Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene.

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

Frequency and biochemical expression of C282Y/H63D hemochromatosis (HFE) gene mutations in the healthy adult population in Italy.

BACKGROUND/AIMS: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. METHODS: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. RESULTS: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. CONCLUSIONS: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established.

Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin 1 in iron deficiency and iron overload.

BACKGROUND & AIMS: Imbalances of iron homeostasis are accompanied by alterations of intestinal iron absorption. The identification of divalent-metal transporter 1 (DMT1) and ferroportin 1 (FP1) has improved our understanding of transmembrane iron trafficking. To gain insight into the regulatory properties of these transporters in the duodenum, we studied their expression in patients with hereditary hemochromatosis (HFE-associated and non-HFE-associated), secondary iron overload, and iron deficiency. METHODS: DMT1, FP1 messenger RNA (mRNA), and protein expression were analyzed in duodenal biopsy specimens from patients by means of TaqMan real-time polymerase chain reaction, Western blotting technique, and immunohistochemistry. RESULTS: DMT1 and FP1 mRNA levels are positively correlated with each other in all patient groups (P < 0.001). Moreover, DMT1 and FP1 mRNA levels were significantly increased in patients with iron deficiency, HFE and non-HFE hemochromatosis, whereas they were unchanged in patients with secondary iron overload. Alterations in DMT1 and FP1 mRNA levels were paralleled by comparable changes in the duodenal expression of these proteins. In patients with normal iron status or iron deficiency, significant negative correlations between DMT1, FP1 mRNA, and serum iron parameters were found, which were absent in subjects with primary hemochromatosis. CONCLUSIONS: DMT1 and FP1 are centrally involved in iron uptake/transfer in the duodenum and in the adaptive changes of iron homeostasis to iron deficiency and overload.

Ursodeoxycholic acid complexation with 2-hydroxypropyl-beta-cyclodextrin increases ursodeoxycholic acid biliary excretion after single oral administration in rats.

Complexation of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) improves the water solubility and the dissolution rate of UDCA and may therefore increase its bioavailability. We compared the amount and the rate of biliary excretion of UDCA and biliary lipid secretion after a single oral administration of UDCA in 3 different pharmaceutical formulations [UDCA-HPbetaCD ('urso-beta-cyclodextrin'), UDCA suspension and UDCA capsule] at 3 different dosages each, in 11 groups (2 control groups) of bile fistula rats. UDCA excretion increased with an increase in dose, biliary UDCA recovery and peak secretion were significantly higher after administration of UDCA-HPbetaCD than after UDCA in suspension or capsule. This enhancement of biliary excretion may achieve greater UDCA enrichment in the bile acid pool than conventional pharmaceutical UDCA formulations, this giving to UDCA-HPbetaCD a considerable therapeutical potential.

Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis.

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin.

Hepatic iron toxicity because of iron overload seems to be mediated by lipid peroxidation of biological membranes and the associated organelle dysfunctions. However, the basic mechanisms underlying this process in vivo are still little understood. Gerbils were dosed with weekly injections of iron-dextran alone or in combination with sylibin, a well-known antioxidant, by gavage for 8 weeks. A strict correlation was found between lipid peroxidation and the level of desferrioxamine chelatable iron pool. A consequent derangement in the mitochondrial energy-transducing capability, resulting from a reduction in the respiratory chain enzyme activities, occurred. These irreversible oxidative anomalies brought about a dramatic drop in tissue ATP level. The mitochondrial oxidative derangement was associated with the development of fibrosis in the hepatic tissue. Silybin administration significantly reduced both functional anomalies and the fibrotic process by chelating desferrioxamine chelatable iron.

Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene.

BACKGROUND AND METHODS: Hereditary hemochromatosis in adults is usually characterized by mutations in the HFE gene on the short arm of chromosome 6. Most patients have a substitution of tyrosine for cysteine at position 282 (C282Y). We studied a large family from Italy that includes persons who have a hereditary iron-overload condition indistinguishable from hemochromatosis but without apparent pathogenic mutations in the HFE gene. We performed biochemical, histologic, and genetic studies of 53 living members of the family, including microsatellite analysis of chromosome 6 and direct sequencing of the HFE gene. RESULTS: Of the 53 family members, 15 had abnormal serum ferritin levels, values for transferrin saturation that were higher than 50 percent, or both. Thirteen of the 15 had elevated body iron levels, diagnosed on the basis of the clinical evaluation and liver biopsy, and underwent iron-removal therapy. The other two, both children, did not undergo liver biopsy or iron-removal therapy. None of the 15 members had the C282Y mutation of the HFE gene; 5 of the 15 (as well as 5 healthy relatives) had another mutation of this gene, a substitution of aspartate for histidine at position 63, but none were homozygous for it. No other mutations were found after sequencing of the entire HFE gene for all family members. Microsatellite analysis showed no linkage of the hemochromatosis phenotype with the short arm of chromosome 6, the site of the HFE gene. CONCLUSIONS: Hereditary hemochromatosis can occur in adults who do not have pathogenic mutations in the hemochromatosis gene.

Diacerhein blocks iron regulatory protein activation in inflamed human monocytes.

Iron Regulatory Proteins (IRPs), by modulating expression of ferritin, which stores excess iron in a non toxic form, and transferrin receptor, which controls iron uptake, are the main controller of cellular iron metabolism. During inflammation, modification of IRP activity may affect iron availability, free radical generation and cytokine gene response in inflammatory cells. In the present study we tested the effect of inflammatory stimuli on IRP function in a human monocytic-macrophagic cell line and the possibility of interfering with these pathways by using an antiinflammatory compound, diacerhein (DAR). IRP activity was enhanced by interferon gamma/lipopolysaccarhide (IFN/LPS), and this effect was consistently counteracted by increasing concentrations of DAR. No direct effect of DAR on IRP activity was found in vitro. However, in vivo, similar IRP activation was achieved by exposing cells to nitric oxide (NO) donors and the LPS/IFN-induced activation of IRP was reversed by NO inhibitors. Interestingly, NO-induced IRP activation was efficiently blocked by DAR. These data show for the first time that a clinically useful antiinflammatory compound, DAR, interferes with IRP activation by NO in inflammed human cells.

Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents.

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosis in vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination of in situ hybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that, in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells.

Heterogeneity of hemochromatosis in Italy.

BACKGROUND & AIMS: Patients with hemochromatosis show variable phenotype expression. We evaluated the frequency of hemochromatosis gene (HFE) mutations and the contribution of HFE genotype, ancestral haplotype, ethnic background, and additional factors (alcohol intake, hepatitis viruses, and beta-thalassemia trait) to the severity of iron overload in a large series of Italian patients with a hemochromatosis phenotype. METHODS: HFE genotype was studied in 188 patients. Phenotype evaluation was available in 153 men and 20 women and was based mainly on iron removed. HFE genotype was determined by a polymerase chain reaction restriction assay and ancestral haplotype through D6S265 and D6S105 microsatellite analysis. RESULTS: The frequency of C282Y homozygotes was 64%, with a decreasing gradient from north to south. C282Y homozygotes showed more severe iron overload than the other HFE genotypes. In the same group, ancestral haplotype was associated with a more severe phenotype. Additional factors may favor the development of a relatively mild hemochromatosis phenotype in patients nonhomozygous for the C282Y mutation. CONCLUSIONS: Hemochromatosis in Italy is a nonhomogenous disorder in which genetic and acquired factors are involved. In patients with a single or no HFE mutation, further studies will enable a differentiation between true genetic disorders and interactions between genetic and acquired factors.

Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis.

In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis. To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by light microscopy. Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs. However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.

Inappropriately high iron regulatory protein activity in monocytes of patients with genetic hemochromatosis.

In genetic hemochromatosis (GH), excess iron is deposited in parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells until the later stages of the disease. As iron absorption is inversely related to RE cells stores, a failure of RE to retain iron has been proposed as the basic defect in GH. In RE cells of GH subjects, we examined the activity of iron regulatory protein (IRP), a reliable indicator of the elusive regulatory labile iron pool, which modulates cellular iron homeostasis through control of ferritin (Ft) and transferrin receptor gene expression. RNA-bandshift assays showed a significant increase in IRP activity in monocytes from 16 patients with untreated GH compared with 28 control subjects (1.5-fold) and five patients with secondary hemochromatosis (SH) with similar iron burden (fourfold). In 17 phlebotomy-treated GH patients, IRP activity did not differ from that of control subjects. In both GH and SH monocyte-macrophages, Ft content increased by twofold and the L subunit-rich isoferritin profile was unchanged as compared with controls. IRP activity was still upregulated in vitro in monocyte-derived macrophages of GH subjects but, following manipulations of iron levels, was modulated normally. Therefore, the sustained activity of monocyte IRP found in vivo in monocytes of GH patients is not due to an inherent defect of its control, but is rather the expression of a critical abnormality of iron metabolism, eg, a paradoxical contraction of the regulatory iron pool. By preventing Ft mRNA translation, high IRP activity in monocytes may represent a molecular mechanism contributing to the inadequate Ft accumulation and insufficient RE iron storage in GH.

C/EBP-beta/LAP controls down-regulation of albumin gene transcription during liver regeneration.

Expression of the albumin gene in the liver is controlled by several liver-enriched transcription factors. However, the mechanisms which contribute to its regulation during pathophysiological states, such as liver regeneration, are still little understood. In the present study we found that during liver regeneration down-regulation of albumin mRNA expression is transcriptionally controlled through a minimal element (nucleotide -170 to +22) of the albumin promoter and is observed mainly during the G1 phase of the cell cycle, while high levels of albumin expression are preserved at later time points. Decreased albumin mRNA levels correlate with a dramatic increase in nuclear expression of C/EBP-beta/LAP, a protein known to bind to the D site of the albumin promoter and also to be involved in cell cycle control. In contrast, nuclear expression of other factors such as HNF-1 or C/EBP-alpha, which also have been shown to transcriptionally control albumin expression, is either unchanged or slightly decreased. We show that pre- and post-translational mechanisms are involved in the higher nuclear expression of C/EBP-beta/LAP as early as 1 h after hepatectomy, which also leads to its increased binding toward the D site of the albumin promoter. Finally, in vitro transcription assays with liver nuclear extracts and recombinant C/EBP-beta/LAP demonstrate that C/EBP-beta/LAP can directly down-regulate transcription mediated by the minimal element of the albumin promoter. Additionally the inhibitory role of C/EBP-beta/LAP on the albumin minimal promoter could be confirmed by transfection experiments in hepatoma cells. These results indicate that C/EBP-beta/LAP, while enhancing transcription of cell cycle-related genes and controlling G1/S phase checkpoint, down-regulates a major liver function, i.e. albumin synthesis, to prepare the hepatocyte for entry into the cell cycle.

Antioxidant activity of silybin in vivo during long-term iron overload in rats.

BACKGROUND & AIMS: Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. METHODS: Rats were fed a 2.5% carbonyl-iron diet and 100 mg.kg body wt-1.day-1 silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. RESULTS: Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. CONCLUSIONS: Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.

Molecular and cellular aspects of iron-induced hepatic cirrhosis in rodents.

Hepatic fibrosis and cirrhosis are common findings in humans with hemochromatosis. In this study we investigated the molecular pathways of iron-induced hepatic fibrosis and evaluated the anti-fibrogenic effect of vitamin E. Male gerbils were treated with iron-dextran and fed a standard diet or a alpha-tocopherol enriched diet (250 mg/Kg diet). In gerbils on the standard diet at 6 wk after dosing with iron, in situ hybridization analysis documented a dramatic increase of signal for collagen mRNA around iron foci onto liver fat storing cells (FSC), as identified by immunocytochemistry with desmin antibody. After 4 mo, micronodular cirrhosis developed in these animals, with nonparenchymal cells surrounding hepatocyte nodules and expressing high level of TGF beta mRNA. In this group, in vivo labeling with [3H]-thymidine showed a marked proliferation of nonparenchymal cells, including FSC. In iron-dosed gerbils on the vitamin E-enriched diet for 4 mo, in spite of a severe liver iron burden, a normal lobular architecture was found, with a dramatic decrease of collagen mRNA accumulation and collagen deposition. At the molecular level, a total suppression of nonparenchymal cell proliferation was appreciable, although expression of collagen and TGF beta mRNAs was still present into microscopic iron-filled nonparenchymal cell aggregates scattered throughout the hepatic lobule. In conclusion, our study shows that anti-oxidant treatment during experimental hepatic fibrosis arrests fibrogenesis and completely prevents iron induced hepatic cirrhosis mainly through inhibition of nonparenchymal cell proliferation induced by iron.

Duodenal ferritin synthesis in genetic hemochromatosis.

BACKGROUND/AIMS: The molecular defect of genetic hemochromatosis (GH) is unknown. It is believed that low expression of duodenal ferritin in GH is caused by tissue or cell specific defect of ferritin synthesis. Our study was designed to ascertain whether the control of duodenal ferritin synthesis in GH was defective. METHODS: Expression at the single cell level of H and L ferritin messenger RNAs and protein and activity of the iron regulatory factor, which controls the translation of ferritin messenger RNA, were assessed in 43 duodenal biopsy specimens from individuals with GH, secondary hemochromatosis (SH), anemia, or normal iron balance. RESULTS: Signal for ferritin H and L subunit messenger RNAs was detected in both absorptive and nonabsorptive cells by in situ hybridization, but in 10 of 14 patients with untreated GH, the signal was lower than in patients with SH or normal subjects. However, immunostaining for ferritin protein documented a diffuse/cytoplasmic pattern, whereas a supranuclear/granular staining was found in normal subjects or patients with SH. The spontaneous activity of duodenal iron regulatory factor was consistently higher in patients with GH than in normal subjects or subjects with anemia or SH. CONCLUSIONS: In patients with GH, ferritin gene transcription is preserved in both absorptive and nonabsorptive intestinal cells. Low accumulation of ferritin is not caused by a defective control of ferritin synthesis but by low expression of ferritin messenger RNA and sustained activity of iron regulatory factor.

Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis.

BACKGROUND/AIMS: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity. METHODS: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situ hybridization analyses on liver samples were performed. RESULTS: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and II. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis. CONCLUSIONS: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during long-term iron overload in rodents.

Enhanced hepatic collagen type I mRNA expression into fat-storing cells in a rodent model of hemochromatosis.

In recent years, identifying the hepatic cell type responsible for collagen synthesis in experimental models of postnecrotic or inflammatory fibrosis has been the subject of active investigation. In primary iron overload states, however, hepatic fibrosis and cirrhosis occur without accompanying necroinflammatory phenomena. In this study, we combined morphological, immunological, cell isolation and purification and molecular biological techniques to identify the hepatic cell responsible for enhanced collagen type I gene expression during chronic enteral iron overload in the rat. Ultrastructural analysis of liver tissue sections from iron-loaded rats specifically revealed an altered appearance of fat-storing cells, which showed few if any fat droplets left and increased rough endoplasmic reticulum. In situ hybridization analysis with specific complementary RNA probes identified enhanced signal for collagen type I into nonparenchymal cells in zones 1 and 2, without signal over the background onto iron-laden hepatocytes. Immunocytochemistry with desmin antibodies combined with in situ hybridization on the same tissue sections identified the cells expressing high level of collagen type I transcripts as fat-storing cells. Northern-blot analysis on RNA extracted from various purified cell isolates, confirmed the presence of collagen type I mRNA signal only into the fat-storing cells isolate. Our study shows that in an experimental model of metabolic fibrosis in which the hepatotoxin selectively accumulates into parenchymal cells, fat-storing cells are the main source of enhanced collagen type I gene expression.