Co-factors in liver disease: The role of HFE-related hereditary hemochromatosis and iron

Biochimica et Biophysica Acta 1790 (2009) 663–670

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Review

Co-factors in liver disease: The role of HFE-related hereditary hemochromatosis and iron Daniel F. Wallace, V. Nathan Subramaniam ⁎ Membrane Transport Laboratory, The Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia

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Article history: Received 29 April 2008 Received in revised form 25 July 2008 Accepted 9 September 2008 Available online 20 September 2008 Keywords: Iron Hemochromatosis Liver Alcohol Hepatitis Porphyria cutanea tarda

a b s t r a c t The severity of liver disease and its presentation is thought to be influenced by many host factors. Prominent among these factors is the level of iron in the body. The liver plays an important role in coordinating the regulation of iron homeostasis and is involved in regulating the level of iron absorption in the duodenum and iron recycling by the macrophages. Iron homeostasis is disturbed by several metabolic and genetic disorders, including various forms of hereditary hemochromatosis. This review will focus on liver disease and how it is affected by disordered iron homeostasis, as observed in hereditary hemochromatosis and due to HFE mutations. The types of liver disease covered herein are chronic hepatitis C virus (HCV) infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatocellular carcinoma (HCC) and porphyria cutanea tarda (PCT). © 2008 Elsevier B.V. All rights reserved.

1. Iron homeostasis The liver is the central regulator of iron homeostasis. Research over the last decade has confirmed that the liver is the primary site of expression of many of the molecules responsible for the regulation of iron homeostasis. The hereditary hemochromatosis (HH) associated molecules HFE, transferrin receptor 2 (TfR2), hemojuvelin, hepcidin and ferroportin are all expressed at high levels in the liver. Mouse models of HH, where the genes have been disrupted or mutated all result in hepatic iron overload [1–7]. Constitutive over-expression of hepcidin in the liver results in iron deficiency anemia [8]. Liverspecific deletion of TfR2 and HFE in mice recapitulates the phenotype of HH [9,10]; whereas, deletion of HFE in intestinal enterocytes [11] and macrophages [10] does not disturb iron homeostasis. These studies all suggest a major role for the liver in iron metabolism. Hepcidin is an iron-regulatory hormone produced in hepatocytes in response to iron overload or inflammation [12,13]. Hepcidin functions to reduce serum iron levels by reducing intestinal iron absorption and iron release from macrophages and other cell types [14,15]. Hepcidin achieves this by binding to the iron exporter ferroportin on the surface of cells and inducing its internalisation and degradation [16]; in this way hepcidin can rapidly reduce serum iron levels. All of the molecules mutated in the various forms of HH are involved in either the regulation of hepcidin expression or hepcidin function. Given that the liver plays such a central role in controlling iron homeostasis it is not surprising that liver disease can lead to disrupted iron metabolism. ⁎ Corresponding author. Tel: +617 3362 0179; fax: +617 3362 0191. E-mail address: [email protected] (V.N. Subramaniam). 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.09.002

It is also possible that disturbed iron metabolism, which may be present in carriers of mutations associated with HH, such as the C282Y and H63D mutations of HFE, may result in more severe disease when associated with a coexisting liver disease. 2. Hereditary hemochromatosis Hereditary hemochromatosis (HH) is attributed mainly to mutations in the HFE gene [17]. The majority of cases of HH are due to homozygosity for the C282Y mutation in HFE; this genotype is present in about 1 in 200 people of north European descent. The C282Y mutation is most prevalent in areas with north European ancestry, with an allele frequency of between 5% and 10% in most north European countries [18]. Around 1 in 10 individuals in these populations are heterozygous for the C282Y mutation. Another variant H63D has a high allele frequency in European populations of between 10% and 20% [18]. Unlike C282Y, H63D is also prevalent outside of Europe in North Africa, the Middle East and parts of Asia, although with reduced allele frequencies. The H63D variant has little effect on iron stores, but the frequency is increased in HH patients who are not homozygous for C282Y, suggesting that it does contribute to iron overload in some cases [19]. The H63D homozygous genotype has a prevalence of around 2–3% in European populations; it is rarely associated with iron overload, although can be associated with slightly elevated serum iron indices [20]. The frequency of H63D among many north European HH patients who carry one copy of C282Y ranges from 74 to 100% [18]. This suggests that the compound (C282Y/H63D) heterozygote genotype in particular increases the risk of developing HH. Although homozygosity for C282Y is present in around 1 in 200

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Europeans and most of these have raised iron indices, the clinical penetrance of this genotype is incomplete. Two studies have attempted to estimate the clinical penetrance of C282Y homozygosity with differing results. One study of 41,038 individuals attending a health appraisal clinic in the USA estimated that only 1% of C282Y homozygotes develop frank clinical hemochromatosis [21]. A more recent study of 31,192 Australian individuals estimated a clinical penetrance in males of 28.4% and 1.2% in females [22]. It is clear that the clinical penetrance of C282Y homozygosity is higher in males than in females, however, there is marked variability in penetrance between these two studies. Mutations in other genes encoding proteins involved in the regulation of iron homeostasis such as hepcidin, hemojuvelin, TfR2 and ferroportin also lead to various forms of iron overload collectively termed non-HFE hemochromatosis [23–27]. These disorders lead to increased iron absorption and iron deposition in the liver, pancreas, heart and other tissues. In the liver the majority of iron is deposited in the parenchymal cells although iron accumulation can also occur in the Kupffer cells, especially in ferroportin disease [28]. Collectively the non-HFE hemochromatosis syndromes account for the minority of cases of HH. HFE-related HH is much more common, hence the role of HFE mutations in other liver diseases has been studied more extensively. As described earlier, there is variability in the clinical penetrance of C282Y homozygosity. Therefore, it should be emphasised that the degree of penetrance may account for the variable results of studies examining the impact of HFE mutations on other liver diseases that are described in more detail in the following sections. 3. Iron-induced liver damage In HH there is an underlying defect in the regulation of hepcidin. Most forms of HH result from decreased hepcidin in relation to iron stores and a subsequent increase in the absorption of dietary iron. This increase in iron absorption over time leads to increased iron accumulation in parenchymal tissues. Eventually the expansion in body iron stores can reach a level where it promotes tissue damage. The mechanisms of liver injury resulting from excess iron include the generation of free radicals and increased lipid peroxidation, which, in turn, lead to mitochondrial dysfunction, lysosomal fragility and cell death. Hepatic iron overload as a result of either HH or secondary iron overload can result in hepatic fibrosis, cirrhosis and HCC. The degree of hepatic fibrosis shows a positive association with hepatic iron concentration and duration of exposure to excess iron [29,30]. Environmental and acquired factors such as chronic alcohol consumption, chronic viral hepatitis and steatosis are all thought to be co-factors in iron-induced fibrogenesis [29–31]. In HH it has been demonstrated that excess hepatic iron promotes the activation of hepatic stellate cells, and this can be reversed by iron removal [32]. Another study suggested that there is a sub-morphological inflammatory process occurring in HH liver and this may play a role in the development of iron-induced hepatic fibrosis [33]. The incidence of cirrhosis in C282Y homozygotes is greater among males than females, and population studies have estimated a prevalence in males of between 3% and 18% and in females of between 0.3% and 5% [22,34–36]. In disorders of erythropoiesis (such as beta-thalassemia and sideroblastic anemia), increased iron absorption and tissue iron deposition can also occur. A common factor in the iron-loading anemias is refractory anemia, with a hypercellular bone marrow and ineffective erythropoiesis, with a resultant decrease in hepcidin expression [37]. In disorders of erythropoiesis in which there are concomitant blood transfusions, the iron burden can increase rapidly through the combined impact of increased iron absorption and transfusion-derived iron. Therefore, these patients may present with iron-induced hepatic fibrosis earlier than patients with HFE-related HH [29,30].

4. Iron as a co-factor in disease Iron has been suggested as a co-factor in the pathogenesis of a number of other liver diseases, in addition to HH [38]. The amount of iron associated with these conditions would generally be considered to be at non-toxic levels, certainly much lower than in HH. However, the combination of moderately increased iron levels with an added insult, either genetic or environmental may act synergistically to precipitate disease. Conditions in which iron has been suggested to be a co-factor include liver disease caused by chronic hepatitis C virus infection (CHC), alcohol, non-alcoholic fatty liver disease (NAFLD) and porphyria cutanea tarda (PCT). The observation that a proportion of heterozygotes for HH have elevated serum iron indices and evidence of mildly increased iron stores [39] has led many to propose that heterozygosity for HFE mutations may be a risk factor for the development of other diseases [40]. The role of HFE mutations, and the associated increase in iron in other liver diseases and the pathways through which they may promote liver damage are illustrated in Fig. 1. 5. Chronic HCV infection HCV infection becomes chronic in approximately 74% of people exposed to the virus [41]. The success of HCV in causing persistent infection is probably due to its high mutation rate and existence as multiple quasi-species [42]. Carriers develop chronic liver disease, but the course of disease is variable. Chronic HCV infection (CHC) is characterised by peaks and troughs of clinical and biochemical activity. Approximately 20% develop cirrhosis after 20 years [43]. The reasons why some individuals develop more advanced disease than others are not clear. The genotype of the virus may be important. Genotypes 1 and 4 seem to be less responsive to treatment [43]. Other genetic and environmental factors also play a role in the pathogenesis of CHC. Susceptibility to cirrhosis is more common in males, those infected after the age of 50 years and in patients who consume excessive amounts of alcohol [43]. Serum iron indices and hepatic iron content are often elevated in CHC [44–46]. The role of iron in the pathogenesis of CHC remains

Fig. 1. The role of iron in liver disease. Oxidative stress in the liver can be induced by alcohol, hepatitis C virus or insulin resistance associated with obesity. The presence of excess iron in the liver may exacerbate this oxidative stress and promote liver injury and fibrosis. In some cases oxidative stress may directly promote carcinogenesis. Oxidative stress can depress hepcidin expression in hepatocytes via reduced activity of C/EBPα. The presence of HFE mutations, in particular C282Y can depress hepcidin expression further and increase iron absorption and accumulation in the liver, contributing further to oxidative stress. Porphyria cutanea tarda (PCT) is caused by reduced activity of the enzyme URO-D and the resultant accumulation of uroporphyrins. Uroporphomethene, an inhibitor of URO-D is a product of the iron-dependent oxidation of uroporphyrinogen.

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unclear. The excess iron associated with CHC may be a co-factor, acting with the HCV to contribute to the progression of liver disease in infected subjects. Alternatively the iron deposits may be a byproduct of the necro-inflammatory disease process, and play no part in disease progression. Several studies have suggested that the iron contributes to disease progression. Phlebotomy therapy (sometimes combined with a low iron diet) has been used to reduce iron stores in CHC patients with resultant decreases in serum transaminases, markers of hepatic fibrosis, hepatic DNA damage, and the incidence of HCC [47–50]. Oxidative stress has been proposed as a major contributory factor in causing liver injury in CHC [51], and it is well known that iron can promote oxidative stress. The association of iron and CHC and the high prevalence of HFErelated HH have led to the investigation of HFE mutations as contributing factors in the progression of liver disease in CHC. Many studies have addressed this issue with conflicting results. Some studies have reported an association between HFE mutations and more severe disease [52–58], whereas others have found no such association [59–65]. Some of these studies however, found an association between hepatic iron or raised serum iron indices and more severe disease independent of HFE mutations [60,63,64]. Recent studies utilising mouse models of HCV infection have investigated the role of iron in the pathogenesis of disease. Transgenic mice expressing the HCV polyprotein were fed a high-iron diet and liver pathology was analysed [66]. In mice fed the high-iron diet there was an increase in hepatic steatosis, lipid peroxidation products, mitochondrial injury and risk of hepatocellular carcinoma development compared to mice fed a normal diet [66]. In another study transgenic mice expressing the HCV polyprotein were shown to have increased hepatic and serum iron concentrations and decreased splenic iron [67]. The increased iron loading was associated with reduced hepatic hepcidin expression and increased expression of ferroportin in duodenum, liver and spleen. The mechanism underlying the decrease in hepcidin expression was shown to be related to reduced DNA binding activity of the transcription factor C/EBPα, a known regulator of hepcidin transcription. Increased expression of C/EBP homology protein (CHOP), an inhibitor of C/EBP DNA binding was thought to be responsible for the reduction in C/EBPα activity. An increase in reactive oxygen species was also observed in the HCV polyprotein transgenic mice and this may explain the increased expression of CHOP [67]. In human subjects with CHC a decrease in hepcidin expression relative to serum ferritin or iron stores has been observed and may account for iron overload associated with CHC [68]. 6. Alcoholic liver disease Alcohol is one of the primary causes of liver disease in the world. Most heavy drinkers will develop steatosis, but only a minority of these will go on to develop advanced liver disease, including hepatitis, fibrosis or cirrhosis [69]. The risk increases with cumulative alcohol consumption, but other co-factors, either genetic or environmental also play a role in progression to liver disease [69]. As with CHC an association of alcoholic liver disease (ALD) with iron has been noted [70]. Many patients with ALD have raised transferrin saturation or serum ferritin [71], and stainable iron has been demonstrated in alcoholic livers [72]. The hepatic iron usually has a mixed distribution in both hepatocytes and Kupffer cells; however, the amount of stored iron is not normally in the range of that seen in HH. An autopsy study of three patients with alcoholic end-stage liver disease found increased stainable iron in the liver and in extrahepatic tissues, including the heart and pancreas [73]. It appears that increased iron in association with alcohol can exacerbate liver damage. Both iron and alcohol can cause oxidative stress and increased reactive oxygen species that can cause lipid peroxidation and damage to cells. High alcohol intake can cause more severe liver disease in HH. Several studies have reported that patients with HH who drink excessive amounts of alcohol have a

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higher risk of developing cirrhosis and HHC [74–77]. In a study of 224 C282Y homozygotes, 61% of those who drank excessive amounts of alcohol (N60 g per day) had severe fibrosis or cirrhosis compared to only 7% of those who consumed less than 60 g per day [76]. In a study of HFE-C282Y/H63D compound heterozygotes, a genotype not normally associated with iron overload, nearly all of those with progressive liver disease had other co-morbid factors present including excessive alcohol consumption [78]. The association of alcohol with iron has led some to evaluate the role of heterozygosity for HFE mutations as a co-factor in the progression of ALD. Most studies found no relationship between carriage of HFE mutations and more advanced liver disease in patients with ALD [79–83]. One study identified a modest increase in the frequency of the H63D mutation among patients with advanced ALD compared to healthy controls [84]. Lauret et al. studied a group of 179 patients with alcoholic cirrhosis, although the overall frequency of HFE mutations in this group was similar to controls, the frequency of C282Y heterozygosity among the 43 patients with cirrhosis and HCC was significantly higher than the frequency of this mutation in patients with cirrhosis alone [81]. Some studies of ALD patients have found an association between HFE mutations and elevated serum iron indices [81] or modestly increased hepatic iron concentration [80]. Recently the mechanisms leading to increased iron in alcoholinduced liver injury have been studied. Animal models of ALD have helped to elucidate the connection between alcohol and iron metabolism. In some mouse strains fed an alcohol-containing diet, increases in iron absorption and serum iron have been observed [85]. Alcohol administration has been associated with suppression of hepatic hepcidin expression and is accompanied by a decrease in the DNA binding activity and expression of the transcription factor C/EBPα [86,87]. It has been suggested that the oxidative stress induced by alcohol affects the activity of C/EBPα and ultimately hepcidin expression, as antioxidants were shown to abolish the effect of alcohol on C/EBPα binding activity and hepcidin expression [87]. Alcohol has also been shown to interfere with the IL-6 mediated induction of hepcidin in a liver cell line, suggesting another pathway through which alcohol may affect iron homeostasis [86]. It has also been shown that alcohol abolishes the upregulation of hepcidin in response to iron loading, suggesting that alcohol negates the protective effect of hepcidin in the face of iron overload [88]. 7. Non-alcoholic fatty liver disease Non-alcoholic fatty liver disease or NAFLD is now recognized to be one of the most common forms of liver disease. Increasing levels of obesity have likely contributed to the increasing incidence of NAFLD in the Western world. NAFLD ranges from simple fatty liver alone to nonalcoholic steatohepatitis (NASH). Up to 20% of patients with NASH go on to develop cirrhosis and HCC [89]. The incidence of diabetes and obesity is high among cases of cryptogenic cirrhosis, suggesting that cryptogenic cirrhosis may be an end-stage form of NASH [90]. The reasons why some people with fatty liver go on to develop progressive liver disease are not fully understood. A “two-hit” hypothesis has been proposed, where the accumulation of fat in the liver is the “first hit”, followed by a “second hit” that results in progression to NASH [91]. It has been proposed that in some cases of NASH iron may be a co-factor in progression of the disease, leading to increased oxidative stress. Hyperferritinaemia has been observed in over 20% of patients with hepatic steatosis [92,93]. A raised ferritin is not always associated with iron overload, but may be a marker of histologic damage or due to other causes such as insulin resistance [93]. The presence of raised serum ferritin or hepatic iron concentration can increase the risk of developing NASH [92]. In an Australian study an association was found between increased hepatic iron and degree of fibrosis in NASH [94]. Insulin resistance is the most important risk factor for the development of NASH and the coexistence of iron may contribute to the

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development of insulin resistance [93]. A new syndrome termed insulin resistance associated hepatic iron overload (IR-HIO) has been described in which mild to moderate hepatic iron overload is associated with features of insulin resistance [95]. Mendler et al. studied 161 non-C282Y homozygous patients with unexplained hepatic iron overload; nearly all (94%) had features of the insulin resistance syndrome, either a body mass index N25, diabetes or hyperlipidaemia. There was an increased prevalence of C282Y/H63D compound heterozygotes among the group and a high proportion had either hepatic steatosis or NASH [95]. A link between iron and the progression of NAFLD has been supported by the observation that iron removal by phlebotomy can improve the insulin resistance and liver function in patients with NAFLD [96,97]. The association of NAFLD with iron has led many to study the role of HFE mutations. Some studies found an association between HFE mutations and NASH [92,94,98,99]. George et al. [94] found an increased prevalence of the C282Y mutation (31%) among 51 patients with NASH compared to controls (13%). In two other studies an association between both common HFE mutations and NASH was observed [92,98]. In a larger North American study, an association between heterozygosity for C282Y and more advanced fibrosis and hepatic iron was observed in patients with NASH [99]. Other studies have failed to find an association between HFE mutations, iron and NAFLD [93,100,101]. Recently it was shown that hepcidin expression is suppressed in an animal model of insulin resistance, suggesting that hepcidin may be responsible for disturbances in iron homeostasis leading to IR-HIO and NAFLD [102]. 8. End-stage liver disease and hepatocellular carcinoma Increased serum iron indices have been associated with end-stage liver disease. In a study of 106 cirrhotic patients awaiting transplantation 31% had raised serum iron indices suggestive of HH, but only 12% of these had an elevated hepatic iron index, and none was homozygous for the C282Y mutation [103]. Another study looked at the prevalence of C282Y among 304 liver transplant patients. The frequency of C282Y was the same in transplant patients as compared to controls, suggesting that heterozygosity for C282Y does not increase the risk of developing end-stage liver disease [104]. A study of 918 transplant recipients only identified a minority (19) with increased hepatic iron deposits. Of these only four had a diagnosis of HH, and only two of these were homozygous for C282Y. Among the other 15 without an HH diagnosis one was a C282Y homozygote, but none of the others had HH associated genotypes [105]. This study suggests that in end-stage liver disease, patients can have hepatic iron loading in the range of HH, but without HFE mutations. In these cases other factors are responsible for the iron loading. In a large study of 5224 patients undergoing liver transplantation, iron overload was significantly associated with HCC, regardless of the underlying aetiology of liver disease, suggesting a possible carcinogenic effect of iron in chronic liver disease [106]. In HH the hepatotoxic effects of iron can lead to progressive fibrosis and eventually cirrhosis and HCC. Deaths due to cirrhosis and HCC are significantly more prevalent in patients diagnosed with HH, compared to the general population [107]. HH patients with cirrhosis have a higher risk of developing HCC [107]. HCC normally develops in cirrhotic liver, however, some cases of HCC have occurred in noncirrhotic patients with HH, suggesting a direct role for iron in carcinogenesis [108–111]. A direct role for iron in hepatic carcinogenesis has been supported by animal studies, where pre-neoplastic nodules and HCC in the absence of fibrosis developed in rats fed a high-iron diet [112]. A recent study of HH liver biopsies showed epigenetic alterations of genes characteristically hypermethylated in HCC, suggesting that epigenetic changes due to iron loading are an early event in HH, and may lead to the increased risk of progression to HCC [113]. Some studies have looked at the role of HFE mutations in the pathogenesis of HCC with variable results [81,114–119]. An association

was found between HFE mutations and HCC occurring in non-cirrhotic liver. Of 35 patients with HCC occurring in non-cirrhotic liver 54% had histological hepatic iron loading and among this group 37% had HFE mutations, two being homozygous for C282Y [115]. In contrast, Boige at al. found no association between HFE mutations and HCC occurring in cirrhotic liver [117]. Another study did find an increased prevalence of the C282Y mutation among patients with HCC and cirrhosis compared to patients with cirrhosis alone [118]. The discrepancies between studies may be due to ethnic differences or the underlying aetiology of the liver disease. A recent study found that hepatic iron and C282Y were associated with a higher risk of developing HCC in ALD but not in CHC [119]. 9. Porphyria cutanea tarda Porphyria cutanea tarda (PCT) is the most common disorder of porphyrin metabolism. It results from reduced activity of uroporphyrinogen decarboxylase (URO-D), the fifth enzyme in the heme biosynthetic pathway. URO-D catalyses the conversion of uroporphyrinogen to coproporphyrinogen; in PCT the reduced activity of URO-D and the resultant build up of uroporphyrins leads to disease. Clinical features include photosensitive skin lesions associated with hepatic accumulation and urinary excretion of uroporphyrins. PCT can be either familial or sporadic. The familial form is due to mutations in the URO-D gene, leading to reduced URO-D activity in the liver and erythrocytes [120]. It is inherited as an autosomal dominant trait. There is marked genetic heterogeneity in familial PCT and other factors can influence disease expression [121]. Sporadic PCT is more common and is not caused by mutations in the URO-D gene. In contrast to familial PCT the defect in sporadic PCT is confined to the liver. The aetiology of sporadic PCT appears to be multifactorial, both environmental and genetic factors contribute. These factors are also likely to be responsible for disease penetrance in familial PCT. Alcohol abuse, CHC and oestrogen use are common risk factors for the development of PCT [122,123]. Excess hepatic iron and increased serum iron indices have been associated with PCT [124]. Evidence for the involvement of iron in the pathogenesis of PCT is further supported by the observation that venesection treatment improves the clinical outcome and biochemical signs of the disease [125,126]. The link between iron and PCT has led to the suggestion that heterozygosity for HH may be associated with the expression of PCT [127]. Since the discovery of HFE several studies have found an association between HFE mutations and PCT. Roberts et al. found an increase in homozygosity and heterozygosity for the C282Y mutation among UK patients with sporadic PCT [128]. In an Australian study the C282Y mutation and CHC were found to be associated with PCT, but as risk factors for disease were independent of each other [129]. Several studies have now looked at the role of HFE mutations in the pathogenesis of PCT. Most have found an association of HFE mutations with PCT, with high levels of homozygosity or heterozygosity for C282Y in patients compared to controls [128–142]. Most of these studies involved patient groups from European backgrounds where the frequency of C282Y is high. In a group of Italian PCT patients, no association with C282Y was detected, however, the milder H63D allele frequency was significantly increased among the PCT group [143]. The presence of the H63D allele was not related to iron status in these patients. In a racially diverse South African study there was an association of both HFE mutations with PCT, however, this was only apparent in patients with European backgrounds [136]. Therefore, HFE mutations as risk factors for the development of PCT can only be considered in populations where these mutations are prevalent. Other risk factors for PCT, such as HCV infection and alcohol may be more important in areas where HFE mutations are rare. The role of iron and HFE have been investigated further by the development of animal models for sporadic PCT. Increased hepatic

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uroporphyrin accumulation or increased urinary uroporphyrin was observed in Hfe-null mice fed with either 5-aminolevulinate or 10% ethanol in their diets [144,145]. The genetic background of the mice was also found to be important for the development of uroporphyria [146]. Another model in which mice are heterozygous for a null allele of the Uro-D gene and homozygous for the Hfe-null allele develop uroporphyria with no treatment [147]. Recently the mechanism underlying the clinical phenotype of PCT has been elucidated. A competitive inhibitor of URO-D, uroporphomethene was isolated from the liver extracts of mouse models of PCT [148]. Uroporphomethene is an oxidation product of uroporphyrinogen and was shown to be a potent inhibitor of URO-D. The oxidation reaction of uroporphyrinogen to the inhibitor uroporphomethene is iron dependent, supporting the concept of iron as a co-factor in PCT. Factors other than iron that influence the production of the inhibitor are also likely to play a role in the pathogenesis of PCT. 10. Conclusions Iron may play a role in many other forms of liver disease in addition to its hepatotoxic effects in HH and secondary iron overload. In PCT there is a clear link with iron and in European populations there is a strong association with HFE mutations. Fig. 1 illustrates the potential pathways through which iron may be contributing to oxidative stress and exacerbating liver damage in other forms of liver disease. There is some evidence that excess iron, when present can exacerbate disease severity in liver diseases of various aetiologies and can lead to an increased risk of carcinogenesis. There is accumulating evidence that the most common disease agents, alcohol and HCV, can affect iron homeostasis by decreasing hepcidin expression in the liver. Iron removal by phlebotomy is used to treat PCT, and may also have beneficial effects in some patients with CHC and NAFLD. Heterozygosity for HFE mutations is not consistently associated with more severe liver disease in patients with ALD, CHC or NAFLD, perhaps due in part to the incomplete penetrance of these mutations. Further investigation is needed to delineate the potential therapeutic role of phlebotomy in patients with CHC or NAFLD who do not respond to standard therapy. References [1] X.Y. Zhou, S. Tomatsu, R.E. Fleming, S. Parkkila, A. Waheed, J. Jiang, Y. Fei, E.M. Brunt, D.A. Ruddy, C.E. Prass, R.C. Schatzman, R. O'Neill, R.S. Britton, B.R. Bacon, W.S. Sly, HFE gene knockout produces mouse model of hereditary hemochromatosis, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 2492–2497. [2] R.E. Fleming, J.R. Ahmann, M.C. Migas, A. Waheed, H.P. Koeffler, H. Kawabata, R.S. Britton, B.R. Bacon, W.S. Sly, Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10653–10658. [3] D.F. Wallace, L. Summerville, P.E. Lusby, V.N. Subramaniam, First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin, Gut 54 (2005) 980–986. [4] F.W. Huang, J.L. Pinkus, G.S. Pinkus, M.D. Fleming, N.C. Andrews, A mouse model of juvenile hemochromatosis, J. Clin. Invest. 115 (2005) 2187–2191. [5] V. Niederkofler, R. Salie, S. Arber, Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload, J. Clin. Invest. 115 (2005) 2180–2186. [6] J.C. Lesbordes-Brion, L. Viatte, M. Bennoun, D.Q. Lou, G. Ramey, C. Houbron, G. Hamard, A. Kahn, S. Vaulont, Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis, Blood 108 (2006) 1402–1405. [7] I.E. Zohn, I. De Domenico, A. Pollock, D.M. Ward, J.F. Goodman, X. Liang, A.J. Sanchez, L. Niswander, J. Kaplan, The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease, Blood 109 (2007) 4174–4180. [8] G. Nicolas, M. Bennoun, A. Porteu, S. Mativet, C. Beaumont, B. Grandchamp, M. Sirito, M. Sawadogo, A. Kahn, S. Vaulont, Severe iron deficiency anemia in transgenic mice expressing liver hepcidin, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 4596–4601. [9] D.F. Wallace, L. Summerville, V.N. Subramaniam, Targeted disruption of the hepatic transferrin receptor 2 gene in mice leads to iron overload, Gastroenterology 132 (2007) 301–310. [10] M. Vujic Spasic, J. Kiss, T. Herrmann, B. Galy, S. Martinache, J. Stolte, H.J. Grone, W. Stremmel, M.W. Hentze, M.U. Muckenthaler, Hfe acts in hepatocytes to prevent hemochromatosis, Cell. Metab. 7 (2008) 173–178.

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