Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Iron accumulation and neurotoxicity in cortical cultures treated with holotransferrin

Nonheme iron accumulates in CNS tissue after ischemic and hemorrhagic insults and may contribute to cell loss. The source of this iron has not been precisely defined. After blood-brain barrier disruption, CNS cells may be exposed to plasma concentrations of transferrin-bound iron (TBI), which exceed that in the CSF by over 50-fold. In this study, the hypothesis that these concentrations of TBI produce cell iron accumulation and neurotoxicity was tested in primary cortical cultures. Treatment with 0.5-3 mg/ml holotransferrin for 24 h resulted in the loss of 20-40% of neurons, associated with increases in malondialdehyde, ferritin, heme oxygenase-1, and iron; transferrin receptor-1 expression was reduced by about 50%. Deferoxamine, 2,2′-bipyridyl, Trolox, and ascorbate prevented all injury, but apotransferrin was ineffective. Cell TBI accumulation was significantly reduced by deferoxamine, 2,2′-bipyridyl, and apotransferrin, but not by ascorbate or Trolox. After treatment with ⁵⁵Fe-transferrin, approximately 40% of cell iron was exported within 16 h. Net export was increased by deferoxamine and 2,2′-bipyridyl, but not by apotransferrin. These results suggest that downregulation of transferrin receptor-1 expression is insufficient to prevent iron-mediated death when neurons are exposed to plasma concentrations of TBI. Chelator therapy may be beneficial for acute CNS injuries associated with loss of blood-brain barrier integrity.     

Characterization of a cytochrome b(558) ferric/cupric reductase from rabbit duodenal brush border membranes

Iron and probably also copper are absorbed by the intestine in their reduced form. A b-type cytochrome, Dcytb, has recently been cloned from mouse and has been proposed to be the corresponding reductase. However, the nature of the cytochrome and the reduction reaction remain unknown. Here we describe the isolation and functional characterization of a novel b-type cytochrome from rabbit enterocytes. The 33 kDa heme protein was solubilized from brush border membranes with Triton X-100 and purified by successive ion exchange chromatography and hydrophobic interaction chromatography. Spectroscopic analysis of the heme revealed a b(558) cytochrome. The purified hemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(II). The rate constants, k(1), for these reactions were 1.38 +/- 0.12 and 0.64 +/- 0.16 min(-1), respectively. Cytochrome b(558) may be the rabbit Dcytb homologue. A novel mechanism of how cytochrome b(558) could shuttle electrons from cytoplasmic ascorbate to luminal dehydroascorbate is proposed.     

"Lumen digestion" technique for isolation of aortic endothelial cells from heme oxygenase-1 knockout mice

Endothelial cell dysfunction plays a critical role in the pathogenesis of cardiovascular diseases. Gene targeted mutant, knockout. or transgenic mice are widely used in the laboratory investigation of these disorders. We describe a simple and reproducible "lumen digestion" technique to isolate aortic endothelial cells from mice that would be useful for researchers in endothelial cell biology. We used wild-type, homozygote, or heterozygote heme oxygenase-1 null mice from which the aorta is isolated and removed under anesthesia. After cauterizing all the branches, both ends of the aorta are cannulated using an Intramedic PE-20 tube. After flushing the aorta with phosphate-buffered saline (PBS), the lumen is repeatedly instilled (five times) with 50 microL 0.25% trypsin in PBS, incubated for 2 min, and flushed with PBS. The outflow is collected in endothelial cell media with 20% fetal bovine serum. After centrifugation, the endothelial cells in the pellet are resuspended in media and plated in a 24-well tissue culture dish. Following culture for 2 to 3 weeks, the cells demonstrate typical cobblestone appearance, stain positive for the endothelial marker CD31, and are capable of low-density lipoprotein uptake. Following challenge with oxidized lipids, heme oxygenase-1 deficient endothelial cells demonstrate increased susceptibility to cell injury.     

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Delta cells, but these subunits fail to accumulate. The respiratory defect in coa1Delta cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Delta and shy1Delta cells is enhanced when Mss51 and Cox10 are coexpressed. Shy1 has been implicated in formation of the heme a3-Cu(B) site in Cox1. The interaction between Coa1 and Cox1, and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of newly synthesized Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Delta cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.     

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain and regulates the sleep response. Moreover, PGD₂ is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD₂ significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD₂ treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD₂ may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.     

The novel heme oxygenase-like protein from Plasmodiumfalciparum converts heme to bilirubin IXα in the apicoplast

The metabolic pathways in apicoplasts of human malaria parasites are promising drug targets. The apicomplexan parasites exhibit delayed cell death when their apicoplast is impaired, but the metabolic pathways within apicoplasts are poorly understood. A nuclear-encoded heme oxygenase (HO)-like protein with an apicoplast-targeted bipartite transit peptide was identified in the Plasmodiumfalciparum genome. Purified mature recombinant PfHO protein converted heme into bilirubin IXα as confirmed by high-performance liquid chromatography. In addition, PfHO required an iron chelator such as deferoxamine for complete activity. These observations lead to the conclusion that a novel enzymatic heme degradation system is present in human malaria parasites.     

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.     

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.     

Heme oxygenase 1 overexpression increases iron fluxes in caco-2 cells

Heme oxygenase-1 is a microsomal enzyme that, when induced by stress, protects the cells from oxidative injury. Heme oxygenase-1 participates in the cleavage of the heme ring producing biliverdin, CO and ferrous Fe. The released Fe becomes part of intracellular Fe pool and can be stored in ferritin or released by an iron exporter. The mechanism by which heme enters cells is not completely understood, although it had been suggested that it might be internalized by an endocytosis process. In this study, we expressed a full-length Heme oxygenase-1 cDNA in Caco-2 cells and measured intracellular iron content, heme-iron uptake and transport and immunolocalization of heme oxygenase-1 in these cells. We found that heme oxygenasc-1 expressing cells showed increased apical heme iron uptake and transepithelial transport when compared to control cells. These results suggested that heme oxygenase-1 mediates heme iron influx and efflux in intestinal cells.     

The lipocalin α1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species

α₁-Microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of α₁-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that α₁-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of α₁-microglobulin was up-regulated by these oxidants and silencing of the α₁-microglobulin expression increased the cytosol oxidation. α₁-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to α₁-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, α₁-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that α₁-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway. [BMB Reports 2014; 47(2): 98-103]     

Transport and intracellular accumulation of vitamin C in endothelial cells: relevance to collagen synthesis

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80-100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b₅₆₁ family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g_max = 3.7 corresponding to a highly anisotropic species, and another at g_max = 3.18; these species are similar to those observed in other cytochromes of the b₅₆₁ family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g_max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b₅₆₁ of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Cellular iron depletion weakens induction of heme oxygenase-1 by cadmium

Heme oxygenase-1 is an inducible cytoprotective gene, although its induction by environmental factors is not completely understood. This study aimed to ascertain if specific nutritive factors or related compounds influence heme oxygenase-1 expression. In HCT-116 cells, cadmium increased heme oxygenase-1 enzymatic activity. This effect of cadmium was weaker in cells made iron-deficient with the iron chelator, desferrioxamine, which was associated with repression of heme oxygenase-1 protein and mRNA expression. The repression by desferrioxamine of cadmium-induced heme oxygenase-1 upregulation was reversed upon iron replenishment of the cells. Additionally, it was found that thiol antioxidants inhibited the heme oxygenase-1 upregulation caused by cadmium and also by ethacrynic acid, which each decreased intracellular glutathione as did buthionine sulfoxamine. Interestingly, cadmium and ethacrynic acid increased nuclear translocation of Nrf2 and subsequent heme oxygenase-1 expression, but buthionine sulfoxamine did not. Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin, and a superoxide scavenger (Tiron) inhibited cadmium-induced upregulation of heme oxygenase-1. Diphenyleneiodonium was the most potent and inhibited NADPH-cytochrome P450 reductase as well, whereas apocynin and Tiron did not. It is concluded that adequate amounts of iron, which at the atomic level can serve as the pivotal element of heme in NADPH oxidase, must be present in cells to permit what appears to be thiol redox-sensitive, NADPH oxidase-dependent upregulation of heme oxygenase-1. Thus, these findings are significant because they suggest that cells without adequate iron would be unable to fully express the stress gene, heme oxygenase-1, when confronted with the toxic metal, cadmium.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.