Hepatoprotective role of nitric oxide in an experimental model of chronic iron overload.

Chronic iron overload (CIO) enhances nitric oxide (*NO) production in the liver, which may represent a hepatoprotective mechanism against CIO toxicity. In order to test this hypothesis, the influence of CIO (diet enriched with 3% (wt/wt) carbonyl-iron for 8 weeks) in the absence or presence of the (*)NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on NOS activity, extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation was studied, in relation to ferritin expression and liver morphology. CIO increased liver NOS activity, ERK1/2 phosphorylation, NF-kappaB DNA binding, and ferritin expression, with normal liver histology. These changes were suppressed by combined CIO and L-NAME treatment, with the resulting inflammatory response of the liver. It is concluded that (*)NO response induced by CIO represents a molecular mechanism affording protection against iron toxicity, which is related to both the activation of the ERK/NF-kappaB pathway involving inducible NOS expression and ferritin upregulation, changes that may be interrelated.     

Potentiation of iron accumulation in cardiac myocytes during the treatment of iron overload in gerbils with the hydroxypyridinone iron chelator CP94

Gerbils administered iron dextran are the only animal species which have been shown to develop hemochromatosis of the liver and heart in the same manner as transfusion dependent homozygous thalassemics. The iron chelating hydroxypyridinone, CP94, has been administered prophylactically to iron overloaded gerbils in a dosing regime which favors the formation of bidentate chelated iron, to examine the possibility of additional toxicity being caused to the liver and heart by the bidentate chelated iron complex. Hepatic iron accumulation was inhibited by CP94 administration for up to 6 weeks, but not after 20 weeks. Iron accumulation in the heart was increased significantly after 6 and 20 weeks of chelator treatment. Pathological changes in both organs were markedly more severe after 20 weeks in chelator treated animals. There was a higher incidence of cardiofibrosis and more extensive liver fibrosis in iron overloaded, chelator treated animals after 20 weeks.     

Post-transfusional iron overload in the haemoglobinopathies

In this report, we review the recent advances in evaluation and treatment of transfusional iron overload (IO). Results of the French thalassaemia registry are described. According to the disease, thalassaemia major or sickle cell anaemia, mechanisms and toxicity of iron overload, knowledge about IO long-term outcome and chelation treatment results, respective value of IO markers, differ. The recent tools evaluating organ specific IO and the diversification of iron chelator agents make possible to individualize chelation therapy in clinical practice. The severity of IO and the level of transfusional iron intake, the preferential localization of IO (heart/liver) as well as the tolerance and adherence profiles of the patient can now be taken into account. Introduction of cardiac magnetic resonance imaging for the quantification of myocardial iron and use of oral chelators have already been reported as decreasing the cardiac mortality rate related to IO in thalassaemia major patients. Long-term observation of patients under oral chelators will show if morbidity is also improving via a more continuous control of toxic iron and/or a better accessibility to cellular iron pools.     

A 31P-NMR study on the energy state of rat liver in an experimental model of chronic dietary iron overload

31P-NMR spectroscopy of rat liver perchloric acid extracts was utilized to assess the hepatic energy state in an experimental model of chronic dietary iron overload. Oral administration of iron for a period of 65 days that induces a steady ten-fold increase in hepatic iron concentration causes a significant decrease in the hepatic ATP level not associated with appreciable modifications of ADP and Pi levels. The phosphorylation ratio appears on the average decreased. The values of the energy state parameters revert to the normal if the concentration of iron in the liver is reversed below the critical level upon withdrawal of iron treatment after 45 days for a period of 20 days. The implication of these energy modifications for the pathogenesis of cell damage in the siderosis is discussed.     

Serum ferritin in iron overload

Even with uncomplicated iron overload, serum ferritin which can be identified in the circulating blood by sensitive immunochemical methods has a direct and quantitative correlation to the iron stored in the organism. The relation of stored iron and serum ferritin is not linear, but has an exponential character. The diagnostic function of serum ferritin as an indicator of stored iron, however, is virtually not influenced by it. The indications listed in Tab. 3 can be demarcated for diagnostic application in cases of iron overload. Hitherto, the molecular microheterogenicity of serum ferritin has exercised no essential impact on its diagnostic application. High ferritin concentrations may arise in the circulating blood by a number of disease processes listed in Tab. 4, without the simultaneous existence of a respective iron overload of the tissue. These correlations have to be observed in the diagnostic application of determining serum ferritin as well as in methodical possibilities of fault (high dose hook effect), thus limiting the use of serum ferritin as an indicator of stored iron both in case of iron overload and iron deficiency. As in all isolated laboratory investigations, all other clinical and chemical laboratory information available about the individual patient has to be taken into account in each case for interpreting the serum ferritin concentration.     

The effect of iron overload on the mitochondrial porphyrin level in the hexachlorobenzene induced experimental porphyria

Liver mitochondria isolated from rats treated with hexachlorobenzene plus iron, present a lower content of total porphyrin in respect to that of mitochondria from rats fed hexachlorobenzene alone. The in vitro mitochondrial porphyrin accumulation processes have been studied in mitochondria from iron loaded rats. It has been found that under these conditions the active porphyrin uptake process, which is driven by the K+ transmembrane gradient, is maximally inhibited in the presence of pentachlorophenol at a concentration similar to that found in vivo in the hexachlorobenzene experimental porphyria. By contrast the same degree of inhibition is presented by control mitochondria only in the presence of pentachlorophenol plus valinomycin, a condition which collapses the transmembrane K+ gradient. A strict correlation between porphyrin uptake and K+ concentration has been found in control as well as in iron treated mitochondria. A possible involvement of peroxidative reactions in the mitochondrial membranes has been proposed as a cause of the changes in the permeability properties of the mitochondrial membranes in the experimental chronic hepatic porphyria under conditions of iron overload.     

Analysis of genes implicated in iron regulation in individuals presenting with primary iron overload

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4CG, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.Nucleotide sequence data reported are available in the Genbank database under the assession numbers AJ604512, AJ609539, AJ616848, AJ616847, AJ609540, AJ715523, AJ715524 and AJ715525.     

Normal iron metabolism and the pathophysiology of iron overload disorders

Iron overload disorders represent a heterogenous group of conditions resulting from inherited and acquired causes. If undiagnosed they can be progressive and fatal. Early detection and phlebotomy prior to the onset of cirrhosis can reduce morbidity and normalise life expectancy. We now have greater insight into the complex mechanisms of normal and disordered iron homeostasis following the discovery of new proteins and genetic defects. Here we review the normal mechanisms and regulation of gastrointestinal iron absorption and liver iron transport and their dysregulation in iron overload states. Advances in the understanding of the natural history of iron overload disorders and new methods for clinical detection and management of hereditary haemochromatosis are also reviewed. The current screening strategies target high-risk groups such as first-degree relatives of affected individuals and those with clinical features suggestive of iron loading. Potential ethical, legal and psychosocial issues arising through application of genetic screening programs need to be resolved prior to implementation of general population screening programs.     

Ferritin in erythrocytes and plasma of patients with iron overload

Erythrocyte and plasma ferritin was followed in 13 patients with iron overload undergoing phlebotomies for at least 6 months in comparison with untreated patients and normal males. Plasma ferritin was widely scattered with an average of only twice the normal, whereas erythrocyte ferritin was highly elevated to about twelve times the normal (p less than 0.0001). - The time course of plasma and erythrocyte ferritin during phlebotomy therapy was analyzed in 3 patients with idiopathic hemochromatosis. Three stages were established: 1. plasma ferritin dropped gradually into the normal range while erythrocyte ferritin remained high, 2. appropriate phlebotomies maintained normal plasma ferritin and high erythrocyte ferritin, and indicated a monthly uptake of dietary iron of 150-200 mg at a steady state, 3. at low plasma ferritin levels, erythrocyte ferritin was rapidly decreased by further intensive phlebotomy therapy. Based on the presumed net removal of iron, 1 microgram/l plasma ferritin was equivalent to 3-6 mg of body iron and 1 microgram/l erythrocyte ferritin to somewhat less than 1 mg of body iron. - An elevated erythrocyte ferritin during phlebotomy therapy in iron overload not only depends on body iron stores like plasma ferritin but may also be regulated by the activity of erythropoiesis.     

Chemically-induced formation of an inhibitor of hepatic uroporphyrinogen decarboxylase in inbred mice with iron overload.

An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2',3',4'-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase.     

The effects of changes in inspired oxygen concentration on the experimental production of pulmonary edema in the dog with chronic left ventricular overload.

A twofold increase in left ventricular output was achieved by suturing a Telfon graft between the aorta and left atrium in dogs. Three weeks after surgery the animals were anesthetized and found to have left ventricular end-diastolic pressures averaging 36 mmHg with markedly elevated right ventricular systolic pressures (RVSP). Oxygen breathing resulted in a decrease in left ventricular pressures, RVSP, and arterial pressure in those animals which survived hypoxia. Fifty percent of the shunted dogs subsequently developed fatal pulmonary edema when allowed to breathe 10% oxygen in nitrogen. These animals showed no change in left ventricular function or pulmonary artery pressure (RVSP) in response to pure oxygen administration. It is suggested that there is a gradation of hemodynamic response to pure oxygen depending on the severity of left ventricular overload. In the severest case the 'fixing' of pulmonary hypertension may be due to neurohumoral mechanisms. The subsequent development of pulmonary edema in these animals with hypoxia either involves a change in permeability or a redistribution of hydrostatic pressure within the pulmonary vasculature.     

Stearoyl coenzyme A desaturase 1 expression and activity are increased in the liver during iron overload

In humans, hepatic iron overload can lead to hepatocellular carcinoma development. Iron related dysregulation of hepatic genes could play a role in this phenomenon. We previously found that the carbonyl-iron overloaded mouse was a useful model to study the mechanisms involved in the development of hepatic lesions related to iron excess. The aim of the present study was to identify hepatic genes overexpressed in conditions of iron overload by using this model. A suppressive subtractive hybridization was performed between hepatic mRNAs extracted from control and 3% carbonyl-iron overloaded mice during 8 months. This methodology allowed us to identify stearoyl coenzyme A desaturase 1 (SCD1) mRNA overexpression in the liver of iron loaded mice. The corresponding enzymatic activity was also found to be significantly increased. In addition, we demonstrated that both SCD1 mRNA expression and activity were increased in another iron overload model in mice obtained by a single iron-dextran subcutaneous injection. Moreover, we found, in both models, that SCD1 mRNA was not only influenced by the quantity of iron in the liver but also by the duration of iron overload since SCD1 mRNA upregulation was not detected in earlier stages of iron overload. In addition, we found that cellular repartition likely influenced SCD1 mRNA expression. In conclusion, we demonstrated that iron excess in the liver induced both the expression of SCD1 mRNA and its corresponding enzymatic activity. The level and duration of iron overload, as well as cellular repartition of iron excess in the liver likely play a role in this induction. The fact that the expression and activity of SCD1, an enzyme adding a double bound into saturated fatty acids, are induced in two models of iron overload in mice leads to the conclusion that iron excess in the liver may enhance the biosynthesis of unsaturated fatty acids.     

Iron accumulation in tissues of magnesium-deficient rats with dietary iron overload

The mineral imbalances in magnesium-deficient rats with dietary iron overload were studied. Forty-four male Wister rats were divided into six groups and fed six diets, two by three, fully crossed: magnesium adequate or deficient, and iron deficient, adequate, or excess. The concentrations of iron, magnesium, calcium, and phosphorus in tissues of the rats were measured. The results were as follows: (1) The excess iron intake reinforced the iron accumulation in liver and spleen of magnesium deficient rats; (2) The saturation of iron binding capacity was enormously elevated in the magnesium deficient rats fed excess iron; and (3) Dietary iron deprivation diminished the degree of calcium deposition in kidney of magnesium deficient rats. These results suggest that magnesium-deprived-rats have abnormal iron metabolism losing homeostatic regulation of plasma iron, and magnesium deficient rats with dietary iron overload may be used as an experimental hemochromatosis model.