Numerous pathologies may involve toxic side effects of free heme and hemederived iron. Deficiency of the hemecatabolizing enzyme, heme oxygenase-1 (HO-1), in both a human patient and transgenic knockout mice leads to an abundance of circulating heme and damage to vascular endothelium. Although heme can be directly cytotoxic, the present investigations examine the possibility that hemoglobin-derived heme and iron might be indirectly toxic through the generation of oxidized forms of low-density lipoprotein (LDL). In support, hemoglobin in plasma, when oxidized to methemoglobin by oxidants such as leukocyte-derived reactive oxygen, causes oxidative modification of LDL. Heme, released from methemoglobin, catalyzes the oxidation of LDL, which in turn induces endothelial cytolysis primarily caused by lipid hydroperoxides. Exposure of endothelium to sublethal concentrations of this oxidized LDL leads to induction of both HO-1 and ferritin. Similar endothelial cytotoxicity was caused by LDL isolated from plasma of an HO-1-deficient child. Spectral analysis of the child's plasma revealed a substantial oxidation of plasma hemoglobin to methemoglobin. Iron accumulated in the HO-1-deficient child's LDL and several independent assays revealed oxidative modification of the LDL. We conclude that hemoglobin, when oxidized in plasma, can be indirectly cytotoxic through the generation of oxidized LDL by released heme and that, in response, the intracellular defense-HO-1 and ferritin-is induced. These results may be relevant to a variety of disorders-such as renal failure associated with intravascular hemolysis, hemorrhagic injury to the central nervous system, and, perhaps, atherogenesis-in which hemoglobin-derived heme may promote the formation of fatty acid hydroperoxides. IntroductionAerobic organisms are well endowed with enzymatic oxidant defense systems, which provide protection against activated oxygen species. Damage caused by reactive oxygen can be greatly amplified by "free" redox active iron. 1-2 For example, iron-rich Staphylococcus aureus is 3 orders of magnitude more susceptible to killing by hydrogen peroxide than are iron-poor staphylococci. 3 Conversely, depletion of cellular iron powerfully protects eukaryotic and prokaryotic cells against oxidant challenge. 4-5 One abundant source of potentially toxic iron is heme, and both exogenous and endogenous heme can synergistically enhance oxidantmediated cellular damage. [6][7][8][9][10] Heme, a ubiquitous iron-containing compound, is quite hydrophobic, readily enters cell membranes, and greatly increases cellular susceptibility to oxidant-mediated killing. 8 Heme also acts as a catalyst for the oxidation of low-density lipoprotein (LDL), generating products toxic to endothelium. 9,[11][12] The toxic effects of heme may be important in a number of pathologies. These include not only acute conditions such as intravascular hemolysis (which can lead to renal failure) but also more insidious processes such as atherogenesis in which intralesional deposits of iron (perhaps ...

Objective-We investigated whether red cell infiltration of atheromatous lesions promotes the later stages of atherosclerosis. Methods and Results-We find that oxidation of ferro (FeII) hemoglobin in ruptured advanced lesions occurs generating ferri (FeIII) hemoglobin and via more extensive oxidation ferrylhemoglobin (FeIII/FeIVϭO). The protein oxidation marker dityrosine accumulates in complicated lesions, accompanied by the formation of cross-linked hemoglobin, a hallmark of ferrylhemoglobin. Exposure of normal red cells to lipids derived from atheromatous lesions causes hemolysis and oxidation of liberated hemoglobin. In the interactions between hemoglobin and atheroma lipids, hemoglobin and heme promote further lipid oxidation and subsequently endothelial reactions such as upregulation of heme oxygenase-1 and cytotoxicity to endothelium. Oxidative scission of heme leads to release of iron and a feed-forward process of iron-driven plaque lipid oxidation. The inhibition of heme release from globin by haptoglobin and sequestration of heme by hemopexin suppress hemoglobin-mediated oxidation of lipids of atheromatous lesions and attenuate endothelial cytotoxicity. Conclusion-The interior of advanced atheromatous lesions is a prooxidant environment in which erythrocytes lyse, hemoglobin is oxidized to ferri-and ferrylhemoglobin, and released heme and iron promote further oxidation of lipids. Oxysterols and oxidation products of polyunsaturated fatty acids are present in human atheromatous lesions. 4,5 Atherosclerotic lesions are hazardous regions for nucleated cells, both endothelial cells and, quite probably, incoming macrophages. 6 The major cytotoxic species may be oxidation products of lipids, particularly lipid hydroperoxides (LOOHs), aldehydes, and carbonyls. 6,7 In artificial systems, oxidation of polyunsaturated fatty acids requires reactive transition metals such as iron and copper. Based on our earlier work, 6,8,9 the metal in atheromatous lesions might be iron derived from heme. Nonprotein-bound heme is a particularly deleterious species inasmuch as it is hydrophobic and easily able to enter cell membranes. 10 In previous studies, we found that endothelial cells exposed to oxidized low-density lipoprotein (LDL) upregulated both heme oxygenase-1 (HO-1) and ferritin, 8,9 presumably as a defense mechanism. 6,11-14 Upregulation of HO-1 15 and ferritin H chain 16 in endothelial cells has been reported in the early phase of progression of atherosclerotic lesions. Expression of HO-1 provides protection against atherosclerosis in several experimental models, 17,18 and HO-1 deficiency in humans has been associated with the appearance of vasculature fatty streaks and atheromatous plaques at the age of 6. 19 We tested the hypothesis that heme-iron may accumulate in atherosclerotic lesions by intrusion and lysis of erythrocytes. Liberated hemoglobin is oxidized, and released hemeiron-dependent oxidation of lipids is strongly favored, contributing to further plaque development. Methods Tissue SamplesSpecimens of ...

Iron-derived reactive oxygen species are involved in the pathogenesis of numerous vascular disorders. One abundant source of redox active iron is heme, which is inherently dangerous when it escapes from its physiologic sites. Here, we present a review of the nature of heme-mediated cytotoxicity and of the strategies by which endothelium manages to protect itself from this clear and present danger. Of all sites in the body, the endothelium may be at greatest risk of exposure to heme. Heme greatly potentiates endothelial cell killing mediated by leukocytes and other sources of reactive oxygen. Heme also promotes the conversion of low-density lipoprotein to cytotoxic oxidized products. Hemoglobin in plasma, when oxidized, transfers heme to endothelium and lipoprotein, thereby enhancing susceptibility to oxidant-mediated injury. As a defense against such stress, endothelial cells upregulate heme oxygenase-1 and ferritin. Heme oxygenase opens the porphyrin ring, producing biliverdin, carbon monoxide, and a most dangerous product-redox active iron. The latter can be effectively controlled by ferritin via sequestration and ferroxidase activity. These homeostatic adjustments have been shown to be effective in the protection of endothelium against the damaging effects of heme and oxidants; lack of adaptation in an iron-rich environment led to extensive endothelial damage in humans.

Iron-derived reactive oxygen species are implicated in the pathogenesis of numerous vascular disorders including atherosclerosis, microangiopathic hemolytic anemia, vasculitis, and reperfusion injury. One abundant source of redox active iron is heme, which is inherently dangerous when released from intracellular heme proteins. The present review concerns the involvement of heme in vascular endothelial cell damage and the strategies used by endothelium to minimize such damage. Exposure of endothelium to heme greatly potentiates cell killing mediated by polymorphonuclear leukocytes and other sources of reactive oxygen. Free heme also promotes the conversion of low-density lipoprotein (LDL) into cytotoxic oxidized products. Only because of its abundance, hemoglobin probably represents the most important potential source of heme within the vascular endothelium; hemoglobin in plasma, when oxidized, transfers heme to endothelium and LDL, thereby enhancing cellular susceptibility to oxidant-mediated injury. As a defense against such toxicity, upon exposure to heme or hemoglobin, endothelial cells up-regulate heme oxygenase-1 and ferritin. Heme oxygenase-1 is a heme-degrading enzyme that opens the porphyrin ring, producing biliverdin, carbon monoxide, and the most dangerous product - free redox active iron. The latter can be effectively controlled by ferritin via sequestration and ferroxidase activity. Ferritin serves as a protective gene by virtue of antioxidant, antiapoptotic, and antiproliferative actions. These homeostatic adjustments have been shown effective in the protection of endothelium against the damaging effects of exogenous heme and oxidants. The central importance of this protective system was recently highlighted by a child diagnosed with heme oxygenase-1 deficiency, who exhibited extensive endothelial damage.

These findings show that atorvastatin treatment favorably affected the lipid profile, increasing the activity of HDL-associated PON and decreasing the cytotoxic effect of oxidative stress.

Human serum paraoxonase is physically associated with an apolipoprotein (Apo-A₁) and clusterin-containing high-density lipoprotein (HDL) and prevents low-density lipoprotein from lipid peroxidation. The aim of our study was to determine whether paraoxonase activity or phenotype is altered in patients with chronic renal failure and in hyperlipidemic subjects without renal insufficiency and to compare the values with those of healthy controls. We investigated the serum paraoxonase activity and polymorphism in 119 hemodialyzed uremic patients, 107 patients with primary hyperlipoproteinemia, and in 110 healthy control subjects. The serum paraoxonase activity was significantly decreased both in hyperlipidemic (p < 0.01) and uremic patients (p < 0.001) as compared with controls. On comparison, the serum paraoxonase activity was significantly lower (p < 0.001) in uremic than in hyperlipoproteinemic patients. The HDL and Apo-A₁ levels were as follows: uremic < hyperlipidemic < control. To assess whether the observed reduction in paraoxonase activity was due to HDL and Apo-A₁ level decreases, we standardized the enzyme activity for HDL and Apo-A₁ concentrations. We found that the standardized paraoxonase activity (paraoxonase/HDL ratio) was also lower in the uremic patients (103.3 ± 69.5) as compared with hyperlipidemic patients (137.64 ± 81.0) and controls (194.45 ± 94.45). The standardized values for Apo-A₁ showed a similar tendency: paraoxonase/Apo-A₁ ratio in uremic patients 89.64 ± 47.8, in hyperlipidemic patients 128.12 ± 69.83, and in controls 161.40 ± 47.35. The phenotypic distribution of paraoxonase (AA, AB, BB) did not change significantly in the patient groups. These results suggest that HDL concentration and phenotypic distribution of paraoxonase may not be the only determining factors, but that other as yet undetermined factors could be involved in the enzyme activity changes.

We tested the hypothesis that adaptation of Candida albicans to chronic oxidative stress inhibits the formation of hyphae and reduces pathogenicity. Candida albicans cells were exposed to increasing concentrations of t-butylhydroperoxide (tBOOH), a lipid peroxidation-accelerating agent, and mutants with heritable tBOOH tolerance were isolated. Hypha formation by the mutants was negligible on Spider agar, indicating that the development of oxidative stress tolerance prevented Candida cells from undergoing dimorphic switches. One of the mutants, C. albicans AF06, was five times less pathogenic in mice than its parental strain, due to its reduced germ tube-, pseudohypha- and hypha-forming capability, and decreased phospholipase secretion. An increased oxidative stress tolerance may therefore be disadvantageous when this pathogen leaves blood vessels and invades deep organs. The AF06 mutant was characterized by high intracellular concentrations of endogenous oxidants, reduced monounsaturated and polyunsaturated fatty acid contents, the continuous induction of the antioxidative defense system, decreased cytochrome c-dependent respiration, and increased alternative respiration. The mutation did not influence growth rate, cell size, cell surface, cellular ultrastructures, including mitochondria, or recognition by human polymorphonuclear leukocytes. The selection of oxidative stress-tolerant respiratory Candida mutants may also occur in vivo, when reduced respiration helps the fungus to cope with antimycotic agents.

Over the past few years increasing evidence has suggested the nongenomic effects of thyroid hormone, such as the activation of the signal transduction pathways and the activation of nuclear factor-B by the induction of oxidative stress. The present study was undertaken to investigate the effect of thyroid hormone on human polymorphonuclear leukocytes (PMNLs) which are known as important sources of reactive oxygen species in the circulation. The production of superoxide anion (O 2 ) and the activity of myeloperoxidase were determined in the presence and absence of several inhibitors of the signalling pathway. -Thyroxine (T 4 ), L-3,5,3 -tri-iodothyronine (T 3 ) and L-3,5-di-iodothyronine (T 2 ) stimulated O 2 production in PMNLs in a dose-dependent manner within a few minutes of addition to cells. Thyroid hormone-stimulated O 2 production was partially inhibited by pertussis toxin, an inhibitor of GTP-binding G protein, and was completely abolished by the protein kinase C inhibitors calphostin C and Ro-32-0432, and by a calcium chelator (BAPTA; bis-(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid). Thyroid hormone stimulated myeloperoxidase activity and induced 125 I incorporation into PMNLs. Furthermore, thyroid hormone pre-incubation enhanced O 2 production for n-formyl-methionyl-leucylphenylalanine (FMLP) stimulation. In conclusion, novel nongenomic actions of thyroid hormone, the induction of superoxide anion production and the stimulation of myeloperoxidase activity in PMNLs were demonstrated. The induction of O 2 production requires calcium and is mediated by a pertussis toxin-sensitive G protein via stimulation of protein kinase C(s). These results suggest the existence of a membrane-bound binding site for thyroid hormone in PMNLs and a physiological role for thyroid hormone in the cellular defence mechanisms by stimulating free-radical production.