Dose-dependent effects of chronic iron burden on heart aldehyde and acyloin production in mice

Iron’s chemical structure and its ability to initiate one-electron reactions are properties that cause it to play a major role in the production and metabolism of oxygen free radicals in biological systems. Oxygen free radicals are conjectured to cause cardiac failure in individuals afflicted with disorders of iron overload. We report on the use of both acyloins and aldehydes as markers of oxidative stress in a murine model of chronic iron-overload cardiomyopathy. Twenty mice were randomized to four treatment groups: (1) control (0.2 mL normal saline ip/mouse/d); (2) 100 mg iron (0.05 mL iron dextran/mouse/d); (3) 200 mg iron (0.1 mL iron dexxtran/mouse/d); (4) 400 mg iron (0.2 mL iron dextran/mouse/d). Significant dose-dependent increases in both total heart aldehyde and total heart acyloin concentrations were found. Furthermore, a significant positive correlation existed between the dose of iron administered and each quantified aldehyde and acyloin found in the heart.     

Cytotoxic aldehyde generation in heart following acute iron-loading

Although the mechanism of myocardial failure following acute iron poisoning is not known, excess iron-catalyzed free radical generation is conjectured to play a role. The effects of time (0 to 360 minutes) on total iron concentrations, glutathione peroxidase activity, and cytotoxic aldehyde production in heart of mice (B6D2F1, n = 65) were first investigated following acute iron-loading (20 mg iron dextran i.p./mouse). In a subsequent experiment, the effects of dose (0 to 80 mg iron dextran i.p./mouse, n = 75) on the aforementioned parameters were investigated. Our results show that the concentrations of cytotoxic aldehydes: (1) significantly differ over-time, with corresponding increases in total concentrations of iron (r = 0.93, p < 0.001); and (2) increase parallel to the total dose of iron administered (r = 0.95, p < 0.001). Furthermore, dose-and time-dependent alterations to glutathione peroxidase activity are observed, which is most likely due to an acute up-regulation of the enzyme as an endogenous protective response to increased free radical activity in the heart subsequent to iron-loading. While no single mechanism is likely to account for the complex pathophysiology of acute iron-induced heart failure, our results shown that iron-loading can result in significant free radical generation, as quantified by cytotoxic aldehydes, in heart tissue of mice. This is the first report on the effects of time and dose on cytotoxic aldehyde generation and glutathione peroxidase activity in heart of mice following acute iron-loading.     

Iron toxicity and its possible association with treatment of Cancer: Lessons from hemoglobinopathies and rare,transfusion-dependent anemias

Exposure to elevated levels of iron causes tissue damage and organ failure, and increases the risk of cancer. The toxicity of iron is mediated through generation of oxidants. There is also solid evidence indicating that oxidant stress plays a significant role in a variety of human disease states, including malignant transformation. Iron toxicity is the main focus when managing thalassemia. However, the short- and long-term toxicities of iron have not been extensively considered in children and adults treated for malignancy, and only recently have begun to draw oncologists’ attention. The treatment of malignancy can markedly increase exposure of patients to elevated toxic iron species without the need for excess iron input from transfusion. This under-recognized exposure likely enhances organ toxicity and may contribute to long-term development of secondary malignancy and organ failure. This review discusses the current understanding of iron metabolism, the mechanisms of production of toxic free iron species in humans, and the relation of the clinical marker, transferrin saturation (TS), to the presence of toxic free iron. We will present epidemiological data showing that high TS is associated with poor outcomes and development of cancer, and that lowering free iron may improve outcomes. Finally, we will discuss the possible relation between some late complications seen in survivors of cancer and those due to iron toxicity.     

Myeloperoxidase deficiency attenuates systemic and dietary iron-induced adverse effects

Iron deficiency is routinely treated with oral or systemic iron supplements, which are highly reactive and could induce oxidative stress via augmenting the activity of proinflammatory enzyme myeloperoxidase (MPO). To investigate the extent to which MPO is involved in iron-induced toxicity, acute (24 h) iron toxicity was induced by intraperitoneal administration of FeSO₄ (25 mg/kg body weight) to MPO-deficient (MpoKO) mice and their wild-type (WT) littermates. Acute iron toxicity was also assessed in WT mice pretreated with an MPO inhibitor, 4-aminobenzoic acid hydrazide. Systemic iron administration up-regulated circulating MPO and neutrophil elastase and elevated systemic inflammatory and organ damage markers in WT mice. However, genetic deletion of MPO or its inhibition significantly reduced iron-induced organ damage and systemic inflammatory responses. In contrast to the acute model, 8 weeks of 2% carbonyl iron diet feeding to WT mice did not change the levels of circulating MPO and neutrophil elastase but promoted their accumulation in the liver. Even though both MpoKO and WT mice displayed similar levels of diet-induced hyperferremia, MpoKO mice showed significantly reduced inflammatory response and oxidative stress than the WT mice. In addition, WT bone-marrow-derived neutrophils (BMDN) generated more reactive oxygen species than MPO-deficient BMDN upon iron stimulation. Altogether, genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron supplementation is a promising approach to reduce iron-induced toxicity/side effects in vulnerable population.     

Iron overload following red blood cell transfusion and its impact on disease severity

Transfusion of red blood cells can be a life-saving therapy both for patients with chronic anemias and for those who are critically ill with acute blood loss. However, transfusion has been associated with significant morbidity. Chronic transfusion results in accumulation of excess iron that surpasses the binding capacity of the major iron transport protein, transferrin. The resulting non-transferrin bound iron (NTBI) can catalyze the production of highly reactive oxygen species (ROS) leading to significant and wide spread injury to the liver, heart, and endocrine organs as well as increases in infection. Acute transfusion of red blood cells in critically ill patients likewise has significant effects including increased mortality, prolonged hospital stays, and elevated risk of nosocomial infection. These effects appear to be more profound with increasing age of stored blood. The progressive release of free iron associated with storage time suggests that morbidity following acute transfusion, like that seen in chronic transfusion, may be due in part to elevated levels of NTBI. It is clear that transfusion is necessary in many instances; however, its risks and benefits must be carefully balanced before proceeding to avoid unnecessary iron toxicity.     

Bleomycin-reactive iron in patients with acute non-lymphocytic leukemia.

Bleomycin-reactive iron was detected in the sera of six out of nine adults undergoing intensive chemotherapy for acute non-lymphocytic leukemia. In these individuals the corresponding transferrin saturation ranged from 96% to 113% and the serum ferritin from 775 to 9975 micrograms/l. Nontransferrin-bound iron has been postulated to be a factor in organ toxicity in iron overload conditions such as beta thalassemia and hereditary hemochromatosis by facilitating the production of tissue-damaging free radicals. We propose that bleomycin-reactive iron should be considered as a possible factor in organ dysfunction seen with intensive cancer chemotherapy.     

Reactive Oxygen Species and the Central Nervous System

Radicals are species containing one or more unpaired electrons, such as nitric oxide (NO.). The oxygen radical superoxide (O2.-) and the nonradical hydrogen peroxide (H2O2) are produced during normal metabolism and perform several useful functions. Excessive production of O2.- and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxyl radical (.OH) and other oxidants in the presence of "catalytic" iron or copper ions. An important form of antioxidant defense is the storage and transport of iron and copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e.g., by ischemia or trauma, can cause increased metal ion availability and accelerate free radical reactions. This may be especially important in the brain because areas of this organ are rich in iron and CSF cannot bind released iron ions. Oxidative stress on nervous tissue can produce damage by several interacting mechanisms, including increases in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminum and in damage to the substantia nigra in patients with Parkinson's disease are reviewed. Finally, the nature of antioxidants is discussed, it being suggested that antioxidant enzymes and chelators of transition metal ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be used in the design of antioxidants for therapeutic use.     

Enhancement of adriamycin toxicity by iron chelates is not a free radical mechanism

The possible involvement of metal ions and free radicals in the cytotoxic mechanism of Adriamycin (ADR) was investigated, using a model system ofEscherichia coli cells. It is shown thatE. coli mediated the production of free radicals under anaerobic (ADR-semiquinone) and aerobic (superoxide) conditions. ADR-induced loss of colony-forming ability was enhanced by the addition of iron (Fe) chelates. These observations suggested that a Fenton-type free radical mechanism was responsible for ADR toxicity. However, the mortality rate was essentially unchanged by the exclusion of oxygen. It was also unaffected by the addition of H₂O₂, catalase, or chelating agents. Cu(II), Zn(II) or Mg(II) had no effect on ADR toxicity. ADR and iron chelates did not induce measurable amounts of DNA strand-breaks. These observations suggest a mechanism of ADR-induced cell killing that is enhanced by Fe chelates, but does not directly involve oxygen-derived free radicals.     

Combination of Iron Overload Plus Ethanol and Ischemia Alone Give Rise to the Same Endogenous Free Iron Pool

Iron overload aggravates tissue damage caused by ischemia and ethanol intoxication. The underlying mechanisms of this phenomenon are not yet clear. To clarify these mechanisms we followed free iron (“loosely” bound redox-active iron) concentration in livers from rats subjected to experimental iron overload, acute ethanol intoxication, and ex vivo warm ischemia. The levels of free iron in non-homogenized liver tissues, liver homogenates, and hepatocyte cultures were analyzed by means of EPR spectroscopy. Ischemia gradually increased the levels of endogenous free iron in liver tissues and in liver homogenates. The increase was accompanied by the accumulation of lipid peroxidation products. Iron overload alone, known to increase significantly the total tissue iron, did not affect either free iron levels or lipid peroxidation. Homogenization of iron-loaded livers, however, resulted in the release of a significant portion of free iron from endogenous depositories. Acute ethanol intoxication increased free iron levels in liver tissue and diminished the portion of free iron releasing during homogenization. Similarly to liver tissue, the primary hepatocyte culture loaded with iron in vitro released significantly more free iron during homogenization compared to non iron-loaded hepatocyte culture. Analyzing three possible sources of free iron release under these experimental conditions in liver cells, namely ferritin, intracellular transferrin-receptor complex and heme oxygenase, we suggest that redox active free iron is released from ferritin under ischemic conditions whereas ethanol and homogenization facilitate the release of iron from endosomes containing transferrin-receptor complexes.     

The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis

Sepsis is characterised by a systemic dysregulated inflammatory response and oxidative stress, often leading to organ failure and death. Development of organ dysfunction associated with sepsis is now accepted to be due at least in part to oxidative damage to mitochondria. MitoQ is an antioxidant selectively targeted to mitochondria that protects mitochondria from oxidative damage and which has been shown to decrease mitochondrial damage in animal models of oxidative stress. We hypothesised that if oxidative damage to mitochondria does play a significant role in sepsis-induced organ failure, then MitoQ should modulate inflammatory responses, reduce mitochondrial oxidative damage, and thereby ameliorate organ damage. To assess this, we investigated the effects of MitoQ in vitro in an endothelial cell model of sepsis and in vivo in a rat model of sepsis. In vitro MitoQ decreased oxidative stress and protected mitochondria from damage as indicated by a lower rate of reactive oxygen species formation (P=0.01) and by maintenance of the mitochondrial membrane potential (P<0.005). MitoQ also suppressed proinflammatory cytokine release from the cells (P<0.05) while the production of the anti-inflammatory cytokine interleukin-10 was increased by MitoQ (P<0.001). In a lipopolysaccharide-peptidoglycan rat model of the organ dysfunction that occurs during sepsis, MitoQ treatment resulted in lower levels of biochemical markers of acute liver and renal dysfunction (P<0.05), and mitochondrial membrane potential was augmented (P<0.01) in most organs. These findings suggest that the use of mitochondria-targeted antioxidants such as MitoQ may be beneficial in sepsis.     

Oxidative stress during exercise: Implication of antioxidant nutrients

Research evidence has accumulated in the past decade that strenuous aerobic exercise is associated with oxidative stress and tissue damage in the body. There is indication that generation of oxygen free radicals and other reactive oxygen species may be the underlying mechanism for exercise-induced oxidative damage, but a causal relationship remains to be established. Enzymatic and nonenzymatic antioxidants play a vital role in protecting tissues from excessive oxidative damage during exercise. Depletion of each of the antioxidant systems increases the vulnerability of various tissues and cellular components to reactive oxygen species. Because acute strenuous exercise and chronic exercise training increase the consumption of various antioxidants, it is conceivable that dietary supplementation of specific antioxidants would be beneficial.     

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.     

Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA

We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system. An observed lag phase in DNA nicking suggests a multi-target model of hydroxyl free radical attack on DNA. In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate. These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate. We have investigated the initial rate of OH production with two OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively. We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for OH trapping by salicylate is 21.1 kcal/nmole. These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different. Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction. The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.     

Hemochromatosis heterozygotes may constitute a radiation-sensitive subpopulation

A primary mechanism of radiation-induced DNA damage is by generation of free radicals. Chronically increased oxidative stress from elevated levels of iron in the body may increase radiation sensitivity by decreasing cellular oxygen radical scavenging capability. Hemochromatosis heterozygotes have elevated body iron. Low-level radiation sensitization by iron may be particularly pertinent for risk of breast cancer. Since 10% of the population appears to be heterozygous for the hemochromatosis gene, a radiosensitizing effect would have pervasive implications.     

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe³⁺ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Superoxide Dismutase, Catalase, and alpha-Tocopherol Content of Stored Potato Tubers

Activated oxygen or oxygen free radical mediated damage to plants has been established or implicated in many plant stress situations. The extent of activated oxygen damage to potato (Solanum tuberosum L.) tubers during low temperature storage and long-term storage is not known. Quantitation of oxygen free radical mediated damage in plant tissues is difficult. However, it is comparatively easy to quantitate endogenous antioxidants, which detoxify potentially damaging forms of activated oxygen. Three tuber antioxidants, superoxide dismutase, catalase, and α-tocopherol were assayed from four potato cultivars stored at 3°C and 9°C for 40 weeks. Tubers stored at 3°C demonstrated increased superoxide dismutase activities (up to 72%) compared to tubers stored at 9°C. Time dependent increases in the levels of superoxide dismutase, catalase, and α-tocopherol occurred during the course of the 40 week storage. The possible relationship between these increases in antioxidants and the rate of activated oxygen production in the tubers is discussed.     

Mechanism of cellular uptake and impact of ferucarbotran on macrophage physiology

Superparamagnetic iron oxide (SPIO) nanoparticles are contrast agents used for magnetic resonance imaging. Ferucarbotran is a clinically approved SPIO-coated carboxydextran with a diameter of about 45-60 nm. We investigated the mechanism of cellular uptake of Ferucarbotran with a cell model using the murine macrophage cell line Raw 264.7. We observed a dose-dependent uptake of these SPIO particles by spectrophotometer analysis and also a dose-dependent increase in the granularity of the macrophages as determined by flow cytometry. There was a linear correlation between the side scattering mean value and iron content (P<0.001, R(2) = 0. 8048). For evaluation of the endocytotic pathway of these ingested SPIO particles, different inhibitors of the endocytotic pathways were employed. There was a significant decrease of side scattering counts in the cells and a less significant change in signal intensity based on magnetic resonance in the phenylarsine oxide-treated macrophages. After labeling with SPIO particles, the macrophages showed an increase in the production of reactive oxygen species at 2, 24, and 48 h; a decrease in mitochondrial membrane potential at 24 h; and an increase in cell proliferation at 24 h. We concluded that Ferucarbotran was internalized into macrophages via the clathrin-mediated pathway and can change the cellular behavior of these cells after labeling.     

ADP-Regulation of Mitochondrial Free Radical Production Is Different with Complex I- or Complex II-Linked Substrates: Implications for the Exercise Paradox and Brain Hypermetabolism

In agreement with classic studies, succinate-supplemented rat and pigeon heart and nonsynaptic brain mitochondrial free radical production is stopped by ADP additions causing the stimulation of respiration from State 4 to State 3. Nevertheless, with Complex I-linked substrates, mitochondria produce free radicals in State 3 at rates similar or somewhat higher than during resting respiration. The absence of sharp increases in free radical production during intense respiration is possible due to strong decreases of free radical leak in State 3. The results indicate that Complex I is the main mitochondrial free radical generator in State 3, adding to its already known important generation of active oxygen species in State 4. The observed rate of mitochondrial free radical production with Complex I-linked substrates in the active State 3 can help to explain two paradoxes: (a) the lack of massive muscle oxidative damage and shortening of life span due to exercise, in spite of up to 23-fold increases of oxygen consumption together with the very low levels of antioxidants present in heart, skeletal muscle, and brain; (b) the presence of some degree of oxidative stress during exercise and hyperactivity in spite of the stop of mitochondrial free radical production by ADP with succinate as substrate.     

Intestinal iron absorption during suckling in mammals

The maintenance of appropriate iron levels is important for mammalian health, particularly during the rapid growth period following birth. Too little iron can lead to irreversible damage to the developing central nervous system and too much iron at this point can have adverse long term consequences, possibly due to excessive free radical production. In order to maintain iron levels, intestinal iron absorption is very efficient in young mammals, such that almost all of the iron in breast milk is utilized. However this high level of absorption is unable to be down regulated in response to excess iron as it can be in adults, implying that different regulatory processes are involved during suckling. Various mechanisms have been proposed to explain this high absorption, including enhanced expression of the proteins involved in iron absorption in adults (particularly DMT1 and ferroportin), non-specific uptake via pinocytosis, and the uptake of lactoferrin bound iron by the lactoferrin receptor. However, at present the precise mechanism is unclear. It is possible that all of these components contribute to the high intestinal iron absorption seen during suckling, or a novel, as yet undescribed, mechanism could be involved. This review summarises the evidence for and against each of the mechanisms described above and highlights how little is known about iron homeostasis in this vital stage of development.