IRON-INDUCED LIVER INJURY

PATHOPHYSIOLOGY OF LIVER DISEASE

1089-3261 /00 $15.00

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IRON-INDUCED LIVER INJURY Herbert L. Bonkovsky, MD, and Richard W. Lambrecht, PhD

BASIC ASPECTS OF IRON CHEMISTRY

Iron is essential for life, but both iron deficiency and iron overload pose significant and potentially fatal health risks.64Two key chemical features that make iron essential are its ability to interact with oxygen (and, in some cases, with sulfur) and its ability to change oxidation states readily between the ferrous (Fe[II]) and ferric (Fe [111]) forms. In the presence of hydrogen peroxide, however, these two features of iron chemistry also allow the generation of powerful oxidants by means of Fenton and iron-catalyzed Haber-Weiss reactions (Fig. l).24, Thus, although iron is essential, it is also potentially deleterious, and it is not surprising that the uptake, metabolism, and distribution of this metal are normally tightly regulated. The body of a typical healthy adult man contains between 3 and 4 g of iron,= and that of a normal woman contains somewhat less. Some of this iron (- 1000 mg) is sequestered in the form of storage compounds, mostly in ferritin and hemosiderin.22The majority of iron in the body is in the form of heme and is associated with hemoglobin (- 2300 mg), myoglobin (- 320 mg) or with heme-containing enzymes (- 80 Supported by a grant from USPHS, NIH (DK 38825 to Herbert L. Bonkovsky). The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the University of Massachusetts Medical School, UMass Memorial Health Care, or the US Public Health Service.

From the Departments of Medicine (HLB, RWL), Biochemistry (HLB) and Molecular Biology (HLB) University of Massachusetts Medical School; and the Division of Digestive Disease and Nutrition, The Liver Center, and The Center for Study of Disorders of Iron and Porphyrin Metabolism, UMass Memorial Health Care, Worcester, Massachusetts

CLINICS IN LIVER DISEASE VOLUME 4 NUMBER 2 MAY 2000

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Fe (11)

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mg). These various hemoproteins can increase the reactivity of iron and the rates of the corresponding reactions by a factor of 1000. A Partial List of Mammalian Proteins That Use Heme as a Cofactor or That Bind Heme With High Affinity cata1ase cystathionine beta-synthase guanylate cyclase, soluble heme binding proteins (HBP), high affinity HBP23 hemopexin liver fatty acid binding protein p22 HBP heme oxygenases hemoglobin microsomal cytochromes, including cytochrome P-450 (a large number of isozymes) cytochrome b, mitochondria1 cytochromes, including cytochrome c myeloperoxidase myoglobin nitric oxide synthases sulfite oxidase tryptophan 2,3-dioxygenase Approximately 100 mg of iron can also be found in non-heme enzymes. A particularly interesting example of a dual-function nonheme enzyme is iron regulatory protein-1 (IRP-1)/ cytoplasmic aconitase, which is illustrated in Figure 2.

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Ferritin mRNA

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Figure 2. The molecular regulation of key genes and proteins involved in iron homeostasis. Iron enters dividing cells of higher eukaryotes chiefly by way of the transferrin receptor (TfR). Once inside cells, the iron may be considered to be in 1 of 3 pools, although overlap and interchanges between these pools may occur. Iron is used in a variety of metabolic processes, or iron may be sequestered in ferritin. lntracellular iron also serves to regulate the iron regulatory proteins (IRPs) such that, when iron is abundant, IRP-1 is in its fully iron-loaded [4Fe-4S] state, which has high aconitase enzymatic activity but low affinity for iron regulatory elements (IREs) (IRPOfl).When iron is scarce, an iron is removed from the iron-sulfur cluster, altering the conformation of the IRP and increasing its binding affinity to IREs. This IRP,, state results in decreased translation of ferritin or the erythroid form of ALA synthase (E-ALA synthase) mRNAs and increased stability of the transferrin receptor mRNA by binding to the IRES contained in these transcripts. (From Bonkovsky HL: Disorders of iron overload. In Syllabus of Postgraduate Course of the American Association for the Study of Liver Diseases. Washington, DC, Armed Forces Institute of Pathology, 1998.)

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A Partial List of Mammalian Proteins That Use Non-Heme Iron as a Cofactor or That Are Iron Storage or Transport Proteins ferrochelatase iron regulatory protein-1/ cytoplasmic aconitase iron storage proteins ferritin hemosiderin iron transport proteins transferrin lactoferrin mobilferrin proteins involved in cellular import or export of iron ceruloplasmin (Cu-containing ferroxidase) divalent cation transporter-1 (DCT-1) (natural resistance associated macrophage protein-2 [NRAMP-21) ferrireductase(s) IREG haephestin lipoxygenases phenylalanine hydroxylase ribonucleotide reductase succinate dehydrogenase tyrosine hydroxylase In addition to the iron associated with proteins, the cell also contains a small amount of low-molecular-weight iron. Some of this iron is in the form of free heme, and this regulatory heme pool modulates a variety of enzymes, including 6-aminolevulinic acid synthase and heme oxygenase. MAJOR TOXICITIES OF IRON OVERLOAD

Although excess iron is capable of killing almost all cells, in humans with primary iron overload (e.g., caused by HLA-linked hereditary hematachromosis [HHC]) or secondary iron overload (e.g., caused by multiple transfusions of blood because of aplastic anemia, thalassemias, or dyserythropoietic anemias), the major organs involved in iron-induced injury are the liver, the pancreas, the heart, the joints, and the endocrine glands (Table 1).This article discusses iron-induced injury to the liver. Because the liver is chiefly responsible for taking up excess iron, both as non-heme iron and heme-bound iron, the liver is the principal target organ for toxicity caused by iron overload. For example, in HHC, by far the most common cause of iron overload, a primary genetic defect in the HFE gene34causes reduced expression of HFE protein on the basolateral membrane domain of duodenal enterocytes, causing increased expression of DCT-1, the apical Fez+ importer, and IREG-1, the baso49, 69 As a result, too much iron is taken up into the lateral Fe portal blood, and the transferrin in this blood becomes highly or fully

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Table 1. MAJOR TARGET ORGANS FOR IRON-INDUCED INJURY Organs Liver Pancreas Heart

Joints Endocrine glands

Comments Usually indolent injury; more prevalent in men than women; high risk of development of hepatocellular carcinoma in ironinduced cirrhosis Endocrine (p-cell) failure more prevalent and occurs earlier than exocrine (acinar cell) failure A target of injury especially in young patients with iron overload; main cause of morbidity and mortality in thalassemias; earliest manifestations are diastolic dysfunction (loss of normal compliance) and conduction disturbances (arrhythmias) Second and third metacarpophalangeal joints, ankles, and knees particularly affected, especially in HFE-linked hepatocellular carcinoma; rather nonresponsive to iron reduction therapies p-cells of islets and gonadotropin-producing cells of anterior pituitary most often and severely affected; Leydig’s cells of testes may also be affected; most common clinical features are hypogonadotropic hypogonadism and insulin-dependent diabetes mellitus

saturated with iron. Non-transferrin-bound iron (NTBI) also increases. Hepatocytes remove some of this excess iron from the circulation and render it nontoxic by depositing it into the central core of ferritin molecules. Ferritin synthesis is increased markedly by excess loosely bound regulatory iron in hepatocytes and most other cell types, through interactions of iron regulatory element (IRE) on the 5’-UTR of ferritin mRNA with IRPs (Fig. 2).11, 57 Although DCT-1 mRNA is also expressed in liver, its levels are much lower than in duodenum, and hepatic levels of DCT-1 mRNA or protein have not as yet been shown to be inappropriately elevated in patients with HHC. Indeed, increased expression of DCT-1 on the basolateral (sinusoidal) membrane domains of hepatocytes, if it occurs, in HHC, could be viewed as an appropriate response to increased concentrations of iron in the portal blood. In recent years, it has also been found that hepatocytes have ferritin receptors and that iron-loaded Kupffer’s cells and other monocytes and macrophages secrete iron-rich ferritin. Although the numbers of ferritin receptors on hepatocytes are low compared with the number of transferrin receptors, each ferritin molecule contains 2500 or more iron atoms, so that much iron may still be taken up by this pathway. Hepatocytes also play a principal role in the uptake, metabolism, and detoxification of heme,I2 which, as already described, is even more reactive than non-heme iron and is more likely to increase oxidative stress. The major pathways of iron uptake by hepatocytes are summarized below.

Uptake of Non-heme Iron From transferrin by transferrin receptor-mediated endocytosis Low-molecular-weight NTBI by an electrogenic pump-probably involving divalent cation transporter-1 (DCT-1).

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From serum- or Kupffer’s cell-or monocyte-derived ferritin by receptor-mediated endocytosis. Uptake of Heme Iron From serum heme-hemopexin-perhaps by a hemopexin receptormediated process From serum heme-albumin-perhaps by a heme transporter or channel From serum hemoglobin-haptoglobin complexes-perhaps by a haptoglobin receptor-mediated process Targets and Mechanisms for Iron-Induced Hepatoxicity

In secondary forms of iron overload, Kupffer’s cells and perhaps other sinusoidal lining cells (endothelial cells, hepatic stellate cells) take up and store iron early in the evolution of the disorder, whereas in HHC, the accumulation of iron in liver cells other than hepatocytes occurs late, after the hepatocytes have exhausted their ability to take up, store, and detoxify iron. There is probably a critical point in the life of each iron-storing hepatocyte beyond which iron-induced oxidative stress causes damage or death of the cell, with release of iron into the liver adjacent to the dead or dying cell. This iron, chiefly in the form of ferritin or hemosiderin, is taken up by nearby resident macrophages (Kupffer’s cells). As a result, small foci of Kupffer’s cells containing high concentrations of stainable iron that are seen as hepatic iron overload progress to fibrosis in HHC or in secondary hemochromatosis. Such foci are important markers of advancing hepatic injury in patients with iron overload. It seems likely that Kupffer’s cells phagocytosing the remains of iron-loaded hepatocytes undergo activation and produce and secrete proinflammatory and profibrogenic cytokines, such as tumor necrosis factor-alpha (TNFa), which, in turn, activate and help to transform hepatic stellate cells from their fat-storing phenotype to their activated myofibroblast-like phenotype. It is currently believed that such transformed hepatic stellate cells are the major sources of excess collagen formation in hemochromatosis and in other fibrosing liver Decreases in collagenase or matrix metalloprotease (MMP) activities and increases in tissue inhibitors of metalloproteases (TIMPs) probably also contribute to net increases in collagen deposition. It is also possible that cells other than hepatic stellate cells contribute to excess collagen production in conditions of iron overload. A model for development of hepatic fibrosis in hemochromatosis is shown in Figure 3. In patients with hemochromatosis with cirrhosis, the risk of developing hepatocellular carcinoma (HCC) is about 40%-higher than for patients with cirrhosis of most other origins.64This increased risk is probably caused by the additional oxidative damage to DNA caused by iron overload and by the effects of cirrhosis itself in stimulating the

+

Moadlo

Normal

TNFa and other

Central

r

Figure 3. Development of hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) in hereditary hemochromatosis. Part of a row of hepatocytes with adjacent hepatic sinusoid are shown. The normal is shown in A. 6,Because of excessive gut absorption of iron in HHC, periportal hepatoExcess Fe into hepatocytes cytes become progressively loaded B with iron. C,With sufficient iron loading, critical injury occurs to peri-portal hepatocytes, leading to leakage of cell contents or lysis of cells. The cell debris, including iron-rich ferritin and hemosiderin, is ingested by Kupffer cells, which become activated and release TNFa and other cytokines. These exert Sinusoi autocrine, paracrine, and endocrine effects on other cells, including hepatic stellate cells (HSC). 0, After sufficient Critical damage activation, the HSC undergo transforto hepatocytes ___ mation to myofibroblast-like phenotype and synthesize and secrete collagen and cytokines. After years of such injury and excess collagen formation, cirrhosis may develop. Hepatocellular carcinoma is a possible sequela of cirrhosis, especially in HLA-linked hereditary hemochromatosis. Continuing oxidative damage to DNA caused by iron overload probably plays a role in HCC Kupffer cells development.

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division of hepatocytes to form regenerative nodules. Once cirrhosis has developed in hemochromatosis, it is generally considered to be irreversible, although reduction of its clinical and histopathologic severity is still possible with iron depletion measures. In fact, early cirrhosis may rarely be reversible. Unfortunately, in HHC with cirrhosis, the high risk for developing HCC is not affected by iron depletion strategies.

Subcellular Organelles that are Targets for Iron Toxicity The increased oxidative stress caused by low-molecular-weight iron in liver cells may cause oxidative damage to unsaturated lipids, amino acids, proteins, and nucleic acids.3 Thus, all parts of iron-loaded cells may be damaged. Damage to mitochondria, lysosomes, and smooth endoplasmic reticulum (SER) has been particularly studied. Most of these studies have been performed in experimental models of iron overload. For example, in rats it was shown that iron loading leads to decreased hepatic levels of the P-450 cytochromes, which are important enzymes of the SER. Time-course experiments showed that such decreases occurred only after iron loading had produced increased oxidative stress and lipid peroxidation (Fig. 4).6These effects were coupled with induction of heme oxygenase-1, which catalyzes the rate-controlling

Control

1212

2731

4085

Hepatic Nonheme Iron Conc (pgFe/g liver) Figure 4. Effects of iron overload on levels of cytochrome P-450 and lipid peroxidation in rats with experimental hemochromatosis. Male weaniing rats in groups of 6 to 24 were fed a control chow diet or the same diet supplemented with finely divided elemental iron (iron carbonyl). Rats were killed 2 to 4 months after the start of these diets, and portions of liver were used for measurement of nonheme iron concentrations (mean values shown along horizontal axis) and for measurements of microsomal concentrations (conc) of total cytochrome P-450 concentrations(open bars) and conjugated dienes (hatched bars), a measure of lipid peroxidation. Note a threshold for development of significant increases in conjugated diene concentrations and decreases in cytochrome P-450 concentrations. Results are mean m + SE.

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step of heme breakdown and which serves as one of the major ways of protecting cells from the ravages of increased oxidative stress. An increase in lysosomal lability has been found in both experimental models of iron overload and in livers of patients with HHC.54,59 Such lability may be associated with activation of lysosomal proteases and autophagy of affected cells. Redox-active iron chelators, such as ascorbate, may increase these effects of iron at some concentrations. Typically, the dose-response curves are parabolic, with low concentrations of such chelators increasing redox cycling and toxicity of iron, and higher concentrations inhibiting these effects. Mitochondria may be the most important subcellular organelles damaged by iron overload. Several defects in the coupled processes of electron transport and oxidative phosphorylation have been described in mitochondria from iron-loaded animal^.^ Defects in the cytochromeoxidase complex, the final common pathway for electrons and H’ leading to production of water and energy (ATP) appear to be most important. Mitochondria1 damage may reach a critical point, the mitochondrial membrane transition point, beyond which the mitochondria swell uncontrollably, burst, and, in turn, produce cellular death. Indeed, death caused both by apoptosis and by necrosis is now believed to depend on mitochondrial damage, loss of ATP synthesis, and leakage of critical 53 After the proteins, such as cytochrome c, from the mit~chondria.~~, permeability transition point of the mitochondrial membrane has been reached and mitochondrial swelling and disintegration are underway, it is not surprising that cells thus affected will die either by cell swelling, blebbing, and eventual explosion (necrosis) or by a p o p t ~ s i s . ~ ~ Iron as a Contributory Factor in Liver Injury The role of heavy iron overload per se in causing hepatic fibrosis and cirrhosis in HHC is well established. Recent evidence suggests that only mild degrees of hepatic iron loading ( 2 4 times the upper limit of normal), which, in themselves are not toxic, may also potentiate hepatic injury caused by other disorders. Hepatic disorders other than hemochromatosis in which iron is thought to play a contributory role are Alcoholic liver disease Nonalcoholic steatoheyatitis “ASH] Chronic viral hepatitis Porphyria cutanea tarda [PCT] Alpha,-antitrypsin deficiency End-stage liver disease with iron overload (non-HFE linked) Iron and Alcoholic Liver Disease

In an experimental model of alcoholic liver disease (ALD) in rats, modest (twofold to threefold) increases in hepatic iron concentration led

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to synergistic increases in hepatic injury, lipid peroxidation, and fibrosis. In this model, unusually high intakes of dietary fat were provided to the rats, and ethanol was infused with a liquid diet directly into the rats’ Thus, the relevance of the findings to ALD as typically encountered clinically may be questioned. In another study, also involving rats, the combination of ethanol in a liquid Lieber-DiCarli diet, following oral iron-carbonyl (2.5%-3%) for 6 to 9 weeks, produced marked increases in mean serum alanine aminotransferase (ALT) (141 U/L), compared with ethanol (34 U/L) or iron (36 U/L) alone, although all were greater than seen in controls (16 U/L).(j3In contrast with the in the model of Stal et results of Tsukamoto et hepatic glutathione (GSH) levels were increased in ethanol- and iron-fed rats, and no increase in fibrosis could be identified. Alcoholic liver disease is frequently associated with elevations in serum transferrin saturation and ferritin, and sometimes with increases (usually mild) in hepatic iron concentration. Thus, alcohol, even without iron administration or hemochromatosis, may increase iron concentration in serum and liver. Possible reasons for this effect include increased iron absorption from the gut, mild hemolysis, increased hepatic iron uptake from heme or ferritin derived from the reticuloendothelial (RE) system (Kupffer’s cells, splenic macrophages, and so forth), and increased mobilization of iron from ferritin stores into a highly reactive, low-molecular-weight, labile iron pool in hepatocytes, serum, and perhaps other tissues.’* For many years, it has been known that hepatic non-heme iron absorption is increased by alcohol consumption and in alcoholic liver disease.25The pathogenetic causes of this increase have not yet been elucidated. For example, it is not known whether alcohol intake or ALD produce changes in expression of the DCT-2 or R E G genes, the major putative transporters for iron into and out of duodenal enterocytes. Iron and Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis is a chronic liver disease that is being seen with increasing frequency, especially in the United States and other developed countries. Although originally it was particularly associated with severe obesity, hyperglycemia, and hyperlipidemia in women, NASH is now being observed at least as frequently in men, in persons with only moderate degrees of obesity, and in children of both genders.2o,44 A recent autopsy study found NASH in 18.5% of North American adults who were obese and in 2.7% of those of normal A firm diagnosis of NASH requires a liver biopsy that shows some or all of the features of alcoholic hepatitis and a history of no or only slight alcohol intake by the patient. The history must be confirmed independently by close family members or friends, because alcoholic patients frequently deny minimalize their alcohol intake. Recently, a group of hepatologists proposed that the minimal histopathologic criteria for the diagnosis of NASH include moderate to severe steatosis,

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lobular inflammation (especially including polymorphonuclear leukocytes [PMNs]), and Mallory’s bodies located in swollen hepatocytes in zone 111, or perisinusoidal fibrosis resembling a chicken-wire fence in zone III.3s Although the pathogenesis of NASH is not fully understood, it certainly must involve hepatic steatosis, hyperinsulinism, and increased oxidative stress. Because iron increases oxidative stress, as already described, iron in the liver might contribute to the pathogenesis of NASH. Indeed, elevated levels of serum ferritin are found in 55% to 62% of patients with NASH, and increases in serum transferrin saturations also occur relatively frequently, in 6% to 22% of these patients. Mild increases in stainable hepatic iron are also relatively common.16, Recent data from Australia, Europe and North America support a link between iron, HFE gene mutations, and NASH. For example, in a recent study of 51 Australian patients with NASH, a much higher than expected prevalence of homozygosity for the C282Y mutation of the HFE gene was found (7.9% of NASH patients versus 0.67% of controls, P < 0.0005. NASH patients also had an increased prevalence of heterozygosity for the C282Y mutation (23.5% of NASH patients versus 11.5% of controls, P < 0.005). The prevalences of homozygosity or heterozygosity for the H63D mutation were not significantly different between Australian patients with NASH and controls. The NASH patients with the C282Y mutation had higher hepatic concentrations of iron ( P < 0.005), hepatic iron index (HII) P < 0.005), and more stainable hepatic iron (P < 0.005) and fibrosis.37 Investigators in Brittany recently described what they at first thought was a new syndrome of hyperferritinemia and metabolic disorders associated with obesity and hyperin~ulinemia,~~ some of which met the usual diagnostic criteria for NASH. Recent HFE mutational analyses in patients with this iron-dysmetabolic syndrome have shown increased prevalences of both the C282Y and the H63D mutations.*6 In the United States, the authors’ and co-workers’ study of 57 patients with NASHZ0also showed significantly increased prevalences of either HFE mutation and of heterozygosity for the H63D mutation [Fig. 51. The NASH patients with one of the HFE mutations had significantly higher levels of serum ferritin, iron, and transferrin saturation (P < 0.05) and of hepatic iron staining ( P < 0.0018) than those without that mutation. Those patients who carried the C282Y mutation (but not those who carried only the H63D mutation) also had a significantly higher score for hepatic fibrosis than those without the C282Y mutation ( P = 0.02), confirming the results from Australia. The metabolic derangements associated with NASH may also influence iron metabolism and effects of HFE gene mutations. For example, hyperinsulinism is a almost universal feature of human obesity and type 2 diabetes mellitus and is present in most patients with NASH. The acute exposure of cells to insulin rapidly increases the expression of transferrin receptors on cell surfaces, by increasing exocytosis of preexistent intracellular receptors.60Furthermore, the protein product of the

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Figure 5. Increased prevalence of mutations of the HFE gene in patients with NASH from the United States. Values are means f SEM. A, Subjects with either mutation. B, Subjects with C282Y mutation. C,Subjects with H63D mutation. A = differs from control (P=0.008); asterisk = differs from control ( P = O . O O l ) ; plus = differs from control (P=0.03); (IT= differs from control (P=0.01). Stippled bar = homozygous abnormal; hatched bar = heterozygous; solid bar = total. (From Bonkovsky HL, Jawaid Q, Tortorelli K, et al: Non-alcoholic steatohepatitis and iron: Increased prevalence of mutations of the HFE gene in nonalcoholic steatohepatitis. J Hepatol 31:421-429, 1999; with permission.)

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normal HFE gene associates with transferrin receptors on cell membranes and regulates their role in iron uptake by ~ells.3~. 52 The C282Y and H63D mutations abrogate or diminish these effects of the HFE protein35,52 and may thus potentiate hyperinsulinism to increase a metabolically active pool of iron, which may exacerbate liver injury in patients with NASH. In view of these findings and the current unsatisfactory therapeutic alternatives for NASH, it is rational to suggest that iron reduction might be of benefit. Indeed, the author and co-workers have found evidence of biochemical responses (normalization of serum ALT levels) to therapeutic phlebotomies in all four NASH patients whom they have thus far treated by iron reduction.20 Iron and Chronic Viral Hepatitis

Much evidence indicates that iron plays an important role in infectious diseases generally and in chronic hepatitis B and C in particular. The author and co-workers have recently reviewed this subject in detail.'"'* A role for iron in viral hepatitis was first emphasized by Blumberg et a1,O ' who reported that, among 67 patients infected with hepatitis B virus (HBV) who were receiving chronic hemodialysis, the 33 who developed chronic infections had significantly higher levels of serum iron than the 34 who cleared their infections spontaneously. Somewhat later, histopathologic studies of patients with thalassemia showed that hepatic iron loading was correlated with severity of chronic hepatitis B and hepatic fibrosis.', 28 Elevated Serum Iron, Transferrin Saturation, and Serum Ferritin in Chronic Viral Hepatitis

Several investigators have recently noticed a high prevalence of elevated serum iron transferrin saturations and ferritin levels in patients with chronic viral hepatitis. For example, among 80 patients with chronic viral hepatitis (21 with chronic HBV, 46 with hepatitis C virus [HCV], 4 with HBV and hepatitis D virus, and nine with non-B, -C, or -D hepatitis) followed at the National Institutes of Health, 29 of the 80 (36%) had increased serum iron levels, 14 of 78 (18%)had increased serum transferrin saturations, and 21 of 70 (30%) had increased serum ferritin levels.29Elevated serum ferritins were particularly common in men; 20 of 47 (42%) had levels above 300 ng/mL. Nevertheless, only 4 of 25 (14%)had elevated hepatic iron concentrations (< 25 kmol 11300 kg]/g dry weight), and in only 2 was the increase marked, as it is in patients with HHC. Recent results from Israel were somewhat different: increases in serum iron and transferrin saturation were observed only in the 63 patients with chronic HCV and in three patients with both HBV and HCV, but not in the 14 patients with chronic HBV alone.2Mean values of these variables in the 63 patients with chronic HCV were significantly higher (P < 0.001) than in the 14 patients with chronic HBV alone or in

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the 43 patients chronic nonviral liver diseases. Although the mean value of serum ferritin was also higher in patients with chronic HCV, the differences among groups were not statistically significant for this variable. Despite the increased serum irons and transferrin saturations, none of the patients had hepatic iron overload as estimated histochemically. Several groups have reported that a decreased likelihood of response to interferon (IFN) therapy of chronic viral hepatitis (especially chronic hepatitis C) is associated with increased levels of serum ferritin or hepatic iron concentration^.'^ Among the patients who did not respond to IFN therapy, the mean hepatic iron concentrations were within normal limits. Thus, frank iron overload is not required. The author and others have also been impressed with how severe and rapidly progressive chronic hepatitis C is in patients with hemochromatosis. In addition to the total amount of iron in the liver, the lobular and cellular distribution of stainable iron also correlates with (and may influence) response to IFN therapy in patients with chronic hepatitis C. Specifically, stainable iron in sinusoidal lining cells or in portal endothelial or stoma1 cells is associated with a poor response to IFN.7,9, 31, 43 Indeed, the score for stainable portal iron was a significant predictor in a multivariate analysis and was as powerful a predictor of response to IFN as HCV genotype or baseline HCV RNA levels in serum.43 Because of the relationships between iron and chronic hepatitis C and because the therapy for chronic hepatitis C remains so unsatisfactory, the effects of iron reduction on responses to treatment of chronic hepatitis C have been studied. Hayashi et a1 first showed that therapeutic phlebotomy decreased serum ALT levels in previously untreated patients and in patients who had not responded to or who had relapsed after IFN therapy.42van Thiel et a1 reported that iron reduction significantly improved the response to IFN (5 million units [MU] daily) in patients with chronic viral hepatitis who previous had not responded to IFN alone at a dosage of 3 MU three times a week.66Two groups recently reported that, in previously treated noncirrhotic patients with chronic hepatitis C, iron reduction by therapeutic phlebotomy before and during 6 months of IFN therapy (3 MU three times a week) significantly improved the end-of-treatment biochemical response (normalization of serum ALTs) and virologic response (loss of detectable HCV RNA).l9*36 Both of these studies were relatively small (37 and 82 patients, respectively), and the intensity and duration of IFN therapy were low. Thus, although greater improvement in biochemical response persisted for at least 24 weeks after completion of therapy, the sustained virologic responses were not quite significant at the 5% level (2-tailed Ps = 0.07 and 0.08). The sustained virologic response rate is significantly higher, however, if results of the two trials are combined. Thus, in patients with chronic hepatitis C, iron reduction does improve the sustained biochemical response and probably improves the sustained virologic response to low-dose IFN therapy. The data of van Thiel et a1 suggest that the long-term beneficial effects of iron reduction may be improved

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further by the use of higher total doses of IFN for therapy of chronic hepatitis C. Combination therapy of chronic hepatitis C with IFN plus ribavirin has recently been shown to be superior to IFN alone for both initial therapy and retreatment of previously treated patients who responded to and relapsed after treatment with IFN alone.56,58 Nonetheless, fewer than 50% of patients show sustained virologic responses following treatment with IFN plus ribavirin. One reason may be that ribavirin causes hemolytic anemia and thus increases hepatic iron storage.30Long-term therapy with ribavirin may thus prove to be of diminishing benefit. Iron and Porphyria Cutanea Tarda

It has been known for many years that hepatic iron plays a key role in the pathogenesis of PCT. The major mechanisms underlying this effect of iron are summarized in Figure 6: iron may increase the rate of formation of uroporphyrinogen, increase oxidation of this porphyrinogen to the corresponding porphyrin, and, with other factors, give rise to an inhibitor of uroporphyrinogen decarboxylase, the enzyme that normally converts uroporphyrinogen into coproporphyrinogen. Deficient activity of uroporphyrinogen decarboxylase is a regular feature of overt PCT. Sixty percent to 70% of patients with PCT show mild to moderate iron overload, and approximately 10% have increases in the range of hemochromatosis. The major cause for the increase in hepatic and total body iron in PCT is the presence of mutations in the HFE gene that have been associated with HHC. For example, it was reported recently that 73% of 70 North American patients with PCT had one of the defined HFE gene mutations.2I Twenty-three percent of these patients were heterozygous, 19% were homozygous for the C282Y mutation; 23% were heterozygous, and 8% were homozygous for the H63D mutation. Similar results have been reported from Europe and Australia. In addition to iron overload, a number of other risk factors for the development of PCT have been recognized. The most important of these are alcohol abuse, estrogen therapy, and the presence of chronic liver diseases of other cause, particularly alcoholic liver disease and chronic hepatitis C.l4,l5A history of heavy alcohol use is present in most patients with PCT, and is the single most important risk factor; iron overload and infection with hepatitis C virus are not far behind. Not surprisingly, patients who abuse alcohol often have experimented with or abused illicit drugs and have often had experience with intravenous drug use. Thus, they are at increased risk for development of chronic hepatitis C infection. Such patients are at particularly high risk for development of PCT. Exposure to drugs and toxins besides alcohol is also a potential risk factor for development of PCT. The drug most often implicated is estrogen, usually taken by women for birth control, but occasionally taken by men as part of therapy for prostatic carcinoma. Polyhaloge-

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Figure 6. The pathogenesis of the defects in heme metabolism that give rise to porphyria cutanea tarda (PCT). The rate-controlling enzymes for hepatic heme biosynthesis (ALA synthase) and breakdown (heme oxygenase) are shown, as is uroporphyrinogendecarboxylase (UROD), the enzyme that carries out the stepwise decarboxylation of uroporphyrinogen to coproporphyrinogen. UROD activity is decreased in PCT. Iron, especially in concert with cytochrome P450, increases oxidation of porphyrinogens to porphyrins. It also seems to enhance production of a nonporphyrin inhibitor of UROD that probably is derived from uroporphyrinogen. Iron also induces heme oxygenase, causing depletion of a regulatory heme pool and thus derepression of ALA synthase, which further increases the production of uroporphyrinogenand uroporphyrin. (From Bonkovsky HL, Lambrecht RW: Hemochromatosis, iron overload, and porphyria cutanea tarda. ln Barton JC, Edwards CQ [eds]: Hemochromatosis. Cambridge, Cambridge University Press, 2000; with permission.)

nated aromatic hydrocarbons, such as hexachlorobenzene or 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) are well-known causes of PCT. Fortunately, few persons today are exposed to high levels of these highly toxic chemicals.

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As shown in Figure 6, hepatic production of uroporphyrin can be increased by at least three factors, which are not mutually exclusive. 1. The rate of decarboxylation of uroporphyrinogen to coproporphyrinogen (catalyzed by uroporphyrinogen decarboxylase) may be decreased. 2. The rate of oxidation of uroporphyrinogen to uroporphyrin may be increased. 3. The amount of 6-aminolevulinic acid (ALA) synthesized by or imported into hepatocytes may be increased.

In most patients with PCT there does not seem to be any inherited defect in activity of hepatic uroporphyrinogen decarboxylase. Thus, deficient activity of this enzyme seems to be an acquired abnormality. Furthermore, even in those patients in whom there is an inherited deficiency in activity of the decarboxylase (type 2 PCT), most are totally asymptomatic and do not overproduce uroporphyrin. Therefore, a fundamental inherited defect in hepatic uroporphyrinogen decarboxylase activity is neither necessary nor sufficient to cause PCT. Excessive oxidation of uroporphyrinogen or hepatacarboxy-porphyrinogen may cause accumulation of porphyrins, and, if sufficiently severe, the phenotype of PCT. The rate at which this oxidation occurs may be influenced by a number of factors, including the activity of cytochrome P-4501A2, hepatic iron, alcohol, or estrogens. Studies in several experimental models of PCT have established that the porphyrogenic polyhalogenated aromatic hydrocarbons such as hexachlorobenzene or TCDD, which are inducers of cytochrome P-4501A2, give rise to inhibitors of uroporphyrinogen decarboxylase. Formation of these putative inhibitors is enhanced by active iron within hepatocytes. A second line of evidence that indirectly supports a role for excess oxidation of porphyrinogens in the pathogenesis of PCT is that antioxidants, such as ascorbate, can diminish uroporphyrin accumulation in a rat model of PCT.'j* It may be clinically significant that some PCT patients have low plasma levels of ascorbate.61 Hepatic ALA synthase is normally the rate-controlling enzyme for porphyrin, porphyrinogen, and heme biosynthesis in the liver. Induction of or increased ALA synthase may thus play a role in the overproduction of uroporphyrin that is the biochemical hallmark of PCT. Drugs such as phenobarbital or glutethimide, which induce hepatic ALA synthase, may markedly increase hepatic porphyrin accumulation, particularly in the presence of iron.I3 As shown in Figure 6, an increase in oxidative stress produced by iron, alcohol, estrogens, or hepatitis C infection is probably the common and key pathogenic factor for the development of PCT. Iron and Alpha, -antitrypsin Deficiency

It has been suggested that deficiency for alpha,-antitrypsin and hemochromatosis occur more frequently than would be expected from chance association^.^^, 55 There is, however, no known genetic linkage

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between the two conditions, nor would any be expected, because the gene for alpha,-antitrypsin is on chromosome 14, whereas the HFE gene, which is mutated in most patients with HHC, is located on chromosome 6 . It may still be possible, however, that patients with mutations in both genes are much more likely to develop symptoms and signs of liver disease. Indeed, such cases have been described, and the author and colleagues are following a man with chronic hepatitis C infection who is also heterozygous for the C282Y mutation of the HFE gene and whose protease inhibitor phenotype is PiMZ. This patient has had an unusually rapid progression from mild hepatitis to moderate hepatitis with cirrhosis over a period of only 4 years, despite efforts at treatment with IFN and phlebotomy.* These observations suggest the possibility that iron in the liver may contribute to the severity and rapidity of progression of liver disease caused by alpha,-antitrypsin deficiency. It seems possible that something similar may happen in other forms of inherited or acquired liver disease. Iron and End-stage Liver Disease: Serum transferrin saturation and ferritin and hepatic iron concentration are often increased in patients with end-stage liver disease. For example, the hepatic iron index has been reported to be in the range typical of HHC (> 1.9) in more than 7% of patients with end-stage liver disease.47The presence of iron may be associated with a more rapid decline of hepatic function or with progression to hepatocellular carciabout 10% of patients with iron overload and endn ~ m a .32,~41~ Only , stage liver disease are homozygous for the C282Y mutation. In one study, a seemingly increased prevalence (55%)of heterozygosity for the C282Y mutation was found in such patients. This finding was not confirmed in another recent study.6 Thus, excess hepatic iron and increases in serum ferritin and transferrin saturation occur frequently in end-stage liver disease that is not caused by HHC. These occurrences are particularly common in patients with alcoholic or posthepatitic cirrhosis. They are less common in patients with end-stage liver disease resulting from primary biliary cirrhosis or primary sclerosing cholangitis. It is unclear how or why excess iron accumulates in the liver in this setting, but it probably plays a pathogenic role in the production of hepatic fibrosis, progression of cirrhosis, and perhaps in the development of HCC. More data, including HFE mutational analyses and further studies of iron transporters in such patients, will be of great interest. ACKLOWLEDGMENTS We thank Jane Blackwood for typing the manuscript.

References 1. Aldouri MA, Wonke 8,Hoffbrand AV, et al: Iron state and hepatic disease in patients with thalassaemia major, treated with long term subcutaneous desferrioxamine. J Clin Pathol40:1353-1359, 1987

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2. Arber N, Konikoff FM, Moshkowitz M, et al: Increased serum iron and iron saturation without liver iron accumulation distinguish chronic hepatitis C from other chronic liver diseases. Dig Dis Sci 392656-2659, 1994 3. Bacon BR, Britton R S Hepatic injury in chronic iron overload. Role of lipid peroxidation. Chem Biol Interact 70:183-226, 1989 4. Bacon BR, ONeill R, Britton RS: Hepatic mitochondria1 energy production in rats with chronic iron overload. Gastroenterology 105:1134-1140, 1993 5. Bacon BR, Healey JF, Brittenham GM, et al: Hepatic microsomal function in rats with chronic dietary iron overload. Gastroenterology 90:1844-1853, 1986 6. Bacon BR, Powell LW, Adams PC, et al: Molecular medicine and hemochromatosis: At the crossroads. Gastroenterology 116:193-207, 1999 7. Banner BF, Barton AL, Cable EE, et al: A detailed analysis of the Knodell score and other histologic parameters as predictors of response to interferon therapy in chronic hepatitis C. Mod Pathol 8:232-238, 1995 8. Banner BF, Karamitsios N, Smith L, et al: Enhanced phenotypic expression of alpha-lantitrypsin deficiency in an MZ heterozygote with chronic hepatitis C . Am J Gastroenterol 93:1541-1545, 1998 9. Barton AL, Banner BF, Cable EE, et al: Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy. Am J Clin Pathol 103:419424, 1995 10. Blumberg BS, Lustbader ED, Whitford PL: Changes in serum iron levels due to infection with hepatitis B virus. Proc Natl Acad Sci USA 78:3222-3224, 1981 11. Bonkovsky HL: Disorders of iron overload. In Syllabus of Postgraduate Course of the American Association for the Study of Liver Diseases. Washington DC, Armed Forces Institute of Pathology, 1998 12. Bonkovsky HL: Iron and the liver. Am J Med Sci 301:3243, 1991 13. Bonkovsky HL: Mechanism of iron-potentiation of hepatic uroporphyria: Studies in cultured chick embryo liver cells. Hepatology 10:354-364, 1989 14. Bonkovsky HL: Porphyria cutanea tarda and hepatitis C. Viral Hepatitis Reviews 4:75-95, 1998 15. Bonkovsky HL, Lambrecht RW Hemochromatosis, iron overload, and porphyria cutanea tarda. In Barton JC, Edwards CQ (eds): Hemochromatosis. Cambridge, Cambridge University Press, 2000 16. Bonkovsky HL, Obando JV:Role of HFE gene mutations in liver diseases other than hereditary hemochromatosis. Current Gastroenterology Reports 1:3&37, 1999 17. Bonkovsky HL, Banner BF, Rothman AL: Iron and chronic viral hepatitis. Hepatology 25:759-768, 1997 18. Bonkovsky HL, Banner BF, Lambrecht RW, et al: Iron in liver diseases other than hemochromatosis. Semin Liver Dis 16:65-82, 1996 19. Bonkovsky H, Israel J, Fontana R, et al: Iron reduction during interferon therapy of chronic hepatitis C: Initial results of a multicenter, randomized, controlled trial in previously untreated patients [abstract]. Gastroenterology 116:A1191, 1999 20. Bonkovsky HL, Jawaid Q, Tortorelli K, et al: Non-alcoholic steatohepatitis and iron: Increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis. J Hepatol 31:421429, 1999 21. Bonkovsky HL, Poh-Fitzpatrick M, Pimstone N, et al: Porphyria cutanea tarda, hepatitis c, and HFE gene mutations in North America. Hepatology 271661-1669, 1998 22. Bothwell TH, Charlton RW, Motulsky AG: Hemochromatosis. In Scriver CR, Beaudet AL, Sly WS, et al: (eds): The Metabolic and Molecular Basis of Inherited Disease, ed 7. New York, McGraw-Hill, 1995, pp 2237-2269 23. Bothwell TH, Charlton RW, Cook JD, et al: Iron Metabolism in Man. Oxford, Blackwell Scientific, 1979 24. Buettner GR, Jurkiewicz BA: Catalytic metals, ascorbate and free radicals: Combinations to avoid. Radiat Res 145:532-541, 1996 25. Chapman RW, Morgan MY, Bell R, et al: Hepatic iron uptake in alcoholic liver disease. Gastroenterology 8414S147, 1983 26. Deugnier Y, Moirand R, Joanelle AM, et al: HLA-H mutations in patients with iron overload and normal transferrin saturation [abstract]. Hepatology 26:199A, 1997

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27. Deugnier Y, Turlin B, Loreal 0, et al: Iron and neoplasia. J Hepatol 28(suppl):21-25, 1998 28. DeVirgiliis S, Cornacchia G, Sanna G, et al: Chronic liver disease in transfusiondependent thalassemia: Liver iron quantitation and distribution. Acta Haematol 65:3239, 1981. 29. Di Bisceglie AM, Axiotis CA, Hoofnagle JH, et al: Measurements of iron status in patients with chronic hepatitis. Gastroenterology 102:2108-2113, 1992 30. DiBisceglie AM, Bacon BR, Kleiner DE, et al: Increase in hepatic iron stores following prolonged therapy with ribavirin in patients with chronic hepatitis C. J Hepatol 21:1109-1112, 1994 31. Fargion S, Carazzone A, Molteni V, et al: Iron and response to interferon alpha in chronic hepatitis C [abstract]. Hepatology 22:415A, 1995 32. Fargion S, Mattiolo M, Sampietro M, et al: Iron as risk factor for hepatocellular carcinoma [abstract]. J Hepatol28(suppl):93A, 1998 33. Faubion WA, Gores GJ: Death receptors in liver biology and pathobiology. Hepatology 29:14, 1999 34. Feder JN, Gnirke A, Thomas W, et al: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399-408, 1996 35. Feder JN, Tsuchihashi 2, Irrinki A, et al: The hemochromatosis founder mutation in HLA-H disrupts P,-microglobulin interaction and cell surface expression. J Biol Chem 272:14025-14028, 1997 36. Fong T-L, Han SH, Tsai NC-S, et al: A pilot randomized, controlled trial of the effect of iron depletion on long-term response to a-interferon in patients with chronic hepatitis C. J Hepatol28:369-374, 1998 37. George DK, Goldwurm S, MacDonald GA, et al: Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology 114311-318, 1998 38. Goodman Z: The NASH Pathology Working Group: The pathology of non-alcoholic steatohepatitis (NASH) [abstract 301. In Abstracts for Symposium on Non-Alcoholic Steatohepatitis. Bethesda, National Institutes of Health, 1998 39. Gross CN, Irrinki A, Feder JN, et al: Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation. J Biol Chem 273:22068-22074, 1998 40. Gunshin H, Mackenzie 8, Berger W, et al: Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388:482488, 1997 41. H a m HW, Kim CY, London WT, et al: Increased serum ferritin in chronic liver disease: A risk factor for primary hepatocellular carcinoma. Int J Cancer 43:37&379, 1989 42. Hayashi H, Takikawa T, Nishimura N, et al: Improvement of serum aminotransferase levels after phlebotomy in patients with chronic active hepatitis C and excess hepatic iron. Am J Gastroenterol 89:986-988, 1994 43. Ikura Y, Morimoto H, Johmura H, et al: Relationship between hepatic iron deposits and response to interferon in chronic hepatitis C. Am J Gastroenterol 91:1367-1373, 1996 44. James OF, Day CP: Non-alcoholic steatohepatitis (NASH): A disease of emerging identity and importance. J Hepatol 29:495-501, 1998 45. Kaserbacher R, Propst T, Propst A, et al: Association between heterozygous alpha 1antitrypsin deficiency and genetic hemochromatosis. Hepatology 18:707-708, 1993 46. Klausner RD, Rouault TA, Harford JB: Regulating the fate of mRNA: The control of cellular iron metabolism. Cell 7219-28, 1993 47. Ludwig J, Hashimoto E, Porayko MK Hemosiderosis in cirrhosis: A study of 447 native livers. Gastroenterology 112:882-888, 1997 48. Mak IT, Weglicki WB: Characterization of iron-mediated peroxidative injury in isolated hepatic lysosomes. J Clin Invest 75:58-63, 1985 49. McKie AT, Weher K, Brennan K, et al: A novel IRE containing mRNA predominantly expressed in duodenum and placenta encodes a basolateral membrane protein associated with iron transport. In Final Program Abstract Book of World Congress on Iron Metabolism. Sorrento Italy, Bioiron '99, 1999, p 33 50. Moirand R, Mortaji AM, Loreal 0, et al: A new syndrome of liver iron overload with normal transferrin saturation. Lancet 349:9597, 1997

IRON-INDUCED LIVER INJURY

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51. Olaso E, Friedman SL: Molecular regulation of hepatic fibrogenesis. J Hepatol 29:836 847, 1998 52. Parkkila S, Waheed A, Britton RS, et al: Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci U S A 9413198-13202, 1997 53. Pate1 T, Roberts LR, Jones BA, et al: Dysregulation of apoptosis as a mechanism of liver disease: An overview. Semin Liver Dis 18:105-114, 1998 54. Peters TA, Seymour C A Acid hydrolase activities and lysosomal integrity in liver biopsies from patients with iron overload. Clin Sci Mol Med 50:75-78, 1976 55. Rabinovitz M, Gavaler JS, Kelly RH, et al: Association between heterozygous alphalantitrypsin deficiency and genetic hemochromatosis. Hepatology 16:145-148, 1992 56. Reichard 0, Norkrans G, Fryden A, et al: Randomized, double-blind, placebo-controlled trial of interferon a-2b with and without ribavirin of chronic hepatitis C. Lancet 351233-87, 1998 57. Rouault TA, Klausner RD: Iron-sulfur clusters as biosensors of oxidants and iron. Trends in Biochemical Sciences 21:174-177, 1996 58. Schalm S, Hansen BE, Chemello L, et al: Ribavirin enhances the efficacy but not the adverse effects of interferon in chronic hepatitis C. J Hepatol 26:961-966, 1997 59. Seymour CA, Peters TJ: Organelle pathology in primary and secondary hemochromatosis. Br J Haematol 40:239-253, 1979 60. Shepherd PR, Soos MA, Siddle K: Inhibitors of phosphoinositide 3-kinase block exocytosis but not endocytosis of transferrin receptors in 3T3-Ll adipocytes. Biochem Biophys Res Commun 211:535539, 1995 61. Sinclair PR, Gorman N, Shedlofsky SI, et al: Ascorbic acid deficiency in porphyria cutanea tarda. J Lab Clin Med 130:197-201, 1997 62. Sinclair PR, Gorman N, Sinclair JF, et al: Ascorbic acid inhibits chemically-induced uroporphyria in ascorbate-requiring rats. Hepatology 22:565-572, 1995 63. Stal P, Johansson I, Ingelman-Sundberg M, et al: Hepatotoxicity induced by iron overload and alcohol. J Hepatol25:538-546, 1996 64. Tavill A S Hemochromatosis. In Schiff L, Scheff ER (eds): Diseases of the Liver, ed 7. Philadelphia, Lippincott, 1993, pp 669491 65. Tsukamoto H, Home W, Kamimura S, et al: Experimental liver cirrhosis caused by alcohol and iron. J Clin Invest 96:620430, 1995 66. VanThiel DH, Friedlander L, Molloy PJ, et al: Retreatment of hepatitis C interferon nonresponders with larger doses of interferon with and without phlebotomy. Hepatogastroenterology 43:1557-1561, 1996 67. Wanless IR, Lentz JS Fatty liver hepatitis (steatohepatitis) and obesity: An autopsy study with analysis of risk factors. Hepatology 12:1106-1110, 1990 68. Wardman P, Candeias LP: Fenton chemistry: An introduction. Radiat Res 145:523531, 1996 69. Zhou XY, Tomatsu S, Fleming RE, et al: HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A 95:2492-2497, 1998

Address reprint requests to Herbert L. Bonkovsky, MD Division of Digestive Disease and Nutrition UMass Memorial Health Care-University Campus 55 Lake Avenue, North Room S6-737 Worcester, MA 01655