Chelatable Iron Levels Research Articles

β-catenin-IRP2-primed iron availability to mitochondrial metabolism is druggable for active β-catenin-mediated cancer

BackgroundAlthough β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment.MethodsHigh-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers.ResultsIron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation.Conclusionsβ-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer.

Lack of glyoxylate shunt dysregulates iron homeostasis in Pseudomonas aeruginosa

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5' RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

Metal‐specific control of gene expression mediated by Bradyrhizobium japonicumMur and Escherichia coliFur is determined by the cellular context

Bradyrhizobium japonicum Mur and Escherichia coli Fur are manganese- and iron-responsive transcriptional regulators, respectively, that belong to the same protein family. Here, we show that neither Mur nor Fur discriminate between Fe(2+) and Mn(2+) in vitro nor is there a metal preference for conferral of DNA-binding activity on the purified proteins. When expressed in E. coli, B. japonicum Mur responded to iron, but not manganese, as determined by in vivo promoter occupancy and transcriptional repression activity. Moreover, E. coli Fur activity was manganese-dependent in B. japonicum. Total and chelatable iron levels were higher in E. coli than in B. japonicum under identical growth conditions, and Mur responded to iron in a B. japonicum iron export mutant that accumulated high levels of the metal. However, elevated manganese content in E. coli did not confer activity on Fur or Mur, suggesting a regulatory pool of manganese in B. japonicum that is absent in E. coli. We conclude that the metal selectivity of Mur and Fur depends on the cellular context in which they function, not on intrinsic properties of the proteins. Also, the novel iron sensing mechanism found in the rhizobia may be an evolutionary adaptation to the cellular manganese status.

Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells.

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]4 cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]4 cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]4 cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with (•)NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged (•)NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief (•)NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1-2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief (•)NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of (•)NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of (•)NO.

Iron-metabolic function and potential antibacterial role of Hepcidin and its correlated genes (Ferroportin 1 and Transferrin Receptor) in Turbot (Scophthalmus maximus)

Antimicrobial peptide plays an important role in fish immunity. The small molecular antimicrobial peptide Hepcidin in turbot was studied and reported in this paper. The Ferroportin 1 (FPN1) and Transferrin Receptor (TFR) genes, which are related to Hepcidin, were cloned in turbot. The characteristics of Hepcidin and its related genes were studied, including an analysis of the expression patterns and cloning of the Hepcidin promoter, the relationship between Hepcidin and NF-κB and the regulation of iron-metabolism. The results showed that the promoter of SmHepcidin contains the binding sites of NF-κB, and NF-κB may directly or indirectly receive feedback signals from SmHepcidin. In the liver, spleen and kidney, in which there was an increased SmHepcidin expression level, SmFPN1 dramatically decreased and SmTFR was also either decreased or exhibited no obvious change after bacterial/viral infection and an injection of exogenous Hepcidin protein. RNAi experiments in turbot kidney cells confirmed the expression changes of these gene patterns. Furthermore, the administration of exogenous Hepcidin protein, which regulates the level of chelatable iron in cells, further confirmed the function of Hepcidin in iron metabolism. It is speculated that the rapidly increased expression of SmHepcidin may induce changes in the expression of related genes, and that the in vivo chelatable iron concentration which participates in the antibacterial process was also changed when exogenous pathogens are present in turbot. It is suggested that SmHepcidin plays a defensive role against pathogenic infection.

ROLE OF CHELATABLE IRON VERSUS MYOGLOBIN IN OXIDATIVE STRESS AFTER CRUSH TRAUMA

ROLE OF CHELATABLE IRON VERSUS MYOGLOBIN IN OXIDATIVE STRESS AFTER CRUSH TRAUMA

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δ yfh1 sensitivity to menadione. Finally, a Δ sod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

Heme oxygenase-1: molecular mechanisms of gene expression in oxygen-related stress.

Disturbances of intracellular redox equilibrium may alter eukaryotic gene expression patterns in the manifestation of an adaptive stress response. The inducible heme oxygenase-1 gene, ho-1, responds dramatically to changes in cellular redox potential provoked by multiple agents (oxidants, xenobiotics, reactive oxygen species, nitric oxide, and ultraviolet-A radiation) as well as deviations in oxygen tension in excess or deficit of normal physiological levels. This dual response to hyperoxic and hypoxic states renders ho-1 an intriguing model system for studying oxygen-regulated gene expression. The complexation or depletion of reduced glutathione apparently represents an underlying mechanism by which oxidants trigger the response. Chelatable iron levels also influence the induction of ho-1 as evidenced by the inhibitory effects of iron-chelating compounds. Redox-sensitive protein kinase cascades (e.g., mitogen-activated protein kinases) participate in ho-1 regulation. Recent progress in understanding ho-1 transcription has identified two distal enhancer regions (E1, E2) in the mouse ho-1 gene that mediate the response to many inducing conditions. This review will examine the potential roles of iron, glutathione, and reactive oxygen species in the upstream events leading to ho-1 activation following oxygen related stress.

The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload

Lipid peroxidation and organelle dysfunction are important factors in hepatic iron toxicity. The form of the intracellular iron responsible for these abnormalities is still unknown. In order to investigate the iron species inducing cell injury, the level of chelatable iron in the liver mitochondria isolated from rats fed a 2.5% carbonyl iron diet for 12 weeks was measured by EPR spectroscopy. The presence of lipid peroxidation products and the energy transducing capacility of the mitochondrial inner membrane was evaluated in parallel. The total iron concentration in the liver mitochondria from iron fed rats progressively increased up to 6 weeks, almost reaching a steady-state. By contrast the level of chelatable iron in mitochondrial fraction transiently increased at about 3–6 weeks of treatment. The induction of lipid peroxidation and a large decrease of ATP occurred at the same time. The enhancement of the energy dissipating calcium cycling was in parallel revealed by studying the mitochondria membrane potential. These results gave experimental evidence to the proposal that the chelatable iron level plays a critical role in initiating organelle dysfunction, at least in this experimental model.

Iron Acquired from Transferrin by K562 Cells Is Delivered into a Cytoplasmic Pool of Chelatable Iron(II)

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37 degrees C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (< 25 degrees C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2'-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 microM cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2'-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55Fe, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-55Fe complexes into organic solvent. Virtually all of the accumulated 55Fe in the chelatable pool could be complexed by iron(II) chelators. The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 microM, and its mean transit time through the chelatable pool was 1-2 h. We conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II). The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.

Circulating levels of Chelatable Iron as a Possible Stimulator of free Radical Activity: A Preliminary study in Pulmonary Tuberculosis

Circulating levels of Chelatable Iron as a Possible Stimulator of free Radical Activity: A Preliminary study in Pulmonary Tuberculosis

Mechanism of Decrease in Transferrin Receptor Synthesis by Interferon-α Treated Human Lymphoblastoid Cells

To clarify the mechanism of antiproliferative action of interferon-α (IFN-α) in hematological malignancy, we examined the transferrin receptor system in the lymphoblastoid cell line, Daudi cells treated with IFN-α. When cells were cultured with 10(4)U/ml of IFN-α, the number of surface transferrin receptors was decreased to 60% of that seen in the control culture. This decrease was not neutralized by co-incubation with the iron chelator, desferrioxamine (10-200 μM), suggesting that the change in the level of chelatable iron did not account for the decrease in transferrin receptor numbers. When determined by metabolic labeling using (35)S-methionine, IFN-α markedly decreased the rate of transferrin receptor biosynthesis. Uptake of iron and the cellular ferritin content also decreased by 50% when incubated with 10(4)U/ml of IFN-α. These data indicate that IFN-α inhibits transferrin receptor biosynthesis in an iron-independent fashion and the subsequent cellular iron-deficiency state may play a role in the antiproliferative action of IFN-α.

Translational Regulation of Ferritin Synthesis by Iron

This review starts with a description of certain features of mammalian ferritins and their DNA and RNA structures relevant to translational control of ferritin synthesis. Although the amino acid sequences of the two ferritin subunits (H and L) diverge in about 50% of the coding region, their five alpha-helices and the exon sizes of their genes are compatible with the proposition that they diverged from a single ancestral gene. Of particular note is their long 5'-untranslated regions (5'UTRs) which include a 28-nucleotide sequence almost completely identical in the H- and L-subunits of a range of species. This motif near the cap region of the 5'-UTR, which forms a specific stem-loop structure, provides for regulation of the translation of H- and L-ferritin mRNAs. When intracellular levels of chelatable iron are not in excess, a large reserve of H- and L-mRNAs is present in the cell sap, restrained from translation by a protein with an Mr of about 90-100,000 which binds to the stem-loop structure. When excess iron floods the cytosol, this protein/RNA complex appears to dissociate and the 40S ribosome subunit is now able to initiate ferritin protein synthesis so that the dormant mRNAs become active and are transferred to the polyribosomes. The mechanism whereby the binding protein is regulated in response to iron is currently under investigation. The regulatory protein occurs in the cell sap and is present in several interchangeable forms which appear to differ in the redox state of specific sulphydryls within the protein. Under some circumstances, the abundance of these forms appears to be altered by intracellular iron status. It is unclear how iron influences binding of the regulatory protein to ferritin mRNA. Some investigators consider that iron binds in the form of heme to the regulatory protein, for which they offer in vitro evidence. We have examined the role of heme versus inorganic chelatable iron in the regulation of ferritin and heme oxygenase synthesis in rat fibroblasts and hepatoma cells. By manipulating the flow of iron between the intracellular chelatable iron and heme iron pools we have concluded that chelatable iron can act as a regulator of ferritin synthesis in a manner which is independent of heme formation. This conclusion does not exclude a role for heme in some specialized cell types.

Measurement by HPLC of desferrioxamine-available iron in rabbit kidneys to assess the effect of ischaemia on the distribution of iron within the total pool.

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10 microMolar region could be accurately and reproducibly measured using this technique. The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0 degrees C in hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.

Intracellular metabolism of biogenic amines in paraneurons.

Serotonin is one of the representative biogenic amines produced from tryptophan in the gastrointestinal tract as well as in the brain. The pineal gland also synthesizes serotonin, but as an intermediate in the biosynthesis of melatonin. The first enzymic step in the biosynthesis of serotonin is the hydroxylation of L-tryptophan catalyzed by tryptophan 5-monooxygenase. The 5-hydroxy-L-tryptophan (L-5HTP) formed is then decarboxylated to serotonin. Since the second step is catalyzed by non-specific aromatic L-amino acid decarboxylase, whether or not a given cell has the capacity to synthesize serotonin depends on the existence of tryptophan monooxygenase, and regulation of the serotonin biosynthesis is achieved mainly by modulating the activity of the first step enzyme. The activity of tryptophan monooxygenase was detected in extracts of the mouse stomach and intestines, although this activity was as low as approximately 1/20 of that in similar extract of the mouse brainstem. The upper small intestine and colon had a higher level of activity than other parts, and in the upper small intestine the enzyme was found to reside primarily in enterochromaffin cells of the mucosa. The intestinal tryptophan monooxygenase shared a common antigenic character with the enzyme from murine mastocytoma and was immunologically different from the brain enzyme. The enzymic properties were also similar to those of mastocytoma tryptophan monooxygenase which requires loosely-bound functional iron (Fe2+) for the activity. It seemed likely that the activity of tryptophan monooxygenase in paraneurons such as mastocytoma and enterochromaffin cells depends on available ferrous iron, and is therefore regulated, at least in part, by the cytosolic level of chelatable iron.

Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells.

Acute administration of iron to rats has been previously shown to induce liver ferritin synthesis by increasing the translation of inactive cytoplasmic ferritin mRNAs for both heavy (H) and light (L) subunits by mobilizing them onto polyribosomes. In this report rat hepatoma cells in culture are used to explore the relationship of this response to intracellular iron levels. After adding iron as ferric ammonium citrate to the medium, latent ferritin H- and L-mRNAs were extensively transferred to polyribosomes, accompanied by increased uptake of [35S]methionine into ferritin protein. Because total cellular levels of L- and H-mRNA were not significantly changed by exposure to iron, the increased ferritin mRNAs on polyribosomes most probably come from an inactive cytoplasmic pool, consistent with the inability of actinomycin-D and of cordycepin to inhibit iron-induced ferritin synthesis. When deferoxamine mesylate, an intracellular iron chelator, was added after the addition of iron to the medium, ferritin mRNA on the polyribosomes was reduced, while the free messenger pool increased, and ferritin synthesis diminished. In contrast, the extracellular iron chelator diethylenetriaminepentaacetic acid failed to inhibit the induction of ferritin protein synthesis. Addition of iron in the form of hemin also caused translocation of mRNA to polyribosomes, a response that could be similarly quenched by deferoxamine. Because hemin does not release chelatable iron extracellularly, we conclude that the level of chelatable iron within the cell has a regulatory role in ferritin synthesis through redistribution of the messenger RNAs between the free mRNA pool and the polyribosomes.

Hemin, chelatable iron, and the regulation of transferrin receptor biosynthesis.

We have examined the mechanism by which hemin regulates the expression of the human transferrin receptor. Previous work led to the suggestion that the regulatory signal is provided by heme (Ward J. H., Jordan, I., Kushner, J. P., and Kaplan, J. (1984) J. Biol. Chem. 259, 13235-13240). We demonstrated that hemin regulates the expression of the receptor via alterations in the rate of receptor biosynthesis. However, this effect can be completely abolished by addition of desferrioxamine, an intracellular iron chelator. Competition curves demonstrate that desferrioxamine and hemin affect the same intracellular iron pool. Since the chelator cannot remove iron from heme, we propose that hemin acts simply by delivering iron to a chelatable iron pool and that levels of chelatable iron provide the regulatory signal for expression of the transferrin receptor gene.

Long Term Results of Venesection Therapy in Idiopathic Haemochromatosis

Observations on the clinical effects of venesection therapy in 85 treated, as compared with 26 untreated, patients with idiopathic haemochromatosis showed decreased pigmentation and hepatomegaly together with a return to normal tests of liver function in half the patients who had abnormal tests at presentation. Control improved in 28 per cent of those patients with diabetes mellitus, although some patients developed it during the period of observation, despite venesection. Portal hypertension, testicular atrophy and arthropathy were not improved. In only 12 patients was there sufficient reaccumulation of iron after the initial course of venesection to merit further treatment. Rates of iron accumulation in these patients varied between 1-4 mg and 4-8 mg per day and chelatable iron levels were noted to be inappropriately high in relation to body iron stores during the early stages of the reaccumulation period. Life table data shows that the percentage survival five and ten years after diagnosis was 66 and 32 per cent respectively for the treated patients, and 18 and 6 per cent respectively for the untreated patients, both statistically highly significant differences (p less than 0-01). Possible clinical differences such as age of presentation, the presence of diabetes mellitus, cirrhosis, clinical hepatic failure and hepatoma between the treated and untreated groups that might otherwise have weighted survival in favour of the treated group were corrected by the use of covariant analysis. This gave mean log survival values of 4-15 and 2-88 for the treated and untreated patients respectively, equivalent to 63-4 months and 17-8 months, a highly significant difference (p less than 0-01). Ten patients, all of whom had cirrhosis at the time of diagnosis, died of malignant hepatoma between three and 15 years after completing venesection therapy. There was also a high rate of death from neoplasms in a variety of other sites--22 per cent in the venesected group, strikingly higher than that rate predicted for a similarly aged population using national cancer mortality rates.