Iron-loaded Hepatocytes Research Articles

Iron-triggered signaling via ETS1 and the p38/JNK MAPK pathway regulates Bmp6 expression.

BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.

In vitro identification and characterisation of iron chelating catechol-containing natural products and derivatives.

Iron is an essential component for multiple biological processes. Its regulation within the body is thus tightly controlled. Dysregulation of iron levels within the body can result in several disorders associated with either excess iron accumulation, including haemochromatosis and thalassaemia, or iron deficiency. In cases of excess body iron, therapy involves depleting body iron levels either by venesection, typically for haemochromatosis, or using iron chelators for thalassemia. However, the current chelation options for people with iron overload are limited, with only three iron chelators approved for clinical use. This presents an opportunity for improved therapeutics to be identified and developed. The aim of this study was to examine multiple compounds from within the Davis open access natural product-based library (512 compounds) for their ability to chelate iron. In silico analysis of this library initially identified nine catechol-containing compounds and two closely related compounds. These compounds were subsequently screened using an in vitro DNA breakage assay and their ability to chelate biological iron was also examined in an iron-loaded hepatocyte cellular assay. Toxicity was assessed in hepatocyte and breast cancer cell lines. One compound, RAD362 [N-(3-aminopropyl)-3,4-dihydroxybenzamide] was able to protect against DNA damage, likely through the prevention of free radicals generated via the Fenton reaction; RAD362 treatment resulted in decreased ferritin protein levels in iron-loaded hepatocytes. Lastly, RAD362 resulted in significantly less cell death than the commonly used iron chelator deferoxamine. This is the first study to identify compound RAD362 as an iron chelator and potential therapeutic.

Sensing of Liver Iron Content Requires Cell-Cell Communication between Hepatocytes and Liver Sinusoidal Endothelial Cells

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

An Unusual Case of Intrauterine Symptomatic Neonatal Liver Failure

We present an unusual case of neonatal liver failure. Isolated ascites was diagnosed in a female fetus at week 34 gestational age upon routine ultrasound. In the 35th week of gestation a cesarean section was carried out after puncture of fetal ascites. After birth the patient showed symptoms and complications of acute liver failure with portal hypertension. High serum ferritin concentrations, MRI findings compatible with tissue iron overload and no evidence for infectious disease or inborn errors of metabolism suggested possible neonatal hemochromatosis (NH). HFE gene mutation analysis studies of the child and parents were negative. An anti-oxidative and iron chelating therapy was introduced, followed by clinical stabilisation of the newborn and normalisation of liver function. The liver biopsy at 4 month of age showed mild fibrosis with a few iron-loaded hepatocytes and macrophages. At 2 years of age the child was virtually healthy. The clinical course of our patient indicates that the pathological changes in the liver being associated with presumptive NH may be reversible when NH is diagnosed early and antioxidative and chelating therapy is immediately initiated.

Regulatory effects of tumor necrosis factor-alpha and interleukin-6 on HAMP expression in iron loaded rat hepatocytes

To study the effect of iron and proinflammatory cytokines on the expression of HAMP and other iron regulatory genes in primary rat hepatocytes. Primary hepatocytes from rats fed a control or iron-enriched diet were plated on extracellular matrix and incubated with inflammatory stimuli in the presence or absence of serum. Cells were also incubated with desferrioxamine or ferric ammonium citrate. mRNA levels were determined by Real-Time PCR. Hepatocytes from control rats increased their HAMP expression during culturing, whereas the opposite was seen in hepatocytes from carbonyl-iron loaded animals. In the presence of serum, tumor necrosis factor-alpha, lipopolysaccharide and interleukin-6 increased HAMP expression in hepatocytes from both control and iron-loaded rats. Under serum-free conditions only tumor necrosis factor-alpha increased HAMP mRNA levels. Desferrioxamine and ferric ammonium citrate decreased HAMP gene expression. Tumor necrosis factor-alpha significantly increased mRNA levels of TfR2 and decreased those of DMT1 and IREG1. HAMP expression differs in cultured as compared with freshly isolated hepatocytes, and decreases in iron-loaded hepatocytes in serum free-media, suggesting that additional serum factors influence HAMP expression. Tumor necrosis factor-alpha regulates the mRNA levels of HAMP, IREG1, DMT1 and TfR2 in cultured hepatocytes from both iron-loaded and control animals.

The molecular basis of ferroportin-linked hemochromatosis.

Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1, and MTP1) result in hemochromatosis type IV, a disorder with a dominant genetic pattern of inheritance and heterogeneous clinical presentation. Most patients develop iron loading of Kupffer cells with relatively low saturation of plasma transferrin, but others present with high transferrin saturation and iron-loaded hepatocytes. We show that known human mutations introduced into mouse Fpn-GFP generate proteins that either are defective in cell surface localization or have a decreased ability to be internalized and degraded in response to hepcidin. Studies using co-immunoprecipitation of epitope-tagged Fpn and size-exclusion chromatography demonstrated that Fpn is multimeric. Both WT and mutant Fpn participate in the multimer, and mutant Fpn can affect the localization of WT Fpn, its stability, and its response to hepcidin. The behavior of mutant Fpn in cell culture and the ability of mutant Fpn to act as a dominant negative explain the dominant inheritance of the disease as well as the different patient phenotypes.

Modulation of stellate cell proliferation and gene expression by rat hepatocytes: effect of toxic iron overload

Mechanisms by which hepatocytes and transdifferentiated hepatic stellate cells (HSC) initiate liver fibrosis in chronic iron toxicity are unknown. This study was to determine if factors in media from control and iron-loaded rat hepatocyte cultures modulate HSC gene expression and proliferation. Conditioned medium (CM) from both control and iron-loaded hepatocytes increased serum-stimulated DNA synthesis by HSC to 140% of control values ( P<0.05). Heating CM (15 min, 80 °C) caused a suppression of DNA synthesis that was partially reversed by a TGF-β-neutralizing antibody. Addition of TGF-β 1 reproduced the suppression. Levels in HSC of mRNA for collagen type I, collagen type IV, TGF-β, and plasminogen activator inhibitor-1 were unaffected by exposure to CM but increased significantly when CM from iron-loaded hepatocytes was heat-treated. In HepG2 cell cultures, iron loading increased total (but not activated) TGF-β secretion into the medium approximately 2-fold. We conclude that increased secretion of latent TGF-β by hepatocytes injured by iron is a potential factor influencing fibrogenic behavior of HSC.

Expression of E-Cadherin and Other Paracellular Junction Genes Is Decreased in Iron-Loaded Hepatocytes

Iron overload in the liver may occur in the clinical conditions hemochromatosis and transfusion-dependent thalassemia or by long-term consumption of large amounts of dietary iron. As iron concentrations increase in the liver, cirrhosis develops, and subsequently the normal architecture of the liver deteriorates. The underlying mechanisms whereby iron loading of hepatocytes leads to the pathology of the liver are not understood. Similarly, a direct relationship between the expression levels of paracellular junction genes and altered hepatocellular physiology has been reported; however, no relationship has been identified between iron loading and the expression of paracellular junction genes. Here, we report that the expression of numerous paracellular junction genes was decreased in iron-loaded hepatocytes, leading to increased cellular permeability, increased baculovirus-mediated gene transfer, and decreased gap junction communication. Iron loading of hepatocytes resulted in decreased E-cadherin promoter activity and subsequently decreased E-cadherin mRNA and protein expression. The data presented in this study describe a clear relationship between iron overload and decreased expression of paracellular junction genes in hepatic cells of rat and human origin.

894 Expression of paracellular junction genes in iron-loaded hepatocytes

894 Expression of paracellular junction genes in iron-loaded hepatocytes

Iron Overload Decreases the Protective Effect of Tumour Necrosis Factor-α on Rat Hepatocytes Exposed to Oxidative Stress

Background: Intracellular iron can participate in the formation of free radicals, leading to liver cell damage. This may be prevented by the ability of ferritin to oxidize and store iron. Tumour necrosis factor alpha (TNF- α ) has been shown to increase the ferritin synthesis. In the liver, cytokines are secreted by activated Kupffer cells and T-lymphocytes. The aim of this study was to investigate the effects of TNF- α on normal and iron-loaded rat hepatocytes exposed to oxidative stress. Methods: Primary cultures of hepatocytes from rats fed a normal rat chow or a carbonyl iron-enriched diet were incubated with TNF- α before incubation with tert -butyl hydroperoxide. Malondialdehyde concentrations, activities of lactate dehydrogenase, ferritin H and manganese superoxide dismutase mRNA and ferritin H protein were analysed. The total amounts of glutathione and chelatable iron were measured. Results: TNF- α diminished the concentrations of malondialdehyde and activities of lactate dehydrogenase in hepatocytes exposed to tert -butyl hydroperoxide. This was seen in hepatocytes from normal but not iron-loaded animals. The transcription of manganese-superoxide dismutase mRNA was increased in both cell types, whereas total glutathione contents of cells were unaffected. The transcription and translation of ferritin H was induced in cells from normal but not from iron-loaded animals. The amount of chelatable iron was significantly lowered only in hepatocytes from normal rats. Conclusions: TNF- α protects rat hepatocytes from normal but not iron-loaded rats from oxidative stress. The protection may be due to an induction of the ferritin synthesis.

New 8-hydroxyquinoline and catecholate iron chelators: influence of their partition coefficient on their biological activity

Four new hexadendate chelators, three hydroxyquinoline-based, Csox, O-Trensox, Cox750, and one catecholate-based CacCam—which have comparable skeletal structures and pFe, but widely different partition coefficients, (Kpart), 0.01, 0.02, 1 and 3.2 respectively, have been tested for their iron chelating efficacy in vitro by two methods. First, by their ability to remove iron from ferritin in solution or second, to remove iron from iron-loaded hepatocytes in vitro. Our objective was to ascertain the importance of Kpart and pFe, on the biological efficiency of the molecule. Previous studies proposed that an ideal value of Kpart of 1 should give maximum biological activity. Mobilization of iron by Csox and CacCAM from ferritin was similar and furthermore more efficient than desferrioxamine B. In the iron-loaded hepatocyte cultures, the three hydroxyquinoline chelators, although showing diversity in terms of lipophilicity, appeared to be very similar in their capacity to chelate iron. CacCAM, the unique catecholate, was the most efficient of the molecules tested, as well as being the least toxic in the cellular model despite having the lowest value of pFe. In conclusion, the use of the partition coefficient and pFe, as tools for predicting biological activity of iron chelators should be not generalized. Further studies are required in order to understand the influence of the structure on the biological activity of the molecule.

The effects of dietary iron overload on fumonisin B1-induced cancer promotion in the rat liver

The present study was performed to determine whether excess hepatic iron modulates the cancer-initiating and promoting properties of FB1. Thirty-eight male F344 rats were divided into four dietary treatment groups: (i) control diet (AIN, n=8); (ii) FB1 250 mg/kg diet (FB1, n=10); (iii) 1–2% carbonyl iron (CI, n=10); or (iv) FB1 plus iron loading (FB1/CI, n=10) for 5 weeks (2×2 factorial design). Hepatic iron concentrations in iron-loaded animals at 5 weeks were 444±56 (CI) and 479±80 μmol/g dry weight (FB1/CI) (mean±SEM). All the FB1-fed rats, in the presence or absence of CI, developed a toxic hepatitis with a 4-fold rise in serum alanine transaminase (ALT) levels. FB1 appeared to augment iron-induced hepatic lipid peroxidation, as measured by the generation of thiobarbituric acid reacting substances (TBARS) in liver homogenates (P<0.0001). Morphometric analysis showed that FB1 caused a significantly greater mean±SEM number of ‘enzyme-altered’ foci and nodules per cm2 (5.34±1.42 vs. 1.50±0.52, P<0.05), as well as a greater area (%) of liver occupied by foci and nodules (0.33±0.12% vs. 0.05±0.03%, P<0.001), compared with FB1/CI. The addition of FB1 to dietary iron loading caused a shift in distribution of iron from hepatocytes to Kupffer cells, probably due to phagocytosis of necrotic iron-loaded hepatocytes. In conclusion, (i) FB1 appears to cause toxicity in the liver independently from effects on lipid peroxidation; (ii) FB1 has a potentiating effect on iron-induced lipid peroxidation; and (iii) dietary iron loading appears to protect against the cancer promoting properties of FB1, possibly due to a stimulatory effect of iron on hepatocyte regeneration.

Enhanced γ-Glutamyl Transpeptidase Expression and Selective Loss of CuZn Superoxide Dismutase in Hepatic Iron Overload

Liver injury caused by iron overload is presumed to involve lipid peroxidation and the formation of products such as 4-hydroxynonenal (4HNE), which has been implicated in hepatic fibrogenesis. Cellular antioxidants that modulate the formation and detoxification of compounds such as 4HNE may represent important protective mechanisms involved in the response to iron overload. This study examines the relationship between 4HNE, collagen content, and antioxidant defenses in the livers of rats fed carbonyl iron for 10 weeks. Iron-loading resulted in significant increases in iron (8.8-fold), 4HNE (1.7-fold), and hydroxyproline (1.5-fold). Total glutathione content was unchanged by iron, but γ-glutamyl transpeptidase activity (GGT) increased sixfold and CuZn superoxide dismutase (CuZnSOD) activity decreased >90%. GGT colocalized with iron deposition and was associated with increased GGT mRNA. Decreased CuZnSOD activity was paralleled by a reduction in CuZnSOD protein on Western blot and immunohistochemistry, but no decrease in CuZnSOD mRNA. Glutathione S-transferase (GST) and Mn superoxide dismutase (Mn SOD) activities were also significantly increased by iron loading. These results demonstrate that iron overload significantly alters the expression of antioxidant enzymes associated with glutathione (GGT and GST) and superoxide metabolism (CuZnSOD and MnSOD). Furthermore, the localized induction of GGT may enhance detoxification of lipid peroxidation-derived aldehydes via glutathione-dependent pathways in iron-loaded hepatocytes. These alterations in antioxidant defenses may represent an adaptive response, limiting accumulation 4HNE, and thus, stimulation of collagen synthesis, accounting for the mild fibrogenic response seen in this model of iron overload.

Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture

Under conditions of iron overload plasma transferrin can be fully saturated and the plasma can transport non-transferrin-bound Fe which is rapidly cleared by the liver. Much of this Fe is complexed by citrate. The aim of the present work was to characterise the mechanisms by which Fe-citrate is taken up by hepatocytes using a rat hepatocyte cell culture model. The cells, after one day in culture, were incubated with 59 Fe -labelled Fe-citrate for varying time periods, then washed and Fe uptake to the membrane and intracellular compartments of the cell was determined by radioactivity measurements. Maximal rates of internalisation of Fe occurred at a Fe:citrate molar ratio of 1:100 or greater, a pH of approximately 7.4 and an extracellular Ca 2+ concentration of 1.0 mM. Fe uptake showed Michaelis–Menten kinetics and was a temperature-dependent process. The K m and V max for Fe internalisation by the cells at 37°C were approximately 7 μM and 2 nmol/mg DNA/min (25×10 6 atoms/cell/min), respectively; and the Arrhenius activation energy was 35 kJ/mol. The transition metals, Zn 2+, Co 2+ and Ni 2+, inhibited Fe uptake when used at 10 and 100 times the concentration of Fe. The rate of Fe internalisation from Fe-citrate was found to be approximately 20 times as great as that from Fe-transferrin with Fe concentrations of 1 and 2.5 μM for both forms of Fe. The rate of Fe uptake by iron-loaded hepatocytes obtained from rats which had been fed carbonyl Fe was not significantly different from that by normal hepatocytes. These experiments show that rat hepatocytes in primary culture have a high capacity to take up non-transferrin-bound Fe in the form of Fe-citrate and that uptake occurs by facilitated diffusion. The iron transport process does not appear to be regulated by cellular Fe levels.

Intracellular distribution of iron (and associated elements) in various cell types of larvae and juveniles of the sea lamprey ( Petromyzon marinus)

The intracellular distribution of iron and other elements was examined in various cell types in larvae and juveniles of the sea lamprey ( Petromyzon marinus) using transmission electron microscopy and energy dispersive x-ray microanalysis. The objective was to establish whether there are cell-type specific relationships between iron and other elements in the iron-rich organs and tissues (adipose tissue, opisthonephric kidneys, dorsal integument, fat column, liver, and posterior intestine) of these two life cycle periods. Iron was localized within either dense bodies (presumptive lysosomes, siderosomes) or in the cytoplasmic matrix of many cell types where it was viewed as haemosiderin/ferritin and ferritin, respectively. Presumptive lysosomes of adipocytes of the nephric folds, dorsal integument, and fat column possessed iron and sulphur and this elemental association was also prevalent in the epithelia of the larval proximal tubules and in the posterior intestine and epidermis of both life periods. Macrophages of the larval haemopoietic tissue (posterior intestine) and of the juvenile opisthonephros, which were described as melanomacrophages because of their granules, possessed iron, sulphur, and calcium. This elemental association was also noted in the presumptive lysosomes of the iron-loaded hepatocytes of the juvenile liver while no elements could be detected in these cells in the larval organ. The variations and similarities in elemental associations between the cell types in each life period and at different life periods is discussed in the context of specific cell functions related to the prevention of iron toxicity. These functions are sequestration of iron and storage as the less toxic haemosiderin (larval adipocytes, macrophages, juvenile hepatocytes) or as part of a process of elimination of excesses of this metal (posterior intestine, dorsal epidermal cells). Due to its unique ability to deal with copious amounts of iron at all periods of the life cycle, the lamprey serves as an important model for studies of iron loading in vertebrates.

Hepatic stellate cell activation in genetic haemochromatosis: Lobular distribution, effect of increasing hepatic iron and response to phlebotomy

Background/Aims: Activated hepatic stellate cells produce increased levels of collagen in animal models of chronic iron overload; however, their role in human genetic haemochromatosis is unknown. This study examined the relationship between hepatic iron concentration and hepatic stellate cell activation in genetic haemochromatosis. Methods: Liver biopsies from 75 patients (55 with haemochromatosis, 14 haemochromatosis patients both pre- and post-phlebotomy and six non iron-loaded disease control subjects) were stained for iron using Perls' Prussian Blue. Thirty biopsies in which there was no evidence of either steatosis or inflammation were subjected to immunohistochemistry for α-smooth muscle actin and desmin and counterstained for iron. Forty-five biopsies demonstrated either steatosis or inflammation, in addition to excess iron. Results: Stellate cells were identified by light microscopy as perisinusoidal cells containing numerous intracellular fat droplets. α-Smooth muscle actin was detected in biopsies with an hepatic iron concentration > 60 μmol/g dry weight. Increasing hepatic iron concentration and hepatic iron index correlated with an increase in α-smooth muscle actin expression ( r=0.81 and 0.72, respectively). Phlebotomy resulted in a significant decrease in α-smooth muscle actin expression. In early disease prior to histological evidence of collagen deposition, whilst activated stellate cells were located in Zone 1, greater numbers were found in Zones 2 and 3 distal to the region of heaviest iron overload. Conclusions: This study has demonstrated for the first time in humans a correlation between hepatic iron concentration and stellate cell activation in haemochromatosis, which is reversed by iron removal. Humoral factors from either iron-loaded hepatocytes or activated Kupffer cells may be responsible for early stellate cell activation in areas of the liver remote from heavy iron loading.

Hepatic mitochondrial energy production in rats with chronic iron overload

Background: Iron overload results in impaired hepatic mitochondrial oxidative metabolism. The current experiments evaluated the effects of iron overload on enzyme activities in the mitochondrial electron transport chain, on hepatic adenine nucleotide levels, and on hepatocellular oxygen consumption. Methods: Hepatic iron overload was produced in rats using dietary carbonyl iron. Hepatic adenine nucleotides were assessed after freeze-clamping, mitochondrial enzyme activities and oxygen consumption were measured in isolated mitochondria, and oxygen consumption in isolated hepatocytes was determined. Results: At a mean hepatic iron concentration of 4630 μg/g, there were no changes in reduced nicotinamide adenine dinucleotide (NADH)-cytochrome c reductase activity (complex I–III), but there was a 35% reduction in succinate-cytochrome c reductase activity (complex II–III), and a 70% decrease in cytochrome c oxidase activity (complex IV). With mild iron loading (2060 μg/g), there was a 28% decrease in hepatic adenosine 5′-triphosphate (ATP) levels with no change in adenosine 5′-diphosphate (ADP) or adenosine 5′-monophosphate (AMP) levels, whereas, at a higher hepatic iron concentration (3170 μg/g), there was a 40% reduction in ATP levels, a 22% decrease in ADP levels, with no change in AMP levels. There was a 48% reduction in oxygen consumption in isolated iron-loaded hepatocytes. Conclusions: Chronic iron overload decreases hepatic mitochondrial cytochrome c oxidase activity, hepatocellular oxygen consumption, and hepatic ATP levels.

Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures

The cytoprotective effect of three flavonoids, catechin, quercetin and diosmetin, was investigated on iron-loaded hepatocyte cultures, considering two parameters: the prevention of iron-increased lipid peroxidation and the inhibition of intracellular enzyme release. These two criteria of cytoprotection allowed the calculation of mean inhibitory concentrations ( ic 50) which revealed that the effectiveness of these flavonoids could be classified as follows: catechin > quercetin > diosmetin. These ic 50 values have been related to structural characteristics of the flavonoids tested. Moreover, the investigation of the capacity of these flavonoids to remove iron from iron-loaded hepatocytes revealed a good relationship between this iron-chelating ability and the cytoprotective effect. The cytoprotective activity of catechin, quercetin and diosmetin could thus be ascribed to their widely known antiradical property but also to their iron-chelating effectiveness. These findings increase further the prospects for the development and clinical application of these potent antioxidants.

Iron overload of the liver by trimethylhexanoylferrocene in rats.

Iron-deficient female Wistar rats were fed a diet, which contained 0.5% trimethylhexanoylferrocene, over a 56-week period. This dietary iron loading resulted in a progressive siderosis and enlargement of the liver with a maximum iron content of 947.0 +/- 148.0 mg (vs. 0.07 +/- 0.04 mg in iron deficiency) and a maximum organ weight of 39.4 +/- 6.6 g (vs. 6.9 +/- 1.4 g in iron-deficient control rats). Up to 43 weeks, whole liver iron rose by increase in iron concentration (max. 28.0 +/- 6.1 mg/g wet weight, w.w.) as well as by enlargement of the organ. Afterwards whole liver iron increased solely by ongoing hepatomegaly. At the commencement of iron loading, stainable iron was almost exclusively stored by hepatocytes equally throughout all areas of the liver lobule. Later, the distribution of iron-loaded hepatocytes became strikingly periportal, and, in addition, Kupffer cells as well as sinus-lining endothelia began to store iron. Animals with a liver iron concentration of more than 10.4 +/- 0.75 mg/g w.w. showed no further increase in ferritin and haemosiderin within hepatocytes. Iron-burdened Kupffer cells/macrophages, however, accumulated permanently, hereby forming intrasinusoidal and portal siderotic nodules and areas. First signs of liver damage such as necrosis of single hepatocytes and mild fibrosis began at a liver iron concentration of 14.7 +/- 1.4 mg/g w.w. With advancement of iron loading, focal necrosis of hepatocytes and iron-burdened macrophages took place, and significant perisinusoidal as well as portal fibrosis developed. Cirrhosis, however, the final stage of impairment in iron overload of the liver in humans, could not be induced in this animal model up to now.

Prevention of Hepatocyte Injury and Lipid Peroxidation by Iron Chelators and α–Tocopherol in Isolated Iron–Loaded Rat Hepatocytes

These experiments were performed to characterize the relationship between lipid peroxidation and hepatocyte viability in iron overload. Hepatocytes were isolated from rats with chronic dietary iron overload and the effects of in vitro iron chelation on lipid peroxidation, cell viability and ultrastructure were studied over a 4-hr incubation period. Cell viability was significantly reduced at 3 and 4 hr in iron-loaded hepatocytes compared with controls and was preceded by an increase in iron-dependent lipid peroxidation. Similarly, extensive degenerative ultrastructural changes were observed in iron-loaded hepatocytes compared with controls after 4 hr of incubation. In vitro iron chelation with either deferoxamine or apotransferrin protected against lipid peroxidation, loss of viability and ultrastructural damage in iron-loaded hepatocytes. The addition of an antioxidant, alpha-tocopherol, also protected against lipid peroxidation and preserved cell viability over a 4-hr incubation. The protective effects of iron chelators and alpha-tocopherol support a strong association between iron-dependent lipid peroxidation and hepatocellular injury in iron overload.