Visualization of non-heme ferric and ferrous iron by highly sensitive non-heme iron histochemistry in the stress-induced acute gastric lesions in the rat

Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.     

Comparative Evaluation of Nephrotoxicity and Management by Macrophages of Intravenous Pharmaceutical Iron Formulations

BackgroundThere is a significant clinical need for effective treatment of iron deficiency. A number of compounds that can be administered intravenously have been developed. This study examines how the compounds are handled by macrophages and their relative potential to provoke oxidative stress.MethodsHuman kidney (HK-2) cells, rat peritoneal macrophages and renal cortical homogenates were exposed to pharmaceutical iron preparations. Analyses were performed for indices of oxidative stress and cell integrity. In addition, in macrophages, iron uptake and release and cytokine secretion was monitored.ResultsHK-2 cell viability was decreased by iron isomaltoside and ferumoxytol and all compounds induced lipid peroxidation. In the renal cortical homogenates, lipid peroxidation occurred at lowest concentrations with ferric carboxymaltose, iron dextran, iron sucrose and sodium ferric gluconate. In the macrophages, iron sucrose caused loss of cell viability. Iron uptake was highest for ferumoxytol and iron isomaltoside and lowest for iron sucrose and sodium ferric gluconate. Iron was released as secretion of ferritin or as ferrous iron via ferroportin. The latter was blocked by hepcidin. Exposure to ferric carboxymaltose and iron dextran resulted in release of tumor necrosis factor α.ConclusionsExposure to iron compounds increased cell stress but was tissue and dose dependent. There was a clear difference in the handling of iron from the different compounds by macrophages that suggests in vivo responses may differ.     

Role of iron in the interaction of red blood cells with methylglyoxal. Modification of L-arginine by methylglyoxal is catalyzed by iron redox cycling.

Diabetes mellitus is characterized by increased methylglyoxal (MG) production. The aim of the present study was to investigate the role of iron in the cellular and molecular effects of MG. A red blood cell (RBC) model and L-arginine were used to study the effects of MG in the absence and presence of iron. Intracellular free radical formation and calcium concentration were measured using dichlorofluorescein and Fura-2-AM, respectively. Effects of MG were compared to the effect of ferrous iron. Reaction of L-arginine with MG was investigated by electron spin resonance (ESR) spectroscopy and by a spectrophotometric method. MG caused an iron dependent oxidative stress in RBCs and an elevation of the intracellular calcium concentration due to formation of reactive oxygen species. Results of co-incubation of MG with ferrous iron in the RBC model suggested an interaction of MG and iron; one interaction was a reduction of ferric iron by MG. A role of iron in the MG-L-arginine reaction was also verified by ESR spectroscopy and by spectrophotometry. Ferric iron increased free radical formation as detected by ESR in the MG-L-arginine reaction; however, ferrous iron decreased it. The reaction of MG with L-arginine yielded a brown product as detected spectrophotometrically and this reaction was catalyzed at a lower rate with ferric iron but at a higher rate with ferrous iron. These data suggest that MG causes oxidative stress in cells, which is due at least in part to ferric iron reduction by MG and to the modification of amino acids e.g. L-arginine by MG, which is catalyzed by iron redox cycling.     

Separate pathways for cellular uptake of ferric and ferrous iron

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.     

Disparate patterns of age-related changes in lipid peroxidation in long-lived naked mole-rats and shorter-lived mice

A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P < 0.005) greater in naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P < 0.001), despite increases in tissue iron (P < 0.05). Contrary to the predictions of the oxidative stress theory, lipid damage levels did not change with age in mole-rats. These data suggest that the patterns of age-related changes in levels of markers of oxidative stress are species specific, and that the pronounced longevity of naked mole-rats is independent of oxidative stress parameters.     

Inhibition effect of ferric iron on the kinetics of ferrous iron

Ferric iron acted as a non-competitive inhibitor for the biological oxidation of ferrous iron and decreased the inhibitory effects of high concentrations of ferrous iron as well as the auto-inhibitive effect the bacterial cells. A previously developed kinetic model for this reaction was modified to incorporate the inhibition effects of ferric iron. © Rapid Science Ltd. 1998     

Asbestos-stimulated changes in nitric oxide and iron metabolism in rats

Under intratracheal asbestos fibers installation it has been investigated NO synthesis in the lung and liver tissues of Wistar rats by EPR method. Asbestos A6-45, sifted through the sieve with size 0.1 mm, has been administrated in a dose of 5 mg/kg. To evaluate the NO synthesis EPR and NO-trap methods have been used. The amplitude of EPR signal "trap-NO" in the lung samples was 12, 16 and 14 times greater than in controls on the 3th, 6th and 10th days after asbestos installation and was corresponding to NO rate of about 2 mkmol/(g x h). In the liver samples of asbestos-stimulated animals the NO level contained in the non-heme iron nitrosyl complexes was about 2 mkmol/g. Thus, the asbestos fibers stimulate NO synthesis not only in the lung tissue, but also in other organs. The obtained data shows that under NO hyperproduction certain changes in iron metabolism take place, such as: the decrease of transferrin iron and the accumulation of ferric iron not bound with transferrin. The accumulation of ferric iron not shielded by proteins is one of the oxidative stress triggers.     

Post-translational self-hydroxylation: a probe for oxygen activation mechanisms in non-heme iron enzymes

Recent years have seen considerable evolution in our understanding of the mechanisms of oxygen activation by non-heme iron enzymes, with high-valent iron-oxo intermediates coming to the forefront as formidably potent oxidants. In the absence of substrate, the generation of vividly colored chromophores deriving from the self-hydroxylation of a nearby aromatic amino acid for a number of these enzymes has afforded an opportunity to discern the conditions under which O2 activation occurs to generate a high-valent iron intermediate, and has provided a basis for a rigorous mechanistic examination of the oxygenation process. Here, we summarize the current evidence for self-hydroxylation processes in both mononuclear non-heme iron enzymes and in mutant forms of ribonucleotide reductase, and place it within the context of our developing understanding of the oxidative transformations accomplished by non-heme iron centers.     

Role of iron in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of iron ions on particulate methane monooxygenase was studied by using the EDTA-treated membranes from Methylosinus trichosporium OB3b. When the membrane was treated with EDTA the activity remained 82% of the as-isolated membranes, and the activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, ferric, ferrous and cupric ions stimulated the activity, indicating those ions were needed for the activity. When propargylamine was added, pMMO activity decreased and also the iron ESR signal decreased. As the ESR signal involves the ferrous nitrosyl complex in EDTA-treated membranes, the active site of pMMO may contain a mononuclear non-heme iron.     

Salmonella acquires ferrous iron from haemophagocytic macrophages

Bacteria harbour both ferrous and ferric iron transporters. We now report that infection of macrophages and mice with a Salmonella enterica Typhimurium strain containing an inactivated feoB‐encoded ferrous iron transporter results in increased bacterial replication, compared to infection with wild type. Inactivation of other cation transporters, SitABCD or MntH, did not increase bacterial replication. The feoB mutant strain does not have an intrinsically faster growth rate. Instead, increased replication correlated with increased expression in macrophages of the fepB‐encoded bacterial ferric iron transporter and also required siderophores, which capture ferric iron. Co‐infection of mice with wild type and a feoB mutant strain yielded a different outcome: FeoB is clearly required for tissue colonization. In co‐infected primary mouse macrophages, FeoB is required for S. Typhimurium replication if the macrophages were IFNγ treated and contain phagocytosed erythrocytes, a model for haemophagocytosis. Haemophagocytes are macrophages that have engulfed erythrocytes and/or leucocytes and can harbour Salmonella in mice. These observations suggest that Salmonella acquires ferrous iron from haemophagocytic macrophages.     

Iron-Induced Oxidative Stress in Erythrocyte Membranes of Non-Insulin-Dependent Diabetic Nigerians

The presence of higher level of endogenous free radical reaction products in the erythrocyte ghost membrane (EGM) of Non-insulin-dependent diabetes mellitus (NIDDM) subjects compared with that of normal healthy controls has been demonstrated. The EGMs of NIDDM subjects were also shown to be more susceptible to exogenously generated oxidative stress than those of normal healthy individuals. The decreased level of reactive thiol groups in the EGM of NIDDM individuals supported this observation. We propose that the presence of significant levels of non-heme iron in the EGM of NIDDM subjects is an indication of the potential for iron-catalysed production of hydroxy and other toxic radicals which could cause continuous oxidative stress and tissue damage. Oxygen free radicals could therefore be responsible for most of the erythrocyte abnormalities associated with non-insulin-dependent diabetes and could indeed be intimately involved in the mechanism of tissue damage in diabetic complications.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

Effect of Allopurinol on the Hepatic and Cerebellar Iron,Selenium, Zinc and Copper Status Following Acute Ethanol Administration to Rats

An acute ethanol load (50mmol/kg, i.p.) induces an increase in the total non-heme iron and in the low-molecular-weight non-heme iron complexes (LMW-Fe) content both in liver and cerebellum. This increase in LMW-Fe is associated with a decrease in some essential trace elements (selenium, zinc, copper) playing a role in the anti-oxidant system. These changes could contribute to the enhancement in lipid peroxidation which occurs at the hepatic and cerebellar level following the ethanol administration.

The administration of allopurinol prior to the ethanol load prevents the changes in non-heme iron and trace elements. This prevention may contribute to the protective effects of allopurinol on the ethanol-induced oxidative stress.     

细菌Rieske非血红素铁环羟化酶在多环芳烃降解中的研究进展

多环芳烃是一种常见的持久性有机污染物,因具有致癌、致突变等毒性而被广泛关注。其微生物降解过程通常由羟化起始,随后脱氢、开环、一步步去除支链,最终进入三羧酸循环。Rieske 非血红素铁环羟化酶(Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases , RHOs , 又称 aromatic ring-hydroxylating dioxygenases) 或细胞色素 P450 氧化酶负责将羟基加成到多环芳烃环上,将疏水性的多环芳烃转化为亲水性的衍生物,这一过程是多环芳烃降解转化的起始步骤,也是关键步骤和限速步骤之一。文中主要介绍 RHOs 的分布、底物特异性、底物识别机制以及研究 RHOs 与多环芳烃的一些技术和方法等,并对 RHOs 在环境修复技术中的潜在应用进行了展望。     

Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry: demonstration of the superior sensitivity of the methods in the liver,spleen, and stomach of the rat

Perfusion-Perls and -Turnbull methods supplemented by the intensification with 3,3-diaminobenzidine (+ DAB) enabled stronger and more extensive staining of nonheme iron than the Perls + and Turnbull + DAB methods carried out on tissue sections fixed with 10% formalin in 0.9% saline or PBS. The section- and perfusion-Perls + DAB methods are not specific for the demonstration of nonheme ferric iron but also stain nonheme ferrous iron. However, owing to its high sensitivity, the perfusion-Perls + DAB method would provide useful information about nonheme iron deposition regardless of oxidation states in normal and pathological conditions. The perfusion-Turnbull + DAB method is specifically demonstrable of nonheme ferrous iron and the results from this method showed significant stores of nonheme ferrous iron in the hepatocytes, Kupffer cells, splenic macrophages, and gastric parietal cells of the rat. Since nonheme ferrous iron is considered to be critically involved in free radical generation, the perfusion-Turnbull + DAB method would visualize such populations of cells that are at risk from free radical damage.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Modeling non-heme iron proteins

Synthetic modeling studies of non-heme iron proteins continue to contribute to our understanding of the mechanism of these proteins. Recently, mononuclear Fe(IV)=O complexes have been prepared and characterized to model the same species that are proposed to be the reactive intermediates in reactions involving mononuclear non-heme iron proteins. Generation of such species for the oxidation of organic substrates has also been demonstrated. Other advances include successful modeling of the structural and functional aspects of diiron non-heme proteins with the use of terphenyl-based carboxylate ligands and the development of several iron-based reagents that catalyze oxidation reactions with the use of various oxidants, including dioxygen.     

Microanalysis of non-heme iron in animal tissues

The method recommended by the Iron Panel of the International Committee for the Standardization in Haematology for measurement of serum iron was adapted for measurement of non-heme iron in animal tissues. The method developed was designed specifically to facilitate measurement of non-heme iron using as little as 10 mg of tissue, in a final reaction volume of 60 microl. In this assay, tissue homogenates are treated with hydrochloric acid and trichloroacetic acid and heated at 95 degrees C. Non-heme iron is released and protein is precipitated. Following centrifugation, iron in the supernatant is reacted with ferrozine in the presence of the reducing agent thioglycolic acid, and the complex is quantified by spectrophotometry. The method was validated by analysis of two Standard Reference Materials (bovine liver), comparing results of this assay against certified values and concentrations determined by flame atomic absorption spectrometry following acid digestion. Results using this method for analysis of non-heme iron in guinea pig tissues (liver, kidney and heart) compared favorably with those obtained using micro-scale adaptations of three published reference methods. The new method was more sensitive, required less time, and was less cumbersome than the three published methods to which it was compared.     

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.