that HOSCN oxidises Cys385 of the [4Fe-4S] cluster. In contrast HOCl damaged multiple sites. Exposure of human coronary artery endothelial cells (HCAEC) to 0-50 micromolar HOCl or 0-150 micromolar HOSCN resulted in an increase in intracellular iron, loss of aconitase activity and a loss of mitochondrial aconitase protein. In contrast cytosolic aconitase was not affected. These data indicate that aconitase - and particularly the mitochondrial form - is a target for MPO-mediated damage with HOSCN showing a selectivity for the [4Fe-4S] cluster and inducing greater iron release. This damage, and the release of iron, may exacerbate oxidative stress in cells at sites of inflammation where active MPO is present.