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Showing 1661–1680 of 2058 publications.
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Bren, Jan Hinrich; Koenig, Kai; Bach, Heiko; Kontush, Anatol S.; Heinle, Helmut; Witting, Paul Kenneth; YlHerttuala, Seppo Pasi Antero; Stocker, Roland; Beisiegel, UlrikeOxidative modification of lipoproteins may trigger and maintain atherogenesis. We compared the effects of different antioxidants (?-tocopherol, probucol, ubiquinone-10) at doses similar to those used in humans in Watanabe Heritable Hyperlipidemic (WHHL) rabbits for 12 months. Aortic lesions were analyzed for their extent and cellular composition of lesions, mean thickness of fibrous caps and density of smooth muscle cells therein, content of antioxidants, non-oxidized and oxidized lipids. Compared to controls, probucol significantly lowered the extent and macrophage content of lesions and increased the existence and smooth muscle cell density of fibrous caps. ?-Tocopherol supplementation increased the aortic content of vitamin E, but had no decreasing effect on either the accumulation of macrophage-specific antigen in the aorta or lesion size. Nevertheless, both probucol and ?-tocopherol significantly decreased in vitro LDL oxidizability, measured under typically strong oxidative conditions. Ubiquinone-10 supplement increased lesion size and the fraction of lesions containing fibrous caps; however, LDL oxidizability remained unaffected by ubiquinone-10 treatment. None of the antioxidants tested lowered oxidized lipids within aortic tissue; however, long-term treatment with probucol provided the most effective anti-atherosclerotic effect, while ?-tocopherol may be pro-atherogenic and ubiquinone-10 exerts ambivalent effects. Our data suggest that (i) widely used oxidation measures, such as ex-vivo LDL oxidizability, do not reflect the degree of atherosclerosis; and (ii) long-term beneficial effects of relatively low doses of antioxidants may be outweighed by high levels of plasma cholesterol in WHHL rabbits. 2002 Elsevier Science Ireland Ltd. All rights reserved.
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Garner, Brett; Priestman, David A.; Stocker, Roland; Harvey, David J.; Butters, Terry D.; Platt, Frances M.The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAc?1-4Gal?1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAc?1-4[NeuNAc?2-3]Gal?1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by Nglycolyl GM2 and the former comprised of NeuNAc?23Gal?1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.
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Upston, Joanne M.; Niu, Xianwa; Brown, Andrew J.; Mashima, Ryuichi; Wang, Hongjie; Senthilmohan, Revathy; Kettle, Anthony James; Dean, Roger T.; Stocker, RolandOxidative modification of low-density lipoprotein is thought to promote arterial lipid accumulation and atherogenesis. Previous studies reported on the presence of certain lipid or protein oxidation products in lesions, although a systematic investigation measuring several oxidation parameters and the accumulation of nonoxidized lipids and antioxidants at various stages of atherosclerosis has not been performed in the same tissue. Using the intimal lipoprotein-containing fraction of human aortic lesions, we demonstrate here that cholesterol accumulated with lesion development and that this increase was already significant at the fatty streak stage. By comparison, cholesterylesters increased significantly only in fibrofatty and more complex lesions that also contained significantly increased amounts of cholesterylester hydro(pero)xides and 27-hydroxycholesterol. Cholesterylester hydroxides were the major lipid oxidation product detected. Despite accumulation of oxidized lipid, ?-tocopherol was also present and maintained at a comparable level over the disease process. Of the oxidized protein moieties measured only o,o-dityrosine increased with disease, although chlorotyrosines were present at relatively high levels in all lesions compared to healthy vessels. Our data show that accumulation of nonoxidized lipid precedes that of oxidized lipid in human aortic lesions.
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Le, Nghia T.V.; Richardson, Des RaymondFerroportin1 is a newly discovered molecule that may play a role in iron export. It is expressed on the basolateral surfaces of mature enterocytes within the duodenum and in macrophages of the spleen and liver. Furthermore, this protein was found to be expressed in placental syncytiotrophoblasts and may be involved in the supply of maternal iron to the fetus. Sequence analysis of ferroportin1 predicts it has ten transmembrane domains, a reductase site and a basolateral localization signal. In addition, the ferroportin1 mRNA transcript contains an iron response element in its 5? untranslated region. This review is focused on the current state of knowledge on ferroportin1 and the medical implications of this discovery. 2002 Elsevier Science Ltd. All rights reserved.
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Edwards, Stacey L.; Wells, Christine A.; Ravasi, Timothy; Hume, David A.; Richardson, Des RaymondNramp2 is a widely expressed metal-ion transporter that is involved in dietary iron absorption in the duodenum and iron uptake from transferrin in peripheral tissues. Nramp1 is a related gene involved in regulation of host pathogen interaction. Nramp2 has at least two alternatively spliced isoforms, one of which contains an iron-responsive element in its 3?-untranslated region. In this study, we investigated the regulation of both isoforms of Nramp2 in activated primary macrophages from mouse strains with wild-type (Bcgr) or mutant (Bcgs) alleles. The Nramp2-IRE and/or -nonIRE transcripts were up-regulated in all mouse strains analyzed after treatment with interferon-? and lipopolysaccharide. cDNA microarray analysis revealed that Nramp2 regulation is controlled discordantly from other iron-regulated genes and classical macrophage-activation genes in different mouse strains. We suggest that Nramp2 is regulated independently of known iron-responsive genes in macrophages, and its function in host defense is unrelated to Nramp1.
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Richardson, Des RaymondFor many years it has been known that neoplastic cells express high levels of the transferrin receptor 1 (TfR1) and internalize iron (Fe) from transferrin (Tf) at a tremendous rate. Considering the high requirement of neoplastic cells for Fe, understanding its metabolism is vital in terms of devising potential new therapies. Apart from TfR1, a number of molecules have been identified that may have roles in Fe metabolism and cellular proliferation. These molecules include transferrin (Tf), the oestrogen-inducible transferrin receptor-like protein, transferrin receptor 2 (TfR2), melanotransferrin (MTf), ceruloplasmin, and ferritin. In the present review these latter molecules are discussed in terms of their potential functions in tumour cell Fe metabolism and proliferation. Further studies are essential to determine the specific roles of these proteins in the pathogenesis of cancer. 2002 Elsevier Science Ireland Ltd.
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Pattison, David I.; Davies, Michael J.; Asmus, Klaus DieterPulse radiolysis techniques have been employed to investigate the one-electron reduction of a variety of chloramines and chloramides. These include models for the side-chain of Lys (6-aminohexanoic acid chloramine and ?-N-acetyl-Lys chloramine), Gly chloramine, ?-alanine chloramine and two models of protein backbone amides, the chloramides of cyclo-(Gly)<inf>2</inf> and cyclo-(Ala)<inf>2</inf>. The molar absorption coefficients and stabilities of these chloramines/amides have been determined. The one-electron reduction of these chloramine/amide species by hydrated electrons occurs with second-order rate constants of the order of 109-1010 M-1 s-1, and results in cleavage of the N-Cl bonds to yield nitrogen-centred radicals and chloride ions (as measured by high performance ion chromatography). The reactivities of the nitrogen-centred radicals have been investigated with the readily oxidisable quenchers, hydroquinone and Trolox. These quenchers were used as models of the in vivo antioxidants, ubiquinol-10 and ?-tocopherol, and react with second-order rate constants between 2 107 and 1 108 M-1 s-1. No evidence was obtained in these pulse radiolysis experiments for a rapid rearrangement of the oxidising nitrogen-centred radicals to reducing carbon-centred radicals, though such reactions have been indicated in previous EPR studies.
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Gatto, Lissa M.; Lyons, Malcolm A.; Brown, Andrew J.; Samman, SamirThis study was designed to investigate the effects of oleic (CIS), palmitic (SAT) and trans fatty acids (TRANS) on cholesterol metabolism. Rats fed the TRANS diet had lower plasma total cholesterol (P < 0.005) and non-HDL-cholesterol (non HDL-C) concentrations (P < 0.005) compared with their CIS-fed counterparts. Plasma HDL-C was highest in rats fed the SAT diet (P = 0.01). An in vivo assay of reverse cholesterol transport (RCT) was performed whereby radiolabeled cholesterol was delivered to the liver as acetylated LDL and the reappearance of label into plasma and HDL was determined. Plasma radioactivity in TRANS-fed rats was lower than in their SAT-fed counterparts (P = 0.01), and consistent with the cholesterol distribution in plasma, the difference was due to lower [3H]-cholesterol in lower density lipoproteins. Despite diet-induced differences in the cholesterol and phospholipid concentrations and fatty acid composition of HDL, the amount of label in HDL did not differ among groups, suggesting that consumption of these diets resulted in HDL populations with similar capacity to participate in RCT. The present findings suggest that dietary trans fatty acids regulate the metabolism of apolipoprotein B-containing lipoproteins in rats and that the effect may be masked in species possessing high plasma cholesteryl ester transfer protein (CETP) activity. These results reinforce the important role of CETP activity in determining the distribution of plasma cholesterol in response to dietary trans fatty acids.
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Richardson, Des Raymond[No abstract available]
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Witting, Paul Kenneth; Travascio, Paola; Sen, Dipankar; Mauk, GrantBoth electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex (?35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of ?250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of ?5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from ?-scission of the primary tert-butyloxyl radical product.
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Hawkins, Clare L.; Davies, Michael J.Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA bases. We show that reaction of HOCl with the exocyclic -NH<inf>2</inf> groups of cytidine, adenosine, and guanosine, and the ring NH groups of all bases, yields chloramines (RNHCl/RR?NCl). These are the major initial products. Chloramine decay can be accelerated by UV light and metal ions, and these reactions, together with thermal decomposition, give rise to nucleoside-derived nitrogen-centered radicals. Evidence is presented for the rapid addition of pyrimidine-derived nitrogen-centered radicals to another parent molecule to give dimers. Experiments with nucleoside mixtures show that the propensity for radical formation is cytidine > adenosine = guanosine > uridine = thymidine. These data are inconsistent with the selectivity of HOCl attack and the stability of the resulting chloramines, but can be rationalized if chlorine transfer between bases is rapid and yields the most stable chloramine, with such transfer preceding radical formation. Thus, though thymidine is the major initial site of chloramine formation, rapid chlorine atom transfer generates cytidine and adenosine chloramines. These reactions rationalize the preferential formation of chlorinated cytidine and adenosine in DNA.
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Davies, Michael J.; Truscott, Roger John WillisProteins comprise approximately 68% of the dry weight of cells and tissues and are therefore potentially major targets for photo-oxidation. Two major types of processes can occur with proteins. The first of these involves direct photo-oxidation arising from the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. The basic principles behind these mechanisms of photo-oxidation of amino acids, peptides and proteins and the potential selectivity of damage are discussed. Emphasis is placed primarily on the intermediates that are generated on amino acids and proteins, and the subsequent reactions of these species, and not the identity or chemistry of the sensitizer itself, unless the sensitizing group is itself intrinsic to the protein. A particular system is then discussed - the cataractous lens - where UV photo-oxidation may play a role in the aetiology of the disease, and tryptophan-derived metabolites act as UV filters. 2001 Elsevier Science B.V. All rights reserved.
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Oram, John F.; Vaughan, Ashley M.; Stocker, Roland?-Tocopherol (?-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting ?-TOH between tissues. Here we show that secretion of ?-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated ?-TOH efflux. ApoA-I lacked the ability to remove ?-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of ?-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the ?-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes ?-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove ?-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of ?-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular ?-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.
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Pattison, David I.; Davies, Michael J.Hypochlorous acid (HOCI) is a potent oxidant, which is produced in vivo by activated phagocytes. This compound is an important antibacterial agent, but excessive or misplaced production has been implicated in a number of human diseases, including atherosclerosis, arthritis, and some cancers. Proteins are major targets for this oxidant, and such reaction results in side-chain modification, backbone fragmentation, and cross-linking. Despite a wealth of qualitative data for such reactions, little absolute kinetic data is available to rationalize the in vitro and in vivo data. In this study, absolute second-order rate constants for the reactions of HOCl with protein side chains, model compounds, and backbone amide (peptide) bonds have been determined at physiological pH values. The reactivity of HOCl with potential reactive sites in proteins is summarized by the series: Met (3.8 107 M-1 s-1) > Cys (3.0 107 M-1 s-1) cystine (1.6 105 M-1 s-1) ? His (1.0 105 M-1 s-1) ?-amino (1.0 x 105 M-1 s-1) > Trp (1.1 x 104 M-1 s-1) > Lys (5.0 103 M-1 s-1) Tyr (44 M-1 s-1) %ap Arg (26 M-1 s-1) > backbone amides (10- 10-3 M-1 s-1) > Gln(0.03 M-1 s-1) Asn (0.03 M-1 s-1). The rate constants for reaction of HOCl with backbone amides (peptide bonds) vary by 4 orders of magnitude with uncharged peptide bonds reacting more readily with HOCl than those in a charged environment. These kinetic parameters have been used in computer modeling of the reactions of HOCl with human serum albumin, apolipoprotein-A1 and free amino acids in plasma at different molar excesses. These models are useful tools for predicting, and reconciling, experimental data obtained in HOCl-induced oxidations and allow estimations to be made as to the flux of HOCl to which proteins are exposed in vivo.
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Gaus, Katharina; Dean, Roger T.; Kritharides, Leonard; Jessup, Wendy K.Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.
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Sleer, Leanne S.; Brown, Andrew J.; Stanley, Keith K.Caveolin is an integral membrane protein that interacts with cholesterol in glycosphingolipid-rich rafts at the cell surface. We have examined the interaction of recombinant His-tagged caveolin-1 with cholesterol and 7-keto cholesterol, the most abundant non-enzymatically formed oxysterol found in oxidised LDL and atheromatous plaque. Our data show that caveolin-1 is able to interact with both sterols. This might have consequences for sterol transport and the signalling properties of cells during atherosclerosis. 2001 Elsevier Science Ireland Ltd. All rights reserved.
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Hazell, Linda J.; Baernthaler, Georg; Stocker, RolandThe oxidative modification of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, and there is evidence that oxidants derived from myeloperoxidase (MPO) contribute to such oxidative damage. Using human iliac arteries we investigated the relationship between lesion stage indicated by the intima-to-media (I/M) ratio and the presence of apolipoprotein B-100 (apoB, a marker for LDL), MPO, and hypochlorite (HOCl)-oxidized proteins identified by immunohistochemistry in the intima, media, and adventitia. More staining for apoB, MPO, and HOCl-oxidized proteins was observed in diseased than healthy vessels. Diseased segments also stained more for the three parameters than healthy segments in the same diseased vessel, highlighting the variability that can occur within a single cross-section of a vessel. However, significant positive correlation between I/M ratio and positive staining for apoB, MPO, and HOCl-oxidized proteins in different segments of individual arteries were apparent in segments with an I/M ratio of > 1.8. Also, the overall extent of intimal staining for apoB, MPO, and HOCl-oxidized proteins increased with increasing I/M ratio. In addition, the extent of apoB staining was greater and appeared at comparatively lower I/M ratios than that of MPO and HOCl-oxidized proteins. Our results support a contribution to atherogenesis of all three parameters assessed, although MPO and HOCl-oxidized proteins appear to participate in the disease process at a later stage than apoB. 2001 Elsevier Science Inc.
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Upston, Joanne M.; Witting, Paul Kenneth; Brown, Andrew J.; Stocker, Roland; Keaney, John F.Oxidized low-density lipoproteins (LDL) are implicated in atherosclerosis. However, large-scale intervention studies designed to test whether antioxidants, such as vitamin E, can ameliorate cardiovascular disease have generated ambivalent results. This may relate to the fact that the mechanism whereby lipid oxidation is initiated in vivo is unknown and the lack of direct evidence for a deficiency of antioxidants in atherosclerotic lesions. Further, there is little evidence to suggest that vitamin E acts as an antioxidant for lipid peroxidation in vivo. Here we tested the antioxidant effect of dietary vitamin E (?-tocopherol) supplementation on intimal proliferation and lipid oxidation in balloon-injured, hypercholesterolemic rabbits. ?-Tocopherol supplementation increased vascular content of ?-tocopherol over 30-fold compared to nonsupplemented and ?-tocopherol-deficient chows. Balloon injury resulted in oxidized lipid deposition in the aorta. Maximum levels of primary lipid oxidation products, measured as hydroperoxides of esterified lipid (LOOH) and oxidized linoleate (HODE), were 0.22 and 1.10 nmol/mg, representing 0.21 and 0.39% of the precursor molecule, respectively. Secondary lipid oxidation products, measured as oxysterols, were maximal at 5.60 nmol/mg or 1.48% of the precursor compound. Vascular HODE and oxysterols were significantly reduced by vitamin E supplementation. However, the intima/media ratio of aortic vessels increased with vitamin E supplementation, suggesting that the antioxidant promoted intimal proliferation. Thus, the study demonstrates a dissociation of aortic lipid oxidation and lesion development, and suggests that vitamin E does not prevent lesion development in this animal model. 2001 Elsevier Science Inc.
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Hawkins, Clare L.; Brown, Bronwyn E.; Davies, Michael J.Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H<inf>2</inf>O<inf>2</inf> and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis. 2001 Academic Press.
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Bernhardt, Paul V.; Chin, Piao; Richardson, Des RaymondLigands of the 2-pyridylcarbaldehyde isonicotinoylhydrazone class show high iron (Fe) sequestering efficacy and have potential as agents for the treatment of Fe overload disease, We have investigated the mechanisms responsible for their high activity, X-ray crystallography studies show that the tridentate chelate 2-pyridylcarbaldehyde isonicotinoylhydrazone undergoes an unexpected oxidation to isonicotinoyl(picollnoyl)hydrazine when complexed with FeIII. In contrast, in the absence of FeIII, the parent hydrazone is not oxidized in aerobic aqueous solution, To examine whether the diacylhydrazine could be responsible for the biological effects of 2-pyridylcarbaldehyde isonicotinoylhydrazone, their Fe chelation efficacy was compared. In contrast to its parent hydrazone, the diacylhydrazine showed little Fe chelation activity, Potentiometric titrations suggested that this might be because the diacylhydrazine was charged at physiological pH, hindering its access across membranes to intracellular Fe pools. In contrast, the Fe complex of this diacylhydrazine was charge neutral, which may allow facile movement through membranes. These data allow a model of Fe chelation for this compound to be proposed: the parent aroylhydrazone diffuses through cell membranes to bind Fe and is subsequently oxidized to the diacylhydrazine complex which then diffuses from the cell. Other diacylhydrazine analogues that were charge neutral at physiological pH demonstrated high Fe chelation efficacy. Thus, for this class of ligands, the charge of the chelator appears to be an important factor for determining their ability to access intracellular Fe. The results of this study are significant for understanding the biological activity of 2-pyridylcarbaldehyde isonicotinoylhydrazone and for the design of novel diacylhydrazine chelators for clinical use.
