Chelator Desferrioxamine Research Articles

Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage.

Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243-1252; Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295-1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride; subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.

Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages.

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1beta (MIP-1beta), RANTES, IFN-gamma-inducible protein-10, and MIP-2 were not affected, and MIP-1alpha was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-gamma and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.

Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of iron salts

Problems caused by the presence of adventitious metals in buffers and reagents are well recognized in studies of metal-catalyzed oxidation reactions. In most cases, metal contamination leads to an increase in rate, and chelating agents are inhibitory. In the present study, however, the rate of copper-catalyzed oxidation of cysteine was found to be increased by buffer purification with Chelex resin or by addition of micromolar concentrations of the specific iron chelator desferrioxamine (DFO). These effects are attributable to inhibition of copper-catalyzed oxidation by adventitious iron. In purified buffer at pH 7.25, containing 0.4 μM copper, cysteine was oxidized at a rate of 32 μM/min. Addition of iron salts to this buffer caused a dose-related decrease in this rate, up to a maximum of 85%. A 50% decrease in rate was recorded at an iron concentration of only 11 nM. Other transition metals were without effect. Similar effects of purification or addition of DFO on the rate of cysteine oxidation were seen in Tris, glycylglycine, Mops, and Pipes buffers. Catalase decreased the rate of cysteine oxidation, but the sensitivity to iron was similar in the presence and absence of catalase. Copper-catalyzed oxidation of cysteamine and reduced glutathione was much less sensitive to inhibition by iron. Our results offer an explanation for the conflicting literature reports of the effects of chelating agents and catalase on cysteine oxidation, and emphasize the need for buffer purification or addition of DFO in studies concerned with the oxidation or cytotoxicity of this thiol. The exceptional sensitivity of copper-catalyzed cysteine oxidation to iron makes this an attractive system for monitoring the iron content of buffers, and may also have application for determining the free iron content of physiological fluids.

Endosomal and lysosomal effects of desferrioxamine: protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest.

The role of endosomal/lysosomal redox-active iron in H2O2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 microM) inhibited cell proliferation in a fluid-phase endocytosis-dependent manner. Flow cytometric analysis of cells exposed to 100 microM DFO for 24 h showed that the cell cycle was transiently interrupted at the G2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.

A phosphomimetic mutation at Ser-138 renders iron regulatory protein 1 sensitive to iron-dependent degradation.

Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs. In iron-replete cells, assembly of a cubane [4Fe-4S] cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase. Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N. M. Brown, S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein, Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1(S138E)) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1(S138E) expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1(S138E) to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1(S138A) is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.

Protective and determining factors for the overall lipid peroxidation in ultraviolet A1-irradiated fibroblasts: in vitro and in vivo investigations.

Lipid peroxidation (LPO) is one major effector mechanism by which ultraviolet (UV) A contributes to photoageing and the promotion of skin cancer. It is a fingerprint of photo-oxidative stress within the skin, and is initiated by several pathways, with different reactive oxygen species (ROS) and iron ions being involved. To elucidate factors involved in UVA1-induced LPO in human dermal fibroblasts and mouse dermis, and the role of antioxidant enzymes in protecting cells against LPO. Using a highly sensitive high-performance liquid chromatography procedure, we measured malondialdehyde (MDA), a specific metabolic tracer molecule for LPO, to determine the overall LPO produced by a given UVA1 dose in vitro and in vivo. By using the iron chelator desferrioxamine (DFO), the hydroxyl radical scavenger dimethylsulphoxide (DMSO) and fibroblasts that specifically overexpress single antioxidant enzymes, we further indirectly assessed the protective effect of manganese superoxide dismutase (MnSOD), catalase and phospholipid hydroperoxide glutathione peroxidase (PHGPx) as well as the relative importance of different ROS and the role of transitional iron for the total amount of LPO induced by a distinct UVA dose. UVA1 irradiation resulted in a time- and dose-dependent increase in MDA levels in vitro, and the in vitro results were shown to have in vivo relevance. Fibroblasts incubated with DFO or DMSO produced lower levels of MDA than controls, as did fibroblasts overexpressing MnSOD, catalase or PHGPx. The cellular iron pool and hydroxyl radicals were the most important determining factors for the total amount of MDA produced after a given UVA1 dose, and PHGPx overexpression had the greatest protective effect against LPO.

Iron chelators for the treatment of iron overload disease: relationship between structure, redox activity, and toxicity.

The success of the iron (Fe) chelator desferrioxamine (DFO) in the treatment of beta-thalassemia is limited by its lack of bioavailability. The design and characterization of synthetic alternatives to DFO has attracted much scientific interest and has led to the discovery of orally active chelators that can remove pathological Fe deposits. However, chelators that access intracellular Fe pools can be toxic by either inhibiting Fe-containing enzymes or promoting Fe-mediated free radical damage. Interestingly, toxicity does not necessarily correlate with Fe-binding affinity or with chelation efficacy, suggesting that other factors may promote the cytopathic effects of chelators. In this review, we discuss the interactions of chelators and their Fe complexes with biomolecules that can lead to toxicity and tissue damage.

P-Terphenyl curtisians protect cultured neuronal cells against glutamate neurotoxicity via iron chelation.

The hyperactivity of ionotropic glutamate receptors has been implicated in the development of the neuronal cell death seen in many neurodegenerative processes including ischemic stroke, traumatic brain injury, and epilepsy. Thus neuronal protection against glutamate-induced neurotoxicity is considered as an appropriate therapeutic strategy for preventing and treating neurodegenerative diseases. Whilst searching for blockers of glutamate-induced toxicity in mouse cortical cells, we isolated p-terphenyl curtisians A - D from the mushroom Paxillus curtisii. Curtisians protected cortical neurons from glutamate-induced toxicity in a dose-dependent manner. Among the glutamate receptor subtypes, curtisians were found to block NMDA receptor-mediated but not AMPA/kainate-mediated cell death. In addition, we found that curtisians exhibited potent antioxidative activity against iron-mediated oxidative damage which was generated by H2O2 neurotoxocity and lipid peroxidation, but no activity was detected in the superoxide, DPPH and ABTS radical scavenging systems, and in protection of N18-RE-105 cells subjected to glutamate-induced glutathione depletion. This effect was likely due to the iron chelating properties of curtisians. The iron chelation ability of curtisians was then further investigated on DNA single strand breakage (SSB) induced by the addition of iron and H2O2, and curtisians prevented DNA SSB like the iron chelator desferrioxamine. These results suggest that the neuroprotective action of curtisians is dependent on their ability to chelate iron as well as to block the NMDA receptor, and that in this context curtisians may be useful as neuroprotective agents against neurological disorders which result in neuronal cell death.

Iron withdrawal strategies fail to prevent the growth of SiHa-induced tumors in mice

Objective Cervical carcinoma is a human papillomavirus (HPV)-associated cancer for which treatment options still mainly rely on surgical procedures, with or without adjuvant radiotherapy and chemotherapy. We have previously shown that the chemically unrelated iron chelators desferrioxamine and deferiprone inhibit the growth and induce the apoptosis of HPV-positive cervical carcinoma cell lines, suggesting that iron chelators may represent a potential therapeutic approach for the treatment of cervical carcinoma. The present study was designed to investigate the effect of iron deprivation on the growth of human cervical carcinoma xenografts in athymic nude mice. Methods Nude mice (nu/nu) of BALB/c background were treated with iron chelators [desferrioxamine (DFO), deferiprone (L1), or starch-DFO conjugate] or were fed with an iron-poor diet 6 weeks prior to subcutaneous injection of Si-Ha cells. These treatments were continued for 5 weeks after injection of the tumor cells. Results Treatment with the maximum tolerated doses of DFO, L1, or starch-DFO conjugate induced no significant iron deprivation in non-iron-overloaded mice, while an iron-poor diet led to a dramatic decrease in serum iron, transferrin iron saturation, and ferritin levels. However, neither iron chelators nor an iron-poor diet could significantly inhibit tumor growth. Conclusion Despite a potent antitumor effect in vitro, iron chelators fail to prevent the growth of cervical carcinoma xenografts in mice. On the basis of these results, clinical trials with iron chelators in patients with cervical carcinoma appear inappropriate.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells.

The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)(29) dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

Bach1 Functions as a Hypoxia-inducible Repressor for the Heme Oxygenase-1 Gene in Human Cells

Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron, carbon monoxide, and bilirubin IXalpha. HO-1 is induced by its substrate heme and various environmental factors, which represents a protective response against oxidative stresses. Here we show that hypoxia represses HO-1 expression in three human cell types but induces it in rat, bovine, and monkey cells, indicating the inter-species difference in the hypoxic regulation of HO-1 expression. The hypoxia-mediated repression of HO-1 expression is consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to interferon-gamma or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by CoCl(2) is associated with reduced expression of Bach1 mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1 gene that is required for repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1 functions as a hypoxia-inducible repressor for the HO-1 gene, thereby contributing to fine-tuning of oxygen homeostasis in human cells.

Lipid Peroxidation-Independent Mechanisms of Vitamin E-Mediated Protection Against Cyclosporine A-Induced Hepatocellular Toxicity and Apoptosis

Cyclosporine A (CsA) produces oxidative stress and apoptosis in rat hepatocytes, but it is not known whether membrane lipid peroxidation plays a role in CsA toxicity. The objective of the present study was to determine whether iron-catalyzed hydroxyl or membrane alkoxyl radical formation is causally involved in the prooxidative cell injury and apoptosis. As previously reported, cultured rat hepatocytes exposed to CsA exhibited concentration-dependent signs of apoptotic cell injury, including chromatin condensation and fragmentation, increased caspase-3 activity, and release of cytosolic lactate dehydrogenase. Addition of the ferric iron chelator desferrioxamine or the novel potent lipophilic iron chelator CGP72670 did not protect against CsA-induced cell death, indicating that iron-catalyzed lipid peroxidation is unlikely to be involved in CsA-mediated apoptosis. In contrast, the classical chain-breaking lipid peroxidation inhibitor α-tocopherol was able to rescue cells from CsA-induced cytotoxicity and apoptosis. α-Tocopherol not only effectively inhibited the production of thiobarbituric acid-reactive substances and the formation of reactive oxygen species, but it also prevented proteins from being oxidized and forming mixed disulfides. Furthermore, α-tocopherol inhibited the cellular uptake of extracellular calcium (45 Ca 2+) into cells, similar to the reducing agent dithiothreitol. By decreasing the extracellular calcium concentrations or by adding calcium channel blockers (diltiazem, nifedipine) or a cell-permeable calcium chelator Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tertraacetic acid (BAPTA), both CsA-induced caspase-3 activation and apoptosis were inhibited, indicating a pivotal role of Ca 2+ in mediating CsA-induced cell injury. These results suggest that α-tocopherol protects from CsA-mediated apoptosis and cytotoxictiy by preventing the oxidation of redox-sensitive Ca 2+ -ATPase. Thus, it is the attenuation of increased calcium influx and, hence, the inhibition of caspase-3 activation, rather than the downstream inhibition of lipid peroxidation, that is a key mechanism of the protection provided by α-tocopherol against CsA-induced cell injury.

Terapia quelante oral com deferiprona em pacientes com sobrecarga de ferro

Apesar da introdução da quelação parenteral de ferro com desferoxamina há mais de trinta anos, 50% dos pacientes com talassemia major morrem antes dos 35 anos de idade, predominantemente por insuficiência cardíaca secundária a sobrecarga de ferro. Embora a desferrioxamina, possa reduzir ou estabilizar o acúmulo de ferro no corpo, um grande número de pacientes não recebe terapia adequada com este quelante devido principalmente à intolerância a um regime que requer administração parenteral prolongada, cinco a sete dias por semana. Para esses pacientes, a deferiprona, um quelante oralmente ativo, surge com uma alternativa de tratamento para o controle da sobrecarga de ferro. Um grande número de estudos clínicos demonstraram a segurança e eficácia da deferiprona. Estima-se que mais de 6 mil pacientes com sobrecarga de ferro já tenham sido tratados com este quelante, com alguns pacientes tomando a droga por mais de dez anos. A quantidade de ferro excretada induzida pela deferiprona é diretamente influenciada pela dose da droga e pelo grau de sobrecarga de ferro do paciente. Recentemente, demonstrou-se que desferrioxamina e deferiprona exibem capacidades quelantes diversas e que, usados simultaneamente ou numa terapia sequencial, promovem uma excreção de ferro adicional ou sinérgica com rápida redução na carga de ferro do corpo. Pela primeira vez é possível considerar esquemas de quelação individualizados baseados nas necessidades individuais de cada paciente.

A novel trivalent cation chelator Feralex dissociates binding of aluminum and iron associated with hyperphosphorylated τ of Alzheimer’s disease

Aluminum (Al(III)) and iron (Fe(III)) are reported to accumulate in neurofibrillary tangles of the Alzheimer’s disease (AD) brain. In these lesions Al (III) and Fe (III) bind with hyperphosphorylated τ (PHFτ), the major constituent of the lesions, and induce its aggregation. It is thought that inhibition and dissociation of such Al (III)/Fe (III) binding associated with PHFτ could slow or halt the τ-related neurofibrillary degeneration in patients with AD. A study, using a previously developed in vitro system in which Al (III) and Fe (III) interact with PHFτ on AD brain sections and on immunoblot membranes showed that the potent Al (III)/Fe (III) chelator desferrioxamine elicited Al (III) chelation when subjected to autoclave heating. Here, the ability of a recently developed chemical chelator Feralex-G to remove PHFτ-bound Al (III)/Fe (III), using reaction conditions at 37 °C, was examined and compared with that of desferrioxamine. Chelation of Fe(III) was achieved by both compounds with no discernible difference in their chelating ability. In contrast, in the present system, the two chelators gave a different Al (III) chelation response. When incubated at 37 °C, desferrioxamine failed to attain notable Al (III) chelation, while Feralex-G displayed efficient Al (III) chelation. Thus, when considering competitive Al (III) removal from brain PHFτ, Feralex-G is a stronger chelator for Al(III) than desferrioxamine. The efficient Al (III) chelation attainable by Feralex-G adds weight to its potential clinical usefulness as a medicine in the aluminum/iron chelation therapy for patients with AD.

Role of Iron and Surface Free Radical Activity of Silica in the Induction of Morphological Transformation of Syrian Hamster Embryo Cells

Using various surface modified silica dusts we previously showed that the samples able to generate reactive oxygen species (ROS) induced transformation of Syrian hamster embryo (SHE) cells. The purpose of the present study was to determine in what measure trace iron on the surface of silica particles is involved in the cellular effects, through the formation of ROS. Min-U-Sil 5 quartz (Q) and an amorphous diatomite earth (DE), as well as the same samples pretreated with the Fe chelators ferrozine (fz, 3 mM) and desferrioxamine (dfx, 0.1 mM), were studied for: (i) morphological transformation; and (ii) aberrant mitotic division of SHE cells, which we reported as one of the possible mechanisms of silica-induced cell transformation. In order to determine the nature of the ROS involved in these effects, the antioxidants superoxide dismutase, catalase and mannitol were added to the treated cells. On a per weight basis, DE appeared more cytotoxic and slightly more transforming than Q. The suspensions of Q and DE preincubated in Fe chelators were significantly less transforming and, in particular Q particles, had reduced potency in inducing abnormal mitoses than the untreated samples. Although the amount of Fe mobilized from the silica particles by dfx was greater than that of Fe(II) mobilized by fz, a decrease in transforming potency was largely achieved after pretreatment of particles with fz. In the presence of antioxidants, both the transformation and mitotic aberration frequencies were significantly decreased. The results show that the iron associated with the surface of particles is implicated in the generation of ROS and is partly responsible for the transforming potency of silicas. This study confirms the role of free radical activity in the induction of cell transformation in vitro, suggesting that oxidative stress is one of the mechanisms of silica carcinogenesis.

Ferric Ions Are Essential for the Biological Activity of the Hormone Glycine-extended Gastrin

Amidated and nonamidated gastrins elicit different biological effects via distinct receptors in different tissues. Amidated gastrin 17 stimulates gastric acid secretion and the development of gastric carcinoids, whereas glycine-extended gastrin 17 stimulates proliferation of the colonic mucosa and the development of colorectal cancers. Because glycine-extended gastrin 17 binds two ferric ions with high affinity (Baldwin, G. S., Curtain, C. C., and Sawyer, W. H. (2001) Biochemistry 40, 10741-10746), we have investigated the identity of the iron ligands and the role of ferric ions in biological activity. Here we report the solution structure of glycine-extended gastrin 17, determined by NMR spectroscopy. The spectral changes observed upon the addition of ferric ions revealed that Glu(7) acted as a ligand at the first ferric binding site, and that Glu(8) and Glu(9) acted as ligands at the second ferric ion binding site. Fluorescence quenching experiments confirmed that a GglyE7A mutant bound only one ferric ion. The inability of this mutant to stimulate proliferation or migration in the IMGE-5 cell line and the observation that the iron chelator desferrioxamine selectively blocked the effects of glycine-extended gastrin 17 indicated that binding of a ferric ion to Glu(7) was essential for biological activity. This is the first report of an essential role for a metal ion in the action of a hormone.

In vitro activation of dibromoacetonitrile to cyanide: role of xanthine oxidase.

Dibromoacetonitrile (DBAN) is a disinfection byproduct of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides evidence for DBAN activation to cyanide (CN(-)) by the hypoxanthine (HX)/xanthine oxidase (XO)/iron (Fe) system in vitro. Optimum conditions for the oxidation of DBAN to CN(-)were characterized. Addition of the sulfhydryl compounds glutathione, N-acetyl- L-cysteine or dithiothreitol significantly enhanced the rate of CN(-)release. A high positive correlation existed between hydroxyl free radical ((*)OH) generation and CN(-) formation. Addition of the (*)OH scavengers mannitol or dimethylthiourea to the reaction mixtures resulted in a significant decrease in the rate of DBAN oxidation. Addition of the antioxidant enzymes catalase or superoxide dismutase resulted in a significant decrease in the rate of DBAN oxidation. The iron chelator desferrioxamine significantly decreased CN(-) formation. The maximum velocity (V(max)) and Michaelis-Menten constant (K(m)) of the reaction were assessed. Allopurinol competitively inhibited the reaction, while folic acid uncompetitively inhibited the reaction. In conclusion, (*)OH generated by the HX/XO/Fe system are implicated in DBAN oxidation. The present results represent a novel pathway for DBAN activation and might be important in explaining DBAN-induced toxicity.

Comparison of effects of oral deferiprone and subcutaneous desferrioxamine on myocardial iron concentrations and ventricular function in beta-thalassaemia

Despite the introduction of the parenteral iron chelator desferrioxamine more than 30 years ago, 50% of patients with thalassaemia major die before the age of 35 years, predominantly from iron-induced heart failure. The only alternative treatment is oral deferiprone, but its long-term efficacy on myocardial iron concentrations is unknown. We compared myocardial iron content and cardiac function in 15 patients receiving long-term deferiprone treatment with 30 matched thalassaemia major controls who were on long-term treatment with desferrioxamine. Myocardial iron concentrations were measured by a new magnetic-resonance T2* technique, which shows values inversely related to tissue iron concentration. The deferiprone group had significantly less myocardial iron (median 34.0 ms vs 11.4 ms, p=0.02) and higher ejection fractions (mean 70% [SD 6.5] vs 63% [6.9], p=0.004) than the desferrioxamine controls. Excess myocardial iron (T2* <20 ms) was less common in the deferiprone group than in the desferrioxamine controls (four [27%] vs 20 [67%], p=0.025), as was severe (T2* <10 ms) iron overload (one [7%] vs 11 [37%], p=0.04). The odds ratio for excess myocardial iron in the desferrioxamine controls versus the deferiprone group was 5.5 (95% CI 1.2-28.8). Conventional chelation treatment with subcutaneous desferrioxamine does not prevent excess cardiac iron deposition in two-thirds of patients with thalassaemia major, placing them at risk of heart failure and its complications. Oral deferiprone is more effective than desferrioxamine in removal of myocardial iron.

15-Deoxy-delta12,14-prostaglandin J2 induces apoptosis of a thyroid papillary cancer cell line (CG3 cells) through increasing intracellular iron and oxidative stress.

Treatment of carcinoma cell lines with 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-gamma, has been reported to induce apoptosis and/or inhibit proliferation. In this study, we investigated the cytotoxic effect and the action mechanisms of 15d-PGJ2 in a thyroid papillary cancer cell line, CG3. The results indicate that 15d-PGJ2 caused cytotoxicity and increased the amount of intracellular reactive oxygen species (ROS) in these cells. Mitochondrial oxidative phosphorylation inhibitors (carbonyl cyanide m-chloro-phenylhydrazone, oligomycin, cyclosporin A and rotenone), NADPH oxidase inhibitor (diphenyleneiodonium), xanthine oxidase inhibitor (allopurinol) and NO synthase inhibitor (N-monomethyl-L-arginine acetate) did not reduce the generation of ROS. However, catalase, N-acetyl-cysteine and the iron chelator desferri-oxamine decreased the intracellular ROS of 15d-PGJ2-treated CG3 cells. Furthermore, 15d-PGJ2 enhanced the accumulation of iron in the CG3 cells. These data suggest that 15d-PGJ2 induces the generation of ROS by enhancing the accumulation of intracellular iron and that the increased oxidative stress may cause apoptosis of CG3 cells.

A novel iron chelator that does not induce HIF-1 activity

Human skin cells (FEK-4) have been shown to undergo an immediate and transient release of low molecular mass iron (LMrFe) when subjected to UVA (320–380 nm) irradiation and this iron may act as a pro-oxidant and increase tissue injury. In order to decrease this transient release of LMrFe, cells were treated with the iron chelators desferrioxamine (DFO) and salicylaldehyde isonicotinoyl hydrazone (SIH). However, although the iron pool decreased, an increase in the DNA binding activity of the hypoxia inducible factor-1 (HIF-1) was observed when DFO and SIH were administered to normal growing FEK-4 cells. The induction of HIF-1 activates the expression of several genes associated with hypoxia and iron homeostasis. HIF-1 induction has also been associated with protection against certain forms of oxidative stress. Therefore, it is difficult to use a conventional HIF-1 activating iron chelator (such as DFO) for mechanistic studies of protection against iron-mediated oxidative stress since any protection observed could be a consequence of either the chelation of LMrFe or the induction of protective genes associated with the hypoxic response. In order to observe the effect of iron chelation on cell function without the induction of hypoxia responsive genes, cells were treated with the novel iron chelator N-(2-hydroxybenzyl)-L-serine (HBSer). Although this compound is an effective iron chelator under the conditions employed in this experiment, it does have a lower iron-binding constant than either DFO or SIH. This may be the major determinant of the observation that the compound does not induce HIF-1 binding or activate HIF-1 responsive transcriptional promoters.