Epigallocatechin gallate markedly enhances formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the reaction of 2'-deoxyguanosine with hypochlorous acid

A tea polyphenol, (-)-epigallocatechin gallate (EGCG), which can scavenge a variety of reactive oxygen species, enhances the yield of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) up to 20-fold in the reaction of 2'-deoxyguanosine with hypochlorous acid (HOCl), compared with the reaction without EGCG. Certain concentrations of EGCG inhibited HOCl-mediated oxidation of 2'-deoxyguanosine to 8-oxo-dG to a limited extent, but efficiently inhibited further oxidation of 8-oxo-dG to spiroiminodihydantoin nucleoside, resulting in the accumulation of 8-oxo-dG in the reaction mixture. Conversely, EGCG inhibited dose-dependently an increase in 8-oxo-dG levels in calf thymus DNA incubated with HOCl. However, addition of HOCl to the DNA preoxidized with an oxidant-generating system (CuCl2, ascorbate, H2O2), led to the extensive loss of 8-oxo-dG due to its further oxidation. EGCG effectively inhibited this HOCl-mediated loss of 8-oxo-dG in the oxidized DNA, resulting in an apparent increase in 8-oxo-dG levels in the oxidized DNA, compared with the levels found without EGCG. The conversion of 8-oxo-dG into other oxidized lesions will inevitably affect recognition by DNA repair enzymes as well as the rates of mutations and DNA synthesis. Thus, our results suggest that as a biomarker of oxidative DNA damage, not only 8-oxo-dG but also the products of its further oxidation should be analyzed.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.     

Inhibition of hypochlorous acid-induced oxidative reactions by nitrite: is nitrite an antioxidant?

Acute and chronic inflammation result in increased nitrogen monoxide (z.rad;NO) formation and the accumulation of nitrite (NO(2)(-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl). At physiologically attainable concentrations, we found that NO(2)(-) significantly inhibits HOCl-mediated DNA strand breakage and ascorbate depletion. HOCl-mediated inactivation of pure alpha(1)-antiproteinase or of the elastase inhibitory capacity of human plasma was inhibited by the addition of NO(2)(-). NO(2)(-) was more effective than ascorbate, GSH, and urate at inhibiting HOCl-mediated toxicity to human HepG2 cells in culture. These data suggest that NO(2)(-) may act in an antioxidant manner by removing HOCl at sites of inflammation where both HOCl and z.rad;NO are overproduced.     

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.     

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H₂O₂-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H₂O₂-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H₂O₂-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.     

Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl.The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain?

Hydrogen sulphide (H(2)S) is a cytotoxic gas that has recently been proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM, and considerably lower H(2)S levels are reported in the brains of Alzheimer's disease (AD) patients. Levels of myeloperoxidase (MPO), an enzyme that catalyses the formation of the oxidant hypochlorous acid (HOCl), are elevated in the prefrontal cortex, hippocampal microglia, and neurons of AD patients where MPO co-localised with beta-amyloid plaques. Recently 3-chlorotyrosine, a bio-marker for MPO activity (and HOCl production), was shown to be elevated threefold in hippocampal proteins from AD patients. Since H(2)S and HOCl are important mediators in brain function and disease, we investigated the effects of H(2)S on HOCl-mediated damage to bio-molecules and to cultured human SH-SY5Y cells. H(2)S significantly inhibited HOCl-mediated inactivation of alpha(1)-antiproteinase and protein oxidation to a comparable extent to reduced glutathione. H(2)S also inhibited HOCl-induced cytotoxicity, intracellular protein oxidation, and lipid peroxidation in SH-SY5Y cells. These data suggest that H(2)S has the potential to act as an inhibitor of HOCl-mediated processes in vivo and that the potential antioxidant action of H(2)S deserves further study, especially since extracellular GSH levels in the brain are very low.     

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.     

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.     

Membrane changes associated with lysis of red blood cells by hypochlorous acid

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidence by ektacytometry, and an increase in K⁺-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, bu complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.     

Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Unregulated uptake of oxidized LDL by the scavenger receptor(s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCI-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25-200 microM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N(alpha)-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25-200 microM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially pro-atherogenic process by scavenging LDL-associated chloramines.     

Evidence for rapid inter- and intramolecular chlorine transfer reactions of histamine and carnosine chloramines: implications for the prevention of hypochlorous-acid-mediated damage

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and Cl(-) by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that is implicated in a wide range of human diseases (e.g., atherosclerosis). Histamine and carnosine have been proposed as protective agents against such damage. However, as recent studies have shown that histidine-containing compounds readily form imidazole chloramines that can rapidly chlorinate other targets, it was hypothesized that similar reactions may occur with histamine and carnosine, leading to propagation, rather than prevention, of HOCl-mediated damage. In this study, the reactions of HOCl with histamine, histidine, carnosine, and other compounds containing imidazole and free amine sites were examined. In all cases, rapid formation (k, 1.6 x 10(5) M(-)(1) s(-)(1)) of imidazole chloramines was observed, followed by chlorine transfer to yield more stable, primary chloramines (R-NHCl). The rates of most of these secondary reactions are dependent upon substrate concentrations, consistent with intermolecular mechanisms (k, 10(3)-10(4) M(-)(1) s(-)(1)). However, for carnosine, the imidazole chloramine transfer rates are independent of the concentration, indicative of intramolecular processes (k, 0.6 s(-)(1)). High-performance liquid chromatography studies show that in all cases the resultant R-NHCl species can slowly chlorinate N-alpha-acetyl-Tyr. Thus, the current data indicate that the chloramines formed on the imidazole and free amine groups of these compounds can oxidize other target molecules but with limited efficiency, suggesting that histamine and particularly carnosine may be able to limit HOCl-mediated oxidation in vivo.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.     

Metabolism of oxidized phosphatidylcholines formed in oxidized low density lipoprotein by lecithin-cholesterol acyltransferase.

The possible involvement of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoyl-2-[1-14C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoyl-2-[1-14C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56 degrees C for 30 min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When 1-palmitoyl-2-[1-14C](9-oxononanoyl) PC and 1-stearoyl-2-[1-14C](5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.     

Modulation of LDL oxidation by 7,8-dihydroneopterin

Human macrophages stimulated with interferon-γ generate neopterin and 7,8-dihydroneopterin which interfere with reactive species involved in LDL oxidation. While neopterin was found to have pro-oxidative effects on copper-mediated LDL oxidation, the influence of 7,8-dihydroneopterin is more complex. This study provides detailed information that 7,8-dihydroneopterin reveals both pro-oxidative and anti-oxidative effects on copper mediated LDL oxidation. 7,8-dihydroneopterin inhibited the oxidation of native LDL effectively monitored by (i) formation of conjugated dienes, (ii) relative electrophoretic mobility (EM) and (iii) specific oxidized epitopes. Using minimally oxidized LDL (mi-LDL) or moderately oxidized LDL (mo-LDL) 7,8-dihydroneopterin changed its antioxidative behavior to a strongly pro-oxidative. Incubation of 7,8-dihydroneopterin with native LDL, mi-LDL or mo-LDL in the absence of copper ions showed that formation of conjugated dienes was more increased in mo-LDL than in mi-LDL while no diene formation was observed with native LDL.

We suggest that 7,8-dihydroneopterin is a modulator for LDL oxidation in the presence of copper ions depending on the “oxidative status” of this lipoprotein.     

Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b5-mediated heme reduction

Myeloperoxidase catalyzes the reaction of chloride ions with H₂O₂ to yield hypochlorous acid (HOCl), which can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl oxidation of HMb affects the rate of ferric heme reduction by cytochrome b₅. For wild-type HMb (HOCl:Mb ratio of 5:1 mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product—sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1 mol:mol, methionine (Met) oxidation was detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order Cys > Met > Trp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant and at a longer incubation time (24 h), consistent with the formation via chlorine-transfer reactions from preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1 mol:mol). These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such heme-Fe³⁺ reduction is critical to the maintenance of function as an oxygen storage protein in tissues.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

Preservation of biological function despite oxidative modification of the apolipoprotein A-I mimetic peptide 4F

Myeloperoxidase (MPO)-derived hypochlorous acid induces changes in HDL function via redox modifications at the level of apolipoprotein A-I (apoA-I). As 4F and apoA-I share structural and functional properties, we tested the hypothesis that 4F acts as a reactive substrate for hypochlorous acid (HOCl). 4F reduced the HOCl-mediated oxidation of the fluorescent substrate APF in a concentration-dependent manner (ED(50) ～ 56 ± 3 μM). This reaction induced changes in the physical properties of 4F. Addition of HOCl to 4F at molar ratios ranging from 1:1 to 3:1 reduced 4F band intensity on SDS-PAGE gels and was accompanied by the formation of a higher molecular weight species. Chromatographic studies showed a reduction in 4F peak area with increasing HOCl and the formation of new products. Mass spectral analyses of collected fractions revealed oxidation of the sole tryptophan (Trp) residue in 4F. 4F was equally susceptible to oxidation in the lipid-free and lipid-bound states. To determine whether Trp oxidation influenced its apoA-I mimetic properties, we monitored effects of HOCl on 4F-mediated lipid binding and ABCA1-dependent cholesterol efflux. Neither property was altered by HOCl. These results suggest that 4F serves as a reactive substrate for HOCl, an antioxidant response that does not influence the lipid binding and cholesterol effluxing capacities of the peptide.