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Crawford DHG, Ramm GA, Bridle KR, Nicoll AJ, Delatycki MB, Olynyk JK. Clinical practice guidelines on hemochromatosis: Asian Pacific Association for the Study of the Liver. Hepatol Int 2023; 17:522-541. [PMID: 37067673 DOI: 10.1007/s12072-023-10510-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Accepted: 02/28/2023] [Indexed: 04/18/2023]
Affiliation(s)
- Darrell H G Crawford
- Faculty of Medicine, The University of Queensland, Brisbane, Australia
- Gallipoli Medical Research Foundation, Brisbane, Australia
| | - Grant A Ramm
- Faculty of Medicine, The University of Queensland, Brisbane, Australia
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
| | - Kim R Bridle
- Faculty of Medicine, The University of Queensland, Brisbane, Australia.
- Gallipoli Medical Research Foundation, Brisbane, Australia.
| | - Amanda J Nicoll
- Department of Gastroenterology, Eastern Health, Box Hill, VIC, Australia
- Monash University, Melbourne, VIC, Australia
| | - Martin B Delatycki
- Bruce Lefroy Centre, Murdoch Children's Research Institute, Melbourne, VIC, Australia
- The University of Melbourne, Melbourne, VIC, Australia
- Victorian Clinical Genetics Services, Parkville, VIC, Australia
| | - John K Olynyk
- Department of Gastroenterology, Fiona Stanley Hospital, Murdoch, WA, Australia
- School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA, Australia
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2
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Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity. Biomolecules 2021; 11:biom11010096. [PMID: 33451048 PMCID: PMC7828500 DOI: 10.3390/biom11010096] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Revised: 01/07/2021] [Accepted: 01/12/2021] [Indexed: 12/14/2022] Open
Abstract
Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.
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Valko M, Jomova K, Rhodes CJ, Kuča K, Musílek K. Redox- and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol 2015; 90:1-37. [DOI: 10.1007/s00204-015-1579-5] [Citation(s) in RCA: 535] [Impact Index Per Article: 53.5] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2015] [Accepted: 08/11/2015] [Indexed: 02/07/2023]
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Baggott JE, Tamura T. Homocysteine, iron and cardiovascular disease: a hypothesis. Nutrients 2015; 7:1108-18. [PMID: 25668155 PMCID: PMC4344578 DOI: 10.3390/nu7021108] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Accepted: 01/27/2015] [Indexed: 12/26/2022] Open
Abstract
Elevated circulating total homocysteine (tHcy) concentrations (hyperhomocysteinemia) have been regarded as an independent risk factor for cardiovascular disease (CVD). However, several large clinical trials to correct hyperhomocysteinemia using B-vitamin supplements (particularly folic acid) have largely failed to reduce the risk of CVD. There is no doubt that a large segment of patients with CVD have hyperhomocysteinemia; therefore, it is reasonable to postulate that circulating tHcy concentrations are in part a surrogate marker for another, yet-to-be-identified risk factor(s) for CVD. We found that iron catalyzes the formation of Hcy from methionine, S-adenosylhomocysteine and cystathionine. Based on these findings, we propose that an elevated amount of non-protein-bound iron (free Fe) increases circulating tHcy. Free Fe catalyzes the formation of oxygen free radicals, and oxidized low-density lipoprotein is a well-established risk factor for vascular damage. In this review, we discuss our findings on iron-catalyzed formation of Hcy from thioethers as well as recent findings by other investigators on this issue. Collectively, these support our hypothesis that circulating tHcy is in part a surrogate marker for free Fe, which is one of the independent risk factors for CVD.
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Affiliation(s)
- Joseph E Baggott
- Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
| | - Tsunenobu Tamura
- Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
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Heli H, Mirtorabi S, Karimian K. Advances in iron chelation: an update. Expert Opin Ther Pat 2011; 21:819-56. [PMID: 21449664 DOI: 10.1517/13543776.2011.569493] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
INTRODUCTION Oxidative stress (caused by excess iron) can result in tissue damage, organ failure and finally death, unless treated by iron chelators. The causative factor in the etiology of a variety of disease states is the presence of iron-generated reactive oxygen species (ROS), which can result in cell damage or which can affect the signaling pathways involved in cell necrosis-apoptosis or organ fibrosis, cancer, neurodegeneration and cardiovascular, hepatic or renal dysfunctions. Iron chelators can reduce oxidative stress by the removal of iron from target tissues. Equally as important, removal of iron from the active site of enzymes that play key roles in various diseases can be of considerable benefit to the patients. AREAS COVERED This review focuses on iron chelators used as therapeutic agents. The importance of iron in oxidative damage is discussed, along with the three clinically approved iron chelators. EXPERT OPINION A number of iron chelators are used as approved therapeutic agents in the treatment of thalassemia major, asthma, fungal infections and cancer. However, as our knowledge about the biochemistry of iron and its role in etiologies of seemingly unrelated diseases increases, new applications of the approved iron chelators, as well as the development of new iron chelators, present challenging opportunities in the areas of drug discovery and development.
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Affiliation(s)
- Hossein Heli
- Islamic Azad University, Science and Research Branch, Department of Chemistry, Fars, 7348113111, Iran
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6
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Chua ACG, Graham RM, Trinder D, Olynyk JK. The regulation of cellular iron metabolism. Crit Rev Clin Lab Sci 2008; 44:413-59. [PMID: 17943492 DOI: 10.1080/10408360701428257] [Citation(s) in RCA: 109] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
While iron is an essential trace element required by nearly all living organisms, deficiencies or excesses can lead to pathological conditions such as iron deficiency anemia or hemochromatosis, respectively. A decade has passed since the discovery of the hemochromatosis gene, HFE, and our understanding of hereditary hemochromatosis (HH) and iron metabolism in health and a variety of diseases has progressed considerably. Although HFE-related hemochromatosis is the most widespread, other forms of HH have subsequently been identified. These forms are not attributed to mutations in the HFE gene but rather to mutations in genes involved in the transport, storage, and regulation of iron. This review is an overview of cellular iron metabolism and regulation, describing the function of key proteins involved in these processes, with particular emphasis on the liver's role in iron homeostasis, as it is the main target of iron deposition in pathological iron overload. Current knowledge on their roles in maintaining iron homeostasis and how their dysregulation leads to the pathogenesis of HH are discussed.
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Affiliation(s)
- Anita C G Chua
- School of Medicine and Pharmacology, University of Western Australia, Fremantle, Western Australia, Australia
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7
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Abstract
The liver plays a central role in iron metabolism. It is the major storage site for iron and also expresses a complex range of molecules which are involved in iron transport and regulation of iron homeostasis. An increasing number of genes associated with hepatic iron transport or regulation have been identified. These include transferrin receptors (TFR1 and 2), a ferrireductase (STEAP3), the transporters divalent metal transporter-1 (DMT1) and ferroportin (FPN) as well as the haemochromatosis protein, HFE and haemojuvelin (HJV), which are signalling molecules. Many of these genes also participate in iron regulatory pathways which focus on the hepatic peptide hepcidin. However, we are still only beginning to understand the complex interactions between liver iron transport and iron homeostasis. This review outlines our current knowledge of molecules of iron metabolism and their roles in iron transport and regulation of iron homeostasis.
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Affiliation(s)
- Ross-M Graham
- School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, PO Box 480, Fremantle 6959, Western Australia, Australia
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8
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Vargas JD, Herpers B, McKie AT, Gledhill S, McDonnell J, van den Heuvel M, Davies KE, Ponting CP. Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric reductases. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2003; 1651:116-23. [PMID: 14499595 DOI: 10.1016/s1570-9639(03)00242-5] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Iron has a variety of functions in cellular organisms ranging from electron transport and DNA synthesis to adenosine triphosphate (ATP) and neurotransmitter synthesis. Failure to regulate the homeostasis of iron can lead to cognition and demyelination disorders when iron levels are deficient, and to neurodegenerative disorders when iron is in excess. In this study we show that three members of the b561 family of predicted ferric reductases, namely mouse cytochrome b561 and mouse and fly stromal cell-derived receptor 2 (SDR2), have ferric reductase activity. Given that a fourth member, duodenal cytochrome b (Dcytb), has previously been shown to be a ferric reductase, it is likely that all remaining members of this family also exhibit this activity. Furthermore, we show that the rat sdr2 message is predominantly expressed in the liver and kidney, with low expression in the duodenum. In hypotransferrinaemic (hpx) mice, sdr2 expression in the liver and kidney is reduced, suggesting that it may be regulated by iron. Moreover, we demonstrate the presence of mouse sdr2 in the choroid plexus and in the ependymal cells lining the four ventricles, through in situ hybridization analysis.
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Affiliation(s)
- J D Vargas
- Department of Human Anatomy and Genetics, University of Oxford, OX1 3QX Oxford, UK
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9
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Levy MA, Tsai YH, Reaume A, Bray TM. Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol 2001; 281:L172-82. [PMID: 11404260 DOI: 10.1152/ajplung.2001.281.1.l172] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.
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Affiliation(s)
- M A Levy
- Department of Human Nutrition, The Ohio State University, Columbus, Ohio 43210, USA
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10
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Abstract
We are interested in learning how iron is safely inserted and stored in ferritin. Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L). In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem. Biophys. Res. Commun. 242, 39-45 (1998)). This results in the protein being expressed at very low levels. This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG). Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit. Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios. Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition.
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Affiliation(s)
- J E Grace
- Biotechnology Center, Utah State University, Logan 84322, USA
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Imbert-Bismut F, Charlotte F, Turlin B, Khalil L, Piton A, Brissot P, Le Charpentier Y, Delattre J, Opolon P, Deugnier Y, Poynard T. Low hepatic iron concentration: evaluation of two complementary methods, colorimetric assay and iron histological scoring. J Clin Pathol 1999; 52:430-4. [PMID: 10562810 PMCID: PMC501429 DOI: 10.1136/jcp.52.6.430] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
AIMS To validate a method of assessment of low hepatic iron concentration based on a biochemical colorimetric assay plus histological scoring. METHODS The within-day and day to day precision of the iron colorimetric assay was determined on frozen rat liver. The coefficient of variation (CV) of iron measurement in two separate samples from the same liver was determined for 21 deparaffinised human biopsies. The intra- and interlaboratory variability of the colorimetric assay and histological scoring were assessed on 38 deparaffinised liver biopsies. RESULTS For the within-day test, the CV was 11% (5.1 (0.6) mumol/g dry weight (dw), mean (SD) iron concentration). For the day to day test, the CV was 19.5% (8.2 (1.6) mumol/g dw). The CV was 14.7% for iron concentration determined in two separate samples from the same liver. By correlation and kappa concordance tests, the intra- and interlaboratory variability of the hepatic iron colorimetric assay and iron histological scoring was slight. Absence of stainable iron corresponded to a liver iron concentration < or = 20 mumol/g dw. CONCLUSIONS A combination of two complementary methods, colorimetric measurement and histological scoring, is an accurate and reliable way of determining low iron concentrations in deparaffinised human liver biopsies. In secondary haemosiderosis, such methods would be essential for investigating the role of low iron overload in fibrogenesis and during the response to antiviral treatment in chronic viral hepatitis.
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Affiliation(s)
- F Imbert-Bismut
- Laboratoire de Biochimie, Hôpital Pitié-Salpêtrière 75013, Paris, France
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12
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Juan SH, Aust SD. Mutational analysis of loading of iron into rat liver ferritin by ceruloplasmin. Arch Biochem Biophys 1999; 361:295-301. [PMID: 9882459 DOI: 10.1006/abbi.1998.0998] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Site-directed mutagenesis was used to investigate the loading of iron into rat liver ferritin by ceruloplasmin. Changes were made in the H chain to investigate the role of tyrosines involved in an inherent ferroxidase activity thought to be involved in the self-loading of iron into ferritin. Mutation Y34F affected the rate of iron loading by ceruloplasmin and incorporation of the oxidized iron into the core. Mutation Y29R (making it analogous to the L chain) had no effect on iron oxidation but slightly decreased core formation. A double mutation in the L chain, to open the alpha-helix bundle channel, and R25Y, making the protein more analogous to the H chain, increased the amount of iron incorporated into the core, again suggesting that this Tyr is involved in ligand exchange for core formation. Additional changes in the L chain involving the BC loop suggest that the entire BC loop is involved in the association of ferritin with ceruloplasmin, increasing its ferroxidase activity and the rate of iron loading into ferritin.
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Affiliation(s)
- S H Juan
- Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA
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Tuccari G, Giuffrè G, Crisafulli C, Barresi G. Immunohistochemical demonstration of lactoferrin in human neoplastic tissues. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1998; 443:337-40. [PMID: 9781378 DOI: 10.1007/978-1-4757-9068-9_42] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- G Tuccari
- Department of Human Pathology, University of Messina, Italy
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14
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Abstract
BACKGROUND/AIMS The endogenous low molecular weight iron chelator, citrate, is considered to be an important contributor to iron transport and the liver the main site of uptake of iron citrate in subjects suffering from diseases of iron overload. Moreover, the citrate-metabolising enzyme, aconitase, is implicated in the regulation of cellular iron metabolism. This study was undertaken to determine the role of citrate and ferric citrate in the uptake of iron by rat hepatocytes. METHODS Cultured rat hepatocytes were incubated (37 degrees C, 15 min) with 100 microM [14C]-citrate in the presence or absence of 1.0 microM 55Fe. Membrane-bound and intracellular radiolabel were separated by incubation with the general protease, Pronase. RESULTS Our results suggest that ferric citrate uptake is mediated by a specific citrate binding site which exhibits a higher affinity for citrate in the presence of iron than in its absence. Citrate was internalised by hepatocytes, with at least 70% being oxidised to CO2 within 15 min. Citrate uptake was pH-dependent, did not require the presence of sodium and increased with increasing iron concentration. Metabolic energy, anion channels, the Na+, K+-ATPase and vesicle acidification do not appear to play a role in uptake of ferric citrate, but functional sulphydryl groups may be involved. CONCLUSIONS The data suggest either that ferric citrate complexes with higher molar ratios of iron to citrate relative to the incubation medium are bound preferentially to the membrane, or that once citrate has delivered its iron to the membrane, the complex dissociates and the components are internalised separately.
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Affiliation(s)
- R M Graham
- Department of Physiology, The University of Western Australia, Perth
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15
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Abstract
We showed previously that ceruloplasmin associates with the H chain of rat liver ferritin during iron loading into ferritin such that the iron oxidized by ceruloplasmin was deposited into ferritin [S.-H. Juan et al. (1997) Arch. Biochem. Biophys. 341, 280-286]. Three synthetic decapeptides derived from domains 2, 4, and 6 of ceruloplasmin, referred to CP-2, CP-4, and CP-6, were utilized to identify a possible binding site on ceruloplasmin for ferritin. Two of the peptides, CP-4 and CP-6, were found to inhibit iron loading into the recombinant ferritin H chain homopolymer (rH-Ft) by ceruloplasmin. The extent of inhibition of iron loading into ferritin by ceruloplasmin by CP-6, but not CP-4, varied with pH, whereas the inhibitory effect remained constant in increasing concentrations of NaCl. The addition of rH-Ft quenched the fluorescence emission of CP-4 and CP-6, but not CP-2. The quenching of fluorescence was used to estimate dissociation constants for the peptides. Iron loading into ferritin in Hepes buffer was not affected in the presence of these peptides. In addition, synthetic peptides corresponding to the BC loop of ferritin H and L chains were utilized to localize an interaction site on ferritin for ceruloplasmin. The BC loop of H chain but not L chain of ferritin stimulated the ferroxidase activity of ceruloplasmin. Only the BC loop of ferritin H chain decreased the amount of iron loading into ferritin by ceruloplasmin.
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Affiliation(s)
- S H Juan
- Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA
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Stäubli A, Boelsterli UA. The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury. THE AMERICAN JOURNAL OF PHYSIOLOGY 1998; 274:G1031-7. [PMID: 9696702 DOI: 10.1152/ajpgi.1998.274.6.g1031] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The labile iron pool (LIP) represents the nonferritin-bound, redox-active iron that has been implicated in oxidative stress and cell injury. Here we examined whether alterations in LIP can be detected in cultured murine hepatocytes and whether increases in LIP are related to the oxidative damage inflicted by the redox cycling drug nitrofurantoin (NFT). Early changes in LIP were monitored with the metal-sensitive fluorescent probe calcein (CA), the fluorescence of which is quenched on binding to iron. Short-term exposure (<1 h) to NFT reduced the CA fluorescence signal by 30%, indicating that the amount of LIP-associated iron had increased. Prolonged exposure (2 h) to NFT caused oxidative cell injury. The addition of the cell-permeable ferrous iron chelator 2,2'-bipyridyl not only prevented the quenching of CA fluorescence but also partially protected from NFT toxicity. It is concluded that reductive stress-induced increase in LIP is an essential event that precedes oxidative cell damage in intact hepatocytes.
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Affiliation(s)
- A Stäubli
- Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach, Switzerland
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Tapia G, Troncoso P, Galleano M, Fernandez V, Puntarulo S, Videla LA. Time course study of the influence of acute iron overload on Kupffer cell functioning and hepatotoxicity assessed in the isolated perfused rat liver. Hepatology 1998; 27:1311-6. [PMID: 9581685 DOI: 10.1002/hep.510270517] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Abstract
This study tested the hypothesis that acute iron overload (500 mg/kg) alters Kupffer cell functioning by promoting free radical reactions associated with the respiratory burst of liver macrophages, assessed in the isolated perfused rat liver under conditions of Kupffer cell stimulation by carbon infusion and inactivation by gadolinium chloride pretreatment. Total serum and hepatic iron levels were markedly enhanced compared with control values 2 to 24 hours after iron treatment. Total liver O2 uptake progressively increased by iron overload reaching a maximum at 6 hours after treatment, an effect that was completely blocked by GdCl3. Concomitantly, carbon-induced GdCl3-sensitive liver O2 uptake was either enhanced by 119% at 2 hours after iron overload, diminished compared with control values at 4 hours, or abolished at 6 hours. Iron-overloaded rats showed a marked increase in liver sinusoidal lactate dehydrogenase efflux at 4 and 6 hours after treatment, an effect that is exacerbated by carbon infusion and reduced (69%-89%) by GdCl3 pretreatment. Both basal and carbon-induced lactate dehydrogenase effluxes returned to control values at 24 hours after iron overload concomitantly with depression of the basal O2 uptake, without development of iron-induced GdCl3-sensitive respiration or Kupffer cell activation by carbon infusion. It is concluded that iron overload induces a derangement in the Kupffer cell functional status represented by early increases in macrophage-dependent respiratory activity, which may contribute to the concomitant liver injury that developed and to the impairment of both hepatic respiration and the macrophage response to particle stimulation observed at later times after treatment.
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Affiliation(s)
- G Tapia
- Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago
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Juan SH, Guo JH, Aust SD. Loading of iron into recombinant rat liver ferritin heteropolymers by ceruloplasmin. Arch Biochem Biophys 1997; 341:280-6. [PMID: 9169016 DOI: 10.1006/abbi.1997.9967] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We have reported previously that the heavy chain of ferritin is required for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for ceruloplasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferritin were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infections Autographa californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the infection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light (L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H chain in the two heteropolymers. The extent of iron incorporation decreased with additional ceruloplasmin. Iron incorporation into rat liver ferritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up to 8:1. Iron loading into horse spleen ferritin, found to have one H chain, appeared similar to that for recombinant ferritin, having only one H chain. Therefore, we propose that the optimal molar ratio of ceruloplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin. These results also suggest that the iron loading channel is contained within a single H chain subunit.
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Affiliation(s)
- S H Juan
- Biotechnology Center, Utah State University, Logan 84322-4705, USA
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19
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Abstract
Recent studies suggest that increased hepatic iron may impair the response to interferon therapy in patients with chronic hepatitis C. We reviewed the records and liver biopsies of 72 patients with chronic hepatitis C to determine the prevalence of iron overload and to evaluate whether there is a correlation between serum and hepatic iron concentrations and activity of liver disease. Patients with other causes of liver disease or iron overload were excluded. Necroinflammatory activity and fibrosis were evaluated using modified Knodell score. Hepatic iron was assessed using Brissot's grading system. Increased serum iron and ferritin levels were found in 29% and 43% patients, respectively. Hepatic iron grades 0, I, II, III, and IV were present in 37%, 35%, 25%, 3%, and 0% of patients, respectively. A significant correlation was found between hepatic iron grade and serum ferritin (P = .0001). There was no correlation between hepatic iron grade and histological activity index or fibrosis score. In summary, we found a high proportion of patients with chronic hepatitis C had mild to moderate increase in hepatic iron content even when patients with alcoholism and recurrent transfusions were excluded. However, very few patients had severely increased iron load.
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Affiliation(s)
- S Haque
- Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA
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Izumi N, Enomoto N, Uchihara M, Murakami T, Ono K, Noguchi O, Miyake S, Nouchi T, Fujisawa K, Marumo F, Sato C. Hepatic iron contents and response to interferon-alpha in patients with chronic hepatitis C. Relationship to genotypes of hepatitis C virus. Dig Dis Sci 1996; 41:989-94. [PMID: 8625774 DOI: 10.1007/bf02091542] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 +/- 100 vs 875 +/- 110 micrograms/g liver, P < 0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection.
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Affiliation(s)
- N Izumi
- Department of Internal Medicine, Musashino Redcross Hospital, Tokyo, Japan
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21
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Arber N, Konikoff FM, Moshkowitz M, Baratz M, Hallak A, Santo M, Halpern Z, Weiss H, Gilat T. Increased serum iron and iron saturation without liver iron accumulation distinguish chronic hepatitis C from other chronic liver diseases. Dig Dis Sci 1994; 39:2656-9. [PMID: 7995192 DOI: 10.1007/bf02087705] [Citation(s) in RCA: 73] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
One hundred twenty-three patients with chronic liver diseases of various etiologies were evaluated for their iron status. The patients were divided into four distinct groups: chronic hepatitis C (63), chronic hepatitis B (14), B + C (3) and nonviral chronic liver diseases (43). In 107 patients (87%) the chronic liver disease was confirmed by biopsy. Mean serum iron (+/- SD) levels in the above four groups were: 166 +/- 62, 103 +/- 52, 142 +/- 48, and 115 micrograms/dl; iron-binding capacity was 346 +/- 80, 325 +/- 72, 297 +/- 27, and 374 +/- 75 micrograms/dl, and iron saturation 50 +/- 18, 32 +/- 16, 48 +/- 16, and 28 +/- 10%, respectively. Serum ferritin, increased in all four groups, was highest in HCV; however, no evidence of hepatic iron accumulation could be found in any of the patients. There were no significant differences in liver function parameters measured in the four groups. We conclude that serum iron, iron saturation, and ferritin are increased in patients with hepatitis C in comparison to hepatitis B or other nonviral, nonhemochromatotic liver diseases. The increased iron status in hepatitis C patients is not manifested by increased liver iron. Awareness of these distinct features of chronic hepatitis C is essential in the diagnosis and treatment of chronic liver diseases.
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Affiliation(s)
- N Arber
- Department of Gastroenterology, Tel-Aviv Medical Center, Ichilov Hospital, Israel
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22
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Mössbauer spectroscopic study of the forms of iron in normal human liver and spleen tissue. ACTA ACUST UNITED AC 1994. [DOI: 10.1007/bf02064626] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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23
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A GTP-binding protein inhibits a gastric housekeeping chloride channel via intracellular production of superoxide. J Biol Chem 1994. [DOI: 10.1016/s0021-9258(17)31533-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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24
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Hiraishi H, Terano A, Razandi M, Pedram A, Sugimoto T, Harada T, Ivey KJ. Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: status of cellular iron in mediating injury. J Cell Physiol 1994; 160:132-4. [PMID: 8021293 DOI: 10.1002/jcp.1041600116] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of 51Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase-induced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose-dependent increase of 51Ci release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe3+) prevented glucose oxidase-induced injury. The presence of phenanthroline (a chelator of divalent Fe2+) prevented glucose oxidase-induced 51Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non-oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe3+ and Fe2+, respectively; and (3) reduction of cellular stored iron (Fe3+) to Fe2+ may be prerequisite for mediation of oxidant-induced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase-derived superoxide.
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Affiliation(s)
- H Hiraishi
- Department of Medicine, Veterans Affairs Medical Center, Long Beach, California 90822
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25
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Badr MZ. Controversial role of intracellular iron in the mechanisms of chemically-induced hepatotoxicity. JOURNAL OF BIOCHEMICAL TOXICOLOGY 1994; 9:25-9. [PMID: 8151629 DOI: 10.1002/jbt.2570090105] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Hepatotoxicity induced by various therapeutic agents, industrial chemicals and environmental pollutants is a well-recognized phenomenon. These chemicals are known to cause liver damage that is localized to either periportal or centrilobular regions of the liver lobule (1-3). Depending on dose, duration, and route of exposure, the resultant liver injury may regress or progress and becomes irreversible (1). Mechanisms involved in this selective, localized toxicity have been the target of extensive research efforts, and many studies produced conflicting results. As depicted in Figure 1, although many investigators implicate iron and lipid peroxidation in this process (4-9), others dispute such assertions (10-12).
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Affiliation(s)
- M Z Badr
- Division of Pharmacology, University of Missouri-Kansas City 64108-2792
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26
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Halliday JW, Ramm GA, Moss D, Powell LW. A new look at ferritin metabolism. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1994; 356:149-56. [PMID: 7887219 DOI: 10.1007/978-1-4615-2554-7_17] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Affiliation(s)
- J W Halliday
- Department of Medicine, University of Queensland/Queensland Institute of Medical Research, Brisbane, Australia
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27
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Hiraishi H, Terano A, Sugimoto T, Harada T, Razandi M, Ivey KJ. Protective role of intracellular superoxide dismutase against extracellular oxidants in cultured rat gastric cells. J Clin Invest 1994; 93:331-8. [PMID: 8282804 PMCID: PMC293772 DOI: 10.1172/jci116964] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
We examined the role of intracellular superoxide dismutase (SOD) as an antioxidant by studying the effect of diethyldithiocarbamate (DDC) on extracellular H2O2-induced damage in cultured rat gastric mucosal cells. 51Cr-labeled monolayers from rat stomachs were exposed to glucose oxidase-generated H2O2 or reagent H2O2, which both caused a dose-dependent increase in 51Cr release. DDC dose-dependently enhanced 51Cr release by hydrogen peroxide, corresponding with inhibition of endogenous SOD activity. This inhibition was not associated either with modulation of other antioxidant defenses, or with potentiation of injury by nonoxidant toxic agents. Enhanced hydrogen peroxide damage by DDC was significantly prevented by chelating cellular iron with deferoxamine or phenanthroline. Inhibition of cellular xanthine oxidase (possible source of superoxide production) by oxypurinol neither prevented lysis by hydrogen peroxide nor diminished DDC-induced sensitization to H2O2. We conclude that (a) extracellular H2O2 induces dose dependent damage to cultured gastric mucosal cells; (b) intracellular SOD plays an important role in preventing H2O2 damage; (c) generation of superoxide seems to occur intracellularly after exposure to H2O2, but independent of cellular xanthine oxidase; and (d) cellular iron mediates the damage by catalyzing the production of more reactive species from superoxide and H2O2, the process which causes ultimate cell injury.
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Affiliation(s)
- H Hiraishi
- Department of Medicine, Veterans Affairs Medical Center, Long Beach, California 90822
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28
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Arce DS, Keen CL. Effects of marginal and severe iron deficiency on hepatic proteins in developing rats are reversible with dietary iron repletion. Reprod Toxicol 1993; 7:61-72. [PMID: 8448418 DOI: 10.1016/0890-6238(93)90011-u] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Transferrin, metallothionein, cytochrome P-450, and the in vitro formation of DNA-benzo[a]pyrene adducts were studied in the offspring of dams that were fed diets moderately or severely deficient in iron (Fe). The study was designed to determine whether Fe deficiency-induced alterations were reversible or if they persisted with post-weaning iron repletion. Throughout gestation and lactation the dams were fed a Control diet = 120 micrograms Fe/g diet, a Marginal Iron diet = 11 micrograms Fe/g diet, or a Low Iron diet = 7 micrograms Fe/g diet. On day 14 of lactation, 4 pups per litter were killed. On day 21, the dams were killed. Half of the remaining pups in each litter were fed their respective diets until they were killed on day 42 (Marginal Iron-Marginal Iron and Low Iron-Low Iron groups). The other half were fed the Control diet (Marginal Iron-Control and Low Iron-Control groups). The dietary intake of the Restricted Fed offspring was matched to rats in the Low Iron-Low Iron group. Offspring in the iron-deficient groups had hematocrits, hemoglobin concentrations, and liver iron levels that were lower than Controls. Day 42 offspring in the iron-deficiency groups had a lower food intake and higher liver zinc and copper levels than Controls. Day 14 Marginal and Low Iron pups had liver metallothionein levels that were lower than Controls. Day 42 Restricted Fed offspring had liver metallothionein levels that were higher than all other groups. Cytochrome P-450 levels and the in vitro formation of benzo[a]pyrene-DNA adducts were higher in Low Iron-Low Iron males than in Control males. Ethoxycoumarin O-deethylase activity was higher in day 42 Low Iron-Low Iron offspring than in Controls. These results show that the iron deficiency-induced alterations were transient, reversible with iron repletion, and in the case of cytochrome P-450 and ethoxycoumarin O-deethylase activity, dependent on the age and sex of the animal.
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Affiliation(s)
- D S Arce
- Biochemical Research and Development, Miles Inc., Berkeley, California 94701
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29
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Hiraishi H, Terano A, Razandi M, Sugimoto T, Harada T, Ivey K. Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells. J Biol Chem 1992. [DOI: 10.1016/s0021-9258(18)42112-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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30
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Di Bisceglie AM, Axiotis CA, Hoofnagle JH, Bacon BR. Measurements of iron status in patients with chronic hepatitis. Gastroenterology 1992; 102:2108-13. [PMID: 1587431 DOI: 10.1016/0016-5085(92)90339-z] [Citation(s) in RCA: 269] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Eighty patients with chronic viral hepatitis were screened for evidence of iron overload. Elevated serum iron values were noted in 36% of cases; serum ferritin values were above normal in 30% of men and 8% of women. Twenty-eight additional patients with chronic hepatitis for whom liver tissue was available for determination of iron content were evaluated to study the significance of iron overload in association with chronic hepatitis. Although 46% had elevated serum iron, ferritin, or transferrin-saturation levels, the hepatic iron concentration was elevated in only four cases, and the hepatic iron index was in the range for hereditary hemochromatosis (greater than 2.0) in only two of these. Serum aspartate aminotransferase activities correlated with serum ferritin levels in these patients, suggesting that ferritin and iron levels were increased in serum because of their release from hepatocellular stores associated with necrosis. Thus, in patients with chronic hepatitis in whom hereditary hemochromatosis is suspected, a liver biopsy should be performed with quantitation of hepatic iron and calculation of the hepatic iron index to confirm the diagnosis.
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Affiliation(s)
- A M Di Bisceglie
- Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland
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31
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Britton RS, Ferrali M, Magiera CJ, Recknagel RO, Bacon BR. Increased prooxidant action of hepatic cytosolic low-molecular-weight iron in experimental iron overload. Hepatology 1990; 11:1038-43. [PMID: 2365281 DOI: 10.1002/hep.1840110620] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
In the iron-loaded liver there may be an increase in the putative intracellular transit pool of iron, components of which could be catalytically active in stimulating lipid peroxidation. To study the levels of low-molecular-weight, catalytically active iron in the liver, cytosolic ultrafiltrates were tested in an assay containing rat liver microsomes and NADPH. Malondialdehyde production was used as an index of lipid peroxidation. This assay system was sensitive enough to detect 0.25 mumol/L ferrous iron; progressive but non-linear increases in malondialdehyde were produced as the iron concentration was increased to 5 mumol/L. Ultrafiltrates from hepatic cytosol of iron-loaded rats had greater prooxidant action than did those from controls. When added to the assay, deferoxamine, an iron chelator, completely suppressed the prooxidant action of hepatic ultrafiltrates, showing that this activity is iron-dependent. Deferoxamine administered intraperitoneally to control animals at a dose of 1 gm/kg completely inhibited the prooxidant effect of hepatic ultrafiltrates prepared from rats killed after 1, 2 and 3 hr. Partial inhibition was observed at 4 hr; by 6 hr the inhibitory effect of deferoxamine was completely lost. Administration of deferoxamine (1 gm/kg intraperitoneally, 1 hr before killing) completely inhibited the prooxidant action of hepatic ultrafiltrates in moderately iron-loaded rats and controls but had no protective effect in heavily iron-loaded rats. These results support the concept that iron overload results in an increase in a hepatic cytosolic pool of low-molecular-weight iron that is catalytically active in stimulating lipid peroxidation. This pool can be chelated transiently in vivo by deferoxamine in moderate, but not heavy, iron overload.
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Affiliation(s)
- R S Britton
- Department of Medicine, Louisiana State University School of Medicine, Shreveport 71130
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32
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Sciot R, Verhoeven G, Van Eyken P, Cailleau J, Desmet VJ. Transferrin receptor expression in rat liver: immunohistochemical and biochemical analysis of the effect of age and iron storage. Hepatology 1990; 11:416-27. [PMID: 2312055 DOI: 10.1002/hep.1840110313] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Hepatic transferrin receptors were studied in normal male rats at 1 to 59 wk after weaning, using immunohistochemical and biochemical techniques. The number of transferrin receptors measured and the intensity of the staining in situ decreased rapidly during the first 10 wk of life and more slowly thereafter. Immunohistochemistry further demonstrated changes in the topographical and (sub)cellular localization of the transferrin receptor. In the young rat livers, staining was almost exclusively present on hepatocytes in acinar zone 2 + 3 in a honeycomb to sinusoidal pattern. With aging, a panacinar heterogeneous and mainly sinusoidal staining of hepatocytes was more frequent. Kupffer cell positivity was more obvious as compared with the young rat livers. The observed changes in transferrin receptor expression may partly be explained by age-dependent alterations in DNA synthesis and proliferative potential of the liver cells. A series of rats were iron loaded with carbonyl iron up to 39 wk and "unloaded" by administration of a normal diet during 20 wk. In these animals, serial histochemical studies showed predominantly parenchymal (7 to 14 wk), mixed parenchymal and reticuloendothelial (39 wk) and almost exclusive reticuloendothelial siderosis (59 wk). In the siderotic livers transferrin receptor numbers tended to be lower than in the controls with significant differences after 14 and 39 wk. Immunohistochemistry showed decreased parenchymal but increased reticuloendothelial transferrin receptor expression with iron load. After the period of unloading, parenchymal transferrin receptors were virtually absent despite the negligible siderosis of these cells. In contrast, siderotic reticuloendothelial cells were intensely positive. These findings support down-regulation of parenchymal transferrin receptor resulting from iron storage. However, the positivity of siderotic reticuloendothelial cells and the absence of re-emergence of parenchymal receptors in conditions of minimal parenchymal and prominent reticuloendothelial siderosis need further elucidation.
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Affiliation(s)
- R Sciot
- Laboratorium voor, Departement Medische Navorsing, Katholieke Universiteit Leuven, Belgium
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33
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Nilsen T, Romslo I. Pyrophosphate-loaded hepatocytes show increased iron uptake from transferrin. Scand J Clin Lab Invest 1990; 50:19-25. [PMID: 2156334 DOI: 10.1080/00365519009091560] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Exogenously added pyrophosphate was found associated with hepatocytes in suspension, particularly in the presence of calcium ions. The cell-associated pyrophosphate was recovered with the stroma fraction. Pyrophosphate thus bound did not affect cellular ATP, leakage of lactate dehydrogenase or trypan blue exclusion. Compared to control cells, pyrophosphate-loaded hepatocytes showed an approximately 70% increase in iron accumulation from transferrin. The excess iron was handled by the pyrophosphate-loaded cells as control cells. The relevance of pyrophosphate to hepatocyte iron uptake is discussed.
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Affiliation(s)
- T Nilsen
- Department of Clinical Chemistry, University of Trondheim, Norway
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34
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Affiliation(s)
- B R Bacon
- Department of Medicine, Louisiana State University School of Medicine, Shreveport 71130-3932
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35
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Britton RS, O'Neill R, Bacon BR. Hepatic mitochondrial malondialdehyde metabolism in rats with chronic iron overload. Hepatology 1990; 11:93-7. [PMID: 2295476 DOI: 10.1002/hep.1840110116] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Peroxidative decomposition of mitochondrial membrane phospholipids with subsequent mitochondrial dysfunction is a postulated mechanism of liver cell injury in parenchymal iron overload. Malondialdehyde is formed when polyunsaturated fatty acids of membrane phospholipids undergo peroxidative decomposition, and it is metabolized by aldehyde dehydrogenase. We studied mitochondrial metabolism of malondialdehyde in rats with chronic dietary iron overload. Hepatic malondialdehyde concentrations were significantly increased in iron-loaded livers, and mitochondrial respiratory control ratios using glutamate as a substrate were decreased by 47% largely owing to reductions in state 3 respiration. When exogenous malondialdehyde was added to mitochondrial fractions, there was significantly less metabolism of malondialdehyde in mitochondria of iron-loaded livers as compared with controls. In addition, there was a 28% decrease in mitochondrial aldehyde dehydrogenase in iron-loaded livers but no change in cytosolic aldehyde dehydrogenase. Increased hepatic malondialdehyde in chronic iron overload may result from a combination of increased production and decreased metabolism of malondialdehyde, both of which may be due to iron-induced mitochondrial lipid peroxidation.
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Affiliation(s)
- R S Britton
- Department of Medicine, Case Western Reserve University School of Medicine, Cleveland Metropolitan General Hospital, Ohio 44109
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36
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Sciot R, de Vos R, van Eyken P, van der Steen K, Moerman P, Desmet VJ. In situ localization of melanotransferrin (melanoma-associated antigen P97) in human liver. A light- and electronmicroscopic immunohistochemical study. LIVER 1989; 9:110-9. [PMID: 2540389 DOI: 10.1111/j.1600-0676.1989.tb00387.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Using an indirect immunoperoxidase technique on frozen sections with the monoclonal antibody 96.5, we investigated the in situ distribution of melanotransferrin, a transferrin (Tf) and transferrin receptor (TfR) related glycoprotein, in human liver. Specimens included normal liver, liver in iron overload, hepatocellular carcinoma, angioma and foetal liver. On light microscopy, immunoreactivity was almost exclusively present on sinusoidal lining cells, apparently endothelial cells; the pattern was similar in normal and in iron-loaded liver. A gradient of more enhanced staining in acinar zone II and III was observed. The endothelial localization of the staining was supported by the positivity of the central vein endothelium and of the angiomas. Immunoelectron microscopy on three liver specimens showed positivity on sinusoidal endothelial cells but not on Ito and Kupffer cells. In addition, positivity on rough endoplasmic reticulum vesicles of some hepatocytes was also present. Four hepatocellular carcinomas showed an intense staining in tumour cells, 3 were weakly positive and 3 were negative. In the foetal livers, the central vein endothelium was positive from 21 weeks of gestation onward and additional positivity of zone III sinusoidal endothelial cells was present from 27 weeks on. The present results show that in the liver melanotransferrin has a localization different from Tf and the TfR. These latter molecules are predominantly localized in parenchymal cells. In addition, there does not appear to be a coordinate regulation secondary to iron storage, between melanotransferrin, Tf and the TfR. The observed gradient in the staining pattern in foetal and adult liver specimens further supports the heterogeneity of the endothelial cell population in the liver and suggests a developmental relationship between endothelial cells of sinusoids and central vein.
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Affiliation(s)
- R Sciot
- Department of Pathology II, University Hospital St. Rafaël, Catholic University of Leuven, Belgium
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37
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Sciot R, van Eyken P, Facchetti F, Callea F, van der Steen K, van Dijck H, van Parys G, Desmet VJ. Hepatocellular transferrin receptor expression in secondary siderosis. LIVER 1989; 9:52-61. [PMID: 2646506 DOI: 10.1111/j.1600-0676.1989.tb00378.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
We investigated the hepatocellular transferrin receptor expression in 55 human liver specimens with secondary siderosis, with an indirect immunoperoxidase technique on frozen sections using 3 monoclonal anti-transferrin receptor antibodies. For comparison, specimens were also stained with the monoclonal antibody BK19.9, recognizing an antigen which is biochemically similar to the transferrin receptor, and with a monoclonal antibody against the epidermal growth factor receptor. The degree of iron overload was estimated semi-quantitatively, taking into account hepatocellular and Kupffer cell iron deposition. In 47 out of 55 specimens hepatocellular transferrin receptor expression was present. The positivity was predominantly localized on hemosiderin-free hepatocytes. With increasing hepatocellular iron deposition, the proportion of cases with absent transferrin receptor immunoreactivity increased. This supports the previously reported disappearance of hepatocellular transferrin receptor expression in primary hemochromatosis cases with severe iron deposition. However, the transferrin receptor negative cases included four specimens in which Kupffer cell iron deposition clearly exceeded hepatocyte iron load. This finding suggests that in addition to hepatocellular iron load other factors may regulate the expression of parenchymal transferrin receptors in iron overload diseases. These may include plasma levels of various iron sources and/or Kupffer cell iron load. The iron deposition did not influence the staining of the hepatocellular epidermal growth factor receptor nor the Kupffer cell staining by the BK19.9 antibody. This confirms the specificity of the findings concerning the behaviour of the transferrin receptor in secondary siderosis.
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Affiliation(s)
- R Sciot
- Department of Pathology, Universitair Ziekenhuis, St. Rafaël, Leuven, Belgium
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38
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Abstract
In both hereditary hemochromatosis and in the various forms of secondary hemochromatosis, there is a pathologic expansion of body iron stores due mainly to an increase in absorption of dietary iron. Excess deposition of iron in the parenchymal tissues of several organs (e.g. liver, heart, pancreas, joints, endocrine glands) results in cell injury and functional insufficiency. In the liver, the major pathological manifestations of chronic iron overload are fibrosis and ultimately cirrhosis. Evidence for hepatotoxicity due to iron has been provided by several clinical studies, however the specific pathophysiologic mechanisms for hepatocellular injury and hepatic fibrosis in chronic iron overload are poorly understood. The postulated mechanisms of liver injury in chronic iron overload include (a) increased lysosomal membrane fragility, perhaps mediated by iron-induced lipid peroxidation, (b) peroxidative damage to mitochondria and microsomes resulting in organelle dysfunction, (c) a direct effect of iron on collagen biosynthesis and (d) a combination of all of the above.
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Affiliation(s)
- B R Bacon
- Department of Medicine, Louisiana State University School of Medicine, Shreveport 71130-3932
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39
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Lombard M, Bomford A, Hynes M, Naoumov NV, Roberts S, Crowe J, Williams R. Regulation of the hepatic transferrin receptor in hereditary hemochromatosis. Hepatology 1989; 9:1-5. [PMID: 2642288 DOI: 10.1002/hep.1840090102] [Citation(s) in RCA: 33] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The liver is the main site of iron accumulation and pathologic sequelae in hereditary hemochromatosis. Whether this is a result solely of inappropriately increased absorption of iron by the gastrointestinal tract or a more generalized regulatory failure of iron balance is unknown. Using immunohistochemical techniques, we have examined the effects of therapeutic changes in liver iron stores on the expression of the hepatic transferrin receptor in hereditary hemochromatosis. Ten patients with untreated hereditary hemochromatosis had no detectable staining for transferrin receptor in their liver biopsies. All had increased hepatic ferritin (mean = 19.9 micrograms per mg protein, range = 1 to 31.7 micrograms per mg protein) and hepatic iron levels (mean = 36.2 micrograms per mg protein, range = 3.6 to 69.9 micrograms per mg protein). In contrast, hepatocyte transferrin receptor was detected in seven patients in whom hepatic iron stores were markedly depleted by venesection (hepatic ferritin mean = 0.32 microgram per mg protein, range = 0.16 to 0.53 microgram per mg protein; hepatic iron mean = 0.98 microgram per mg protein, range = 0.3 to 2.1 micrograms per mg protein). Sequential data from one patient confirmed the reexpression of receptor in response to therapeutic iron depletion, whereas data from another patient studied during treatment illustrated a reciprocal relationship between liver tissue distribution of iron and expression of transferrin receptor. The finding that appropriate physiologic regulation of the hepatic transferrin receptor operates in hereditary hemochromatosis does not support the concept of a generalized defect in receptor-mediated uptake of transferrin-bound iron.
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Affiliation(s)
- M Lombard
- Liver Unit, Kings College Hospital, London, United Kingdom
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De Vos R, Sciot R, van Eyken P, Desmet VJ. Immunoelectron microscopic localization of hepatic transferrin receptors in human liver with and without iron overload. VIRCHOWS ARCHIV. B, CELL PATHOLOGY INCLUDING MOLECULAR PATHOLOGY 1988; 55:11-7. [PMID: 2898829 DOI: 10.1007/bf02896555] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
The expression of transferrin receptors (TfR's) has been investigated in eight liver biopsy specimens (four from patients without demonstrable iron and four from patients with iron storage due to primary hemochromatosis (HC)) using immunoelectron microscopy to demonstrate TfR's by the simultaneous application of two specific monoclonal antibodies (OKT9 and B3/25) to tissue chopper sections. In the four specimens without iron overload, hepatocytes, but not sinusoidal lining cells, stained positively and immunoreactivity was mainly localized in the cytoplasm. Positively stained cisternae of the endoplasmic reticulum indicated synthesis of the TfR. The presence of TfR's on segments and coated invaginations of the sinusoidal membrane and in small, but otherwise unidentified vesicles in the cytoplasm is compatible with endo-/exocytotic transport and recycling of TfR's as demonstrated by biochemical studies. Occasional positively stained material in canalicular lumina together with positively stained canalicular microvilli and pericanalicular vesicles suggest that transcellular transport may be an additional pathway for TfR's. In three biopsies showing severe iron overload due to HC, TfR immunoreactivity was completely absent. The remaining specimen showing HC, exhibited relatively mild iron overload and showed only a few positively stained hepatocytes. This supports the previously reported disappearance of hepatic TfR expression in HC when iron overload is severe.
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Affiliation(s)
- R De Vos
- Department of Medical Research, Catholic University of Leuven, Belgium
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Irving MG, Halliday JW, Powell LW. Association between alcoholism and increased hepatic iron stores. Alcohol Clin Exp Res 1988; 12:7-13. [PMID: 3279862 DOI: 10.1111/j.1530-0277.1988.tb00124.x] [Citation(s) in RCA: 62] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Although alcoholic liver disease is often associated with some increase in hepatic iron stores, it is now established that when gross iron overload is present, this is due to genetic hemochromatosis. Furthermore, there appears to be a critical iron concentration necessary for the induction of hepatic fibrosis. Lipid peroxidation induced by ethanol and/or iron would appear to play a major role in hepatic damage in both humans and experimental animals. Although the exact mechanism(s) of induction of lipid peroxidation by ethanol and iron remains to be elucidated, both toxins can exert a synergistic effect upon hepatic lipid peroxidation. Iron overload has also been shown to stimulate directly hepatocyte and hepatic procollagen mRNA expression, which is further stimulated by ethanol. The observed synergism between iron and alcohol with respect to both hepatic lipid peroxidation and collagen biosynthesis offers a possible explanation of the apparent early onset of fibrosis and cirrhosis in patients with iron overload who have an excessive alcohol intake.
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Affiliation(s)
- M G Irving
- Department of Medicine, University of Queensland, Royal Brisbane Hospital, Australia
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Sciot R, Paterson AC, van Eyken P, Callea F, Kew MC, Desmet VJ. Transferrin receptor expression in human hepatocellular carcinoma: an immunohistochemical study of 34 cases. Histopathology 1988; 12:53-63. [PMID: 2836292 DOI: 10.1111/j.1365-2559.1988.tb01916.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Using a panel of five monoclonal anti-transferrin receptor antibodies, we investigated the transferrin receptor expression in 34 human hepatocellular carcinomas of Belgian (n = 6), Italian (n = 7) and South African (n = 21) origin. For comparison the tumours were also stained with the monoclonal antibody BK 19.9, recognizing an antigen biochemically similar to the transferrin receptor, and with a monoclonal antibody against the epidermal growth factor receptor. Hepatocellular carcinomas express large amounts of transferrin receptors as demonstrated by the intense transferrin receptor immunostaining in 33/34 cases. Differences in staining pattern between and within the tumours were not related to the degree of tumour differentiation, nor to the origin or race of the patient. In 15 cases which included non-tumoural tissue, the tumour was more intensely stained than the surrounding liver parenchyma. The BK 19.9 immunoreactivity was generally weaker and mainly involved stromal cells, except in three cases where an intense staining of the tumour cells was seen. The epidermal growth factor receptor staining was also weaker and only in four cases was the immunoreactivity of the tumour stronger than the surrounding parenchyma. Demonstration of the transferrin receptor may be useful for the detection of malignant foci in liver biopsies. This may be of particular interest in the histological investigation of minute hepatocellular carcinomas.
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Affiliation(s)
- R Sciot
- Laboratory for Histo- and Cytochemistry, Catholic University of Leuven, Belgium
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Britton RS, Bacon BR, Recknagel RO. Lipid peroxidation and associated hepatic organelle dysfunction in iron overload. Chem Phys Lipids 1987; 45:207-39. [PMID: 3319227 DOI: 10.1016/0009-3084(87)90066-1] [Citation(s) in RCA: 66] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Iron overload can have serious health consequences. Since humans lack an effective means to excrete excess iron, overload can result from an increased absorption of dietary iron or from parenteral administration of iron. When the iron burden exceeds the body's capacity for safe storage, the result is widespread damage to the liver, heart and joints, and the pancreas and other endocrine organs. Clear evidence is now available that iron overload leads to lipid peroxidation in experimental animals, if sufficiently high levels of iron are achieved. In contrast, there is a paucity of data regarding lipid peroxidation in patients with iron overload. Data from experiments using an animal model of dietary iron overload support the concept that iron overload results in an increase in an hepatic cytosolic pool of low molecular weight iron which is catalytically active in stimulating lipid peroxidation. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in homogenates of liver samples from both iron-loaded human subjects and experimental animals. Some current hypotheses focus on the possibility that the demonstrated functional abnormalities in organelles of the iron-loaded liver may play a pathogenic role in hepatocellular injury and eventual fibrosis. The recent demonstration that hepatic fibrosis is produced in animals with long-term dietary iron overload will allow this model to be used to further investigate the relationship between lipid peroxidation and hepatic injury in iron overload.
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Affiliation(s)
- R S Britton
- Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106
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Sciot R, Paterson AC, Van den Oord JJ, Desmet VJ. Lack of hepatic transferrin receptor expression in hemochromatosis. Hepatology 1987; 7:831-7. [PMID: 3308665 DOI: 10.1002/hep.1840070507] [Citation(s) in RCA: 69] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The major part of hepatocellular iron is derived from uptake of transferrin-bound iron by means of nonspecific fluid-phase endocytosis and specific, saturable binding on high-affinity transferrin receptors. We investigated the expression of transferrin receptors on hepatocytes in liver biopsies of 22 cases of hemochromatosis (21 primary hemochromatosis and 1 secondary hemochromatosis), using immunohistochemical demonstration of the human transferrin receptor with the specific monoclonal antibody OKT9. Fifty liver biopsies (normal and pathological) without demonstrable iron storage (Perls' stain negative) served as controls. In the controls, membranous and/or cytoplasmic transferrin receptor expression was always present on hepatocytes, albeit in variable numbers and patterns without obvious relation to the underlying liver disease. In 19 of 22 hemochromatosis cases with severe iron overload, OKT9 immunoreactivity on hepatocytes was completely absent. Three hemochromatosis cases showed few hepatocytes positive for OKT9. One showed mild iron overload, while the second, a successfully treated case, was free of iron. The remaining hemochromatosis case was a known alcoholic with severe iron overload. Since OKT9 binding to the transferrin receptor is not blocked by previous binding of transferrin, the findings show that in advanced hemochromatosis hepatocytes do not express transferrin receptors. This finding is in keeping with the inverse relation between transferrin receptor expression and exogenous iron supply in various cell cultures. These results indicate that in hemochromatosis,apparently as a result of progressive iron overload,transferrin receptor expression on hepatocytes disappears.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- R Sciot
- Departement Medische Navorsing, Universitair Ziekenhuis St. Rafaël, Leuven, Belgium
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Pollera CF, Ameglio F, Reina S, Nardi M, Abbolito MR, Parracino C. Changes in serum iron levels following very high-dose cisplatin. Cancer Chemother Pharmacol 1987; 19:257-60. [PMID: 3581420 DOI: 10.1007/bf00252983] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
A four-fold (P less than 0.001) mean increase in iron levels was found in 18 patients (a total of 36 courses of therapy) with ovarian cancer at the end of a 5-day course of cisplatin (40 mg/m2 per day every 4-5 weeks). The kinetics of these modifications began very early (24-48 h after initiation of therapy): they reached their maximum on the 4th-5th day, coinciding with the last drug administration, and basal levels were recovered after the 10th day. A subsequent eight-fold average increase (P less than 0.001) in ferritin serum levels, beginning 2 days after the iron changes, was observed, but showed a slower regression (after the 15th day). Reticulocyte counts were lowered (P less than 0.001) with the same time-course of the iron increases, but returned to pretreatment levels within 2 weeks. Total bilirubin and serum glutamate-pyruvate transaminase showed significantly delayed increases compared with iron. The results are in keeping with a reduced iron utilization by the erythroid precursors, but other mechanisms cannot be excluded. There is no statistical correlation between the early iron increases and the subsequent hemoglobin nadir values.
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Wright TL, Brissot P, Ma WL, Weisiger RA. Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 1986. [DOI: 10.1016/s0021-9258(18)67473-3] [Citation(s) in RCA: 89] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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Sibille JC, Octave JN, Schneider YJ, Trouet A, Crichton R. Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature. EUROPEAN JOURNAL OF BIOCHEMISTRY 1986; 155:47-55. [PMID: 3948880 DOI: 10.1111/j.1432-1033.1986.tb09457.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.
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