Porphyria cutanea tarda (PCT), the most common

Transcription

1 Uroporphyria Caused by Ethanol in Hfe( / ) Mice as a Model for Porphyria Cutanea Tarda Peter R. Sinclair, 1,2,3 Nadia Gorman, 1,2 Heidi W. Trask, 1,2 William J. Bement, 1 Juliana G. Szakacs, 4 George H. Elder, 5 Dominic Balestra, 1,6 Jacqueline F. Sinclair, 1,2,3 and Glenn S. Gerhard 1,7 Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe( / ) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe( / ) but not wild-type mice. The increases in nonheme iron in Hfe( / ) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe( / ) mice seem to be an excellent model for studies of alcohol-mediated PCT. (HEPATOLOGY 2003;37: ) Porphyria cutanea tarda (PCT), the most common human porphyria, manifests as a skin disease and is characterized by massive hepatic production and urinary excretion of uroporphyrin (URO) (see reviews 1-3 ). The disease can be sporadic or familial, but 71% to 81% of U.S. cases are sporadic. 4,5 Most patients with familial PCT are heterozygous for a mutation in the uroporphyrinogen decarboxylase (UROD) gene, leading to a 50% loss of UROD activity. 1-3 In patients with both sporadic and familial PCT, UROD becomes highly inactivated in the liver, resulting in increased accumulation of uroporphyrinogen, which is oxidized to URO. Both sporadic and familial PCT are multifactorial diseases 5 and are Abbreviations: PCT, porphyria cutanea tarda; URO, uroporphyrin; UROD, uroporphyrinogen decarboxylase; ALA, 5-aminolevulinate; ALAS, 5-aminolevulinate synthase; CYP, cytochrome P450. From the 1 VA Medical Center, White River Junction, VT; Departments of 2 Biochemistry, 3 Pharmacology/Toxicology, 6 Medicine, and 7 Pathology, Dartmouth Medical School, Hanover, NH; 4 Department of Pathology, University of Utah Medical School, Salt Lake City, UT; and 5 Department of Medical Biochemistry, University of Wales Medical School, Heath Park, Wales, United Kingdom. Received July 1, 2002; accepted October 30, Supported by funds from the Department of Veterans Affairs and grants from the National Institutes of Health (ES06263 to P.R.S. and AG14731 to G.S.G.). Address reprint requests to: Peter R. Sinclair, Ph.D., VA Medical Center, 215 Main St., White River Junction, VT psinc@dartmouth.edu; fax: This is a US government work. There are no restrictions on its use /03/ $0.00/0 doi: /jhep most commonly precipitated in susceptible individuals by consumption of alcoholic beverages. 2-5 Other precipitants include estrogenic steroids and infection with hepatitis C virus. 1,3,5 The mechanism of action of the precipitants is not known. There is usually a mild hepatic siderosis in patients with PCT, and the disease is responsive to iron depletion by phlebotomy or treatment with chelators of iron. The alternative therapy, low-dose chloroquine, may also be effective by altering hepatic iron pools. 6 Many patients of northern European descent with PCT carry the same C282Y mutation in the HFE gene as found in hereditary hemochromatosis, an iron-storage disease 7,8 characterized by increased duodenal iron absorption and increased hepatic iron levels. In recent studies, 12% to 24% of patients of northern European origin with PCT were found to be homozygous for the C282Y (HFE) mutation. 4,8-10 Other studies with fewer patients have also found an association between PCT and heterozygosity for C282Y. 10 In some countries (e.g., Italy), PCT is associated with other HFE mutations such as H63D. 11 Although the exact role of iron in PCT is unknown, a known effect of the HFE mutation in hemochromatosis is to preferentially increase iron accumulation in hepatic parenchymal cells, 12 the site of URO accumulation. The role of iron in human PCT is also supported by studies in rodents administered polyhalogenated aromatic hydrocarbons to produce uroporphyria. Administration of 351

2 352 SINCLAIR ET AL. HEPATOLOGY, February 2003 iron accelerates this uroporphyria, 13 and iron deficiency in mice protects against this uroporphyria. 14 A single injection of iron dextran, even in the absence of polyhalogenated aromatic hydrocarbons, causes uroporphyria in certain mouse strains after several months. 15 The effect of increases in hepatic iron on uroporphyria is also observed in Hfe( / ) mice, which rapidly become uroporphyric when administered the porphyrin precursor 5-aminolevulinate (ALA). 16 Iron staining is more pronounced in parenchymal cells in Hfe( / ) mice than in wild-type mice with the same genetic background. 16,17 Hfe( / ) mice that are also UROD( / ) become highly uroporphyric by the age of 14 weeks without any additional treatment. 18 These studies suggest an interaction between altered iron metabolism and hepatic UROD inactivation. Although a role for iron in PCT is suggested by observations in humans and animal models, there is much less understanding of the role of alcohol, the most common precipitating factor in PCT. 2,5 There have been several suggested mechanisms for the alcohol effect. For instance, alcohol may induce 5-aminolevulinate synthase (ALAS), the rate-limiting enzyme in porphyrin biosynthesis. 2,3,19 Induction of this enzyme is stimulated by iron, 20 and alcohol may enhance this stimulation. Alternatively, alcohol may have effects on intestinal uptake or other aspects of iron metabolism. 21 Progress in testing these various hypotheses for the effect of alcohol has been severely hampered by the lack of an animal model for alcohol-induced uroporphyria, even though there have been attempts to develop such an animal model. In several rodent studies, attempts to increase URO accumulation by administering ethanol in the drinking water over several weeks have not been successful. 22,23 In the present study, we report an animal model in which uroporphyria was produced in Hfe( / ) mice by long-term treatment with ethanol given in the drinking water. We used this model because long-term treatment with ethanol is analogous to the consumption of alcoholic beverages in PCT. Hepatic URO was increased after 6 to 7 months of treatment with 10% ethanol in Hfe( / ) mice but not in wild-type mice. Hepatic UROD activity was also markedly decreased in ethanol-treated Hfe( / ) mice. There were parallel increases in total hepatic nonheme iron and ALAS activities in ethanol-treated Hfe( / ) mice, associated with increased localization of storage iron in parenchymal cells. These findings suggest biochemical mechanisms by which alcohol and iron may interact synergistically to cause PCT. Materials and Methods Animals. Hfe( / ) mice were derived and maintained in the 129S6/SvEvTac background. 17 Confirmation of the Hfe( / ) genotype was performed as described previously. 17 Wild-type mice of the same 129 substrain were purchased from Taconic (Germantown, NY). Only male mice (8-10 weeks old, g body weight) were used. Animals were housed in standard plastic cages and maintained on 12-hour light/dark cycles with free access to rodent chow (Teklad LM-485 containing 0.02% FeSO 4 ) from Harlan Teklad (Madison, WI). Ethanol was administered in the drinking water at 10% (vol/vol) as the only source of water. For urinary porphyrin composition, urine was collected in the morning as fresh samples from individual animals. All animal protocols were approved by the Animal Use Committees of the VA Medical Center and Dartmouth College as being in conformity with the criteria outlined in the Guide for Care and Use of Laboratory Animals prepared by the National Academy of Sciences (NIH publication 86-23, revised 1985). Preparation of Liver Fractions. Liver homogenates and microsomes were prepared and stored as described previously. 24 The homogenates were centrifuged at 10,000g for 10 minutes. The pellet was used as the mitochondrial/nuclear fraction for determination of ALAS activity. The postmitochondrial cytosol and microsomal fractions were obtained as described 24 and stored frozen at 80 C until analyzed. Antibodies. A polyclonal antibody raised against rat cytochrome P450 (CYP)1A2, which detects both the mouse CYP1A2 and CYP1A1 proteins, and a polyclonal antibody prepared against rat CYP3A were generous gifts from Steve Wrighton (Lilly Research Labs, Indianapolis, IN). A polyclonal antibody prepared against human CYP2E1 was purchased from Oxford Biomedical Inc. (Oxford, MI). Assays. Porphyrin composition in urine and liver homogenates was determined spectrophotofluorometrically and for most samples was confirmed by reverse-phase high-performance liquid chromatography. 25 The ratio of URO to coproporphyrin was determined spectrofluorometrically in urine from individual mice as described previously. 26 Protein concentrations were determined by the method of Lowry et al. 27 using bovine serum albumin as a standard. Microsomal methoxyresorufin demethylase and uroporphyrinogen oxidation were measured as described previously. 28 UROD was assayed in 100,000g supernatant fractions using pentacarboxyporphyrinogen I as substrate as described previously. 16 ALAS activity in the freshly made mitochondrial fraction was determined as described. 29 The presence of CYP1A1, CYP1A2, CYP2E1, and CYP3A proteins in liver homogenates was determined by immunoblotting, following the electrophoretic separation of proteins in liver homogenates as

3 HEPATOLOGY, Vol. 37, No. 2, 2003 SINCLAIR ET AL. 353 described. 16 Immunoreactive proteins were quantitated by scanning digitized blot images with One-Dscan software (Scanalytics Inc., Fairfax, VA). At least 4 different samples were analyzed for each treatment and scanned in triplicate. Nonheme iron was measured by the method of Torrance and Bothwell, as described. 16 Plasma alanine aminotransferase level was determined by the clinical laboratory of the VA Hospital using the automated DADE/ Dimension Clinical Chemistry System. Histology. Portions of liver were fixed in 10% buffered formalin. Fixed sections were stained by both standard hematoxylin-eosin and Perls Prussian blue stain. The latter were counterstained with nuclear fast red (0.1%) for 10 seconds (rather than the usual 4 minutes) to maximize detection of storage iron. 16 Histologic grading was performed separately by 2 pathologists who were in agreement. Iron was separately graded in zones 1 to 3 from 0 to 4 in Kupffer (sinusoidal lining) cells and hepatocytes. 16 The sum of the grades for zones 1 to 3 was used for quantification. Statistical Analyses. Two separate experiments were performed, and the results are combined from these experiments. Results are presented as mean SD. Significance was determined by one-way ANOVA except where the SDs were significantly different, in which case a nonparametric Mann-Whitney test was used. P values less than.05 were considered significant. Results Fig. 1. Hepatic URO content and UROD activities in Hfe( / ) and wild-type mice treated with ethanol. Male wild-type or Hfe( / ) mice were treated with 10% ethanol in drinking water for 6 to 7 months. (A) URO was determined spectrofluorimetrically as described in Materials and Methods. (B) UROD activities were determined in cytosol (100,000g supernatant) using pentacarboxyporphyrinogen I as substrate, as described in Materials and Methods. The values for individual mice are shown, with the bar indicating the mean. *Indicates groups that were significantly different from each other. Numbers of animals per group: wild-type untreated, 3; wild-type treated with ethanol, 5; untreated Hfe( / ), 8;Hfe( / ) treated with ethanol, 9. Ethanol Induction of Uroporphyria in Hfe( / ) Mice. We investigated whether 10% ethanol in the drinking water produces uroporphyria in Hfe( / ) mice. The mice readily drank the ethanol, and all mice drank similar volumes of fluid. Ethanol-treated mice were indistinguishable in appearance and weight from untreated mice during the course of the study (weight gains for untreated and ethanol-treated mice, and g, respectively). To screen for uroporphyria, urine was collected every 3 to 4 weeks and the ratio of URO to coproporphyrin was measured. This ratio has been previously shown to be an effective screen for increases in hepatic URO. 26 This ratio did not increase in ethanoltreated Hfe( / ) mice until after 4 months. After 5 months, most of the ethanol-treated Hfe( / ) mice had URO/coproporphyrin ratios exceeding 1.0, whereas the ratio for the untreated Hfe( / ) or ethanol-treated wildtype animals was less than 0.5 (data not shown). After a further 1 to 2 months, all mice were killed. No mice were lost during the study. Hepatic URO content after 6 to 7 months of ethanol consumption was significantly increased in the ethanoltreated Hfe( / ) mice (Fig. 1A), whereas essentially no change occurred in all the other groups (Fig. 1A). A wide variation in URO values was found among individual ethanol-treated Hfe( / ) mice, and the amounts of URO accumulated were relatively low. These results are consistent with the mice being at the initial stages of uroporphyria. Similar degrees of variation in URO values have been reported in uroporphyria induced by other protocols. 30,31 Hepatic UROD activity was significantly decreased in ethanol-treated Hfe( / ) mice compared with wild-type mice (Fig. 1B). Hepatic URO values correlated inversely with UROD activities (r 0.84). Ethanol consumption produced no decreases in UROD activity in wild-type mice. The decreases in UROD activity in eth-

4 354 SINCLAIR ET AL. HEPATOLOGY, February 2003 Fig. 2. Hepatic nonheme iron and ALAS activities in Hfe( / ) and wild-type mice treated with ethanol. Samples were prepared from the same mice as indicated in the legend to Fig. 1. (A) Nonheme iron was determined in liver homogenates as described in Materials and Methods. (B) ALAS activities were determined on a mitochondrially enriched fraction, as described in Materials and Methods but have been recalculated on a per liver wet weight basis. *,# Indicates groups that were significantly different from each other. Numbers of animals per group for A: wild-type untreated, 3; wild-type treated with ethanol, 5; untreated Hfe( / ), 8;Hfe( / ) treated with ethanol, 9. Numbers of animals per group for B: wild-type untreated, 3; wild-type treated with ethanol, 5; untreated Hfe( / ), 4;Hfe( / ) treated with ethanol, 5. anol-treated Hfe( / ) mice were similar to those seen in uroporphyria produced by other protocols. 16 Although some of the ethanol-treated Hfe( / ) mice had nearnormal hepatic URO and UROD values, there was no correlation between hepatic iron concentration and hepatic URO values. Effect of Ethanol on Hepatic Iron Content and Distribution. As expected from previous studies, 16,17 levels of hepatic nonheme iron in untreated Hfe( / ) mice were significantly greater than in untreated wildtype mice. Ethanol consumption caused a further 50% increase in hepatic nonheme iron in Hfe( / ) mice but had no effect in wild-type mice (Fig. 2A). Histologically, untreated wild-type mice showed some Prussian blue staining in Kupffer cells but no staining in parenchymal cells. No change was produced by ethanol treatment of these mice (Fig. 3A and B; Table 1). In untreated Hfe( / ) mice, staining of storage iron in Kupffer cells was similar to that observed in wild-type mice. However, as observed previously, there was also staining of parenchymal cells 16 (Fig. 3C and Table 1). In the ethanol-treated Hfe( / ) mice, there was a marked increase in staining of parenchymal cells, particularly in the centrilobular zone 3, in addition to the staining of Kupffer cells (Fig. 3D and Table 1). The increase in histologically detected storage iron was consistent with the increases in total nonheme iron. Effect of Ethanol Consumption on Liver Injury. The effect of ethanol consumption on hepatotoxicity was assessed in Hfe( / ) mice by measuring plasma alanine aminotransferase levels after the mice were killed and by histology. No differences in these values were found between untreated and ethanol-treated Hfe( / ) mice (30 7 and 34 3 U/L, respectively). The liver histology of ethanol-treated Hfe( / ) mice showed no abnormalities other than focal macrovesicular steatosis in 2 of 9 of the Hfe( / ) mice treated with ethanol (results not shown). Effect of Ethanol on Hepatic ALAS Activity. To determine whether increases in ALAS activity might contribute to ethanol-mediated uroporphyria in Hfe( / ) mice, the activity of this enzyme was measured in a mitochondrially enriched fraction prepared from wild-type and Hfe( / ) mouse livers (Fig. 2B). Significantly higher activities were seen in untreated Hfe( / ) mice than in untreated wild-type mice (P.05), coincident with the increase in total nonheme iron (Fig. 2A). Treatment with ethanol caused a significant increase in ALAS activity in Hfe( / ) mice (Fig. 2B), paralleling increases in both total nonheme iron (Fig. 2A) and parenchymal cell iron (Fig. 3D). Effect of Ethanol on Hepatic CYP Levels. Because CYP1A2 has been previously implicated in the development of uroporphyria, 26,32,33 we evaluated whether ethanol increased CYP1A2 in Hfe( / ) mice as determined enzymatically by microsomal methoxyresorufin demethylase and uroporphyrinogen oxidase activities 34 and immunochemically by Western blots of liver homogenates. Hepatic microsomal CYP1A2 enzyme activities were not different in ethanol-treated and untreated Hfe( / ) mice (Table 2). There was also no difference in the amount of immunochemically detected CYP1A2 protein in ethanoltreated and untreated wild-type and Hfe( / ) mice (Table 2). However, immunochemically detected CYP2E1 and CYP3A, which are induced by ethanol in several ex-

5 HEPATOLOGY, Vol. 37, No. 2, 2003 SINCLAIR ET AL. 355 Fig. 3. Localization of hepatic iron in wild-type and Hfe( / ) mice treated with ethanol. Mice were treated as described in the legend to Fig. 1. Following Perls staining, sections were counterstained for only 10 seconds to maximize detection of blue-stained iron. 16 (A) Wild-type untreated mice. (B) Wild-type ethanol-treated mice. (C) Hfe( / ) untreated mice. (D) Hfe( / ) ethanol-treated mice. (Original magnification, 400.) perimental systems, 35 were increased about 1.5- and 2.5- fold, respectively, by treatment with ethanol in both Hfe( / ) and wild-type mice. Discussion Hfe Status Table 1. Hepatic Iron Staining With Perls Stain Treatment No. of Mice Kupffer Cells Parenchymal Cells / Untreated EtOH (0 5) 0.2 (0 1) / Untreated 8 5 (2 6) 2.5 (1 5) EtOH 9 5 (3 7) 4 (3 6) NOTE. Where indicated, mice received 10% ethanol in drinking water for 6 to 7 months. The values for Kupffer and parenchymal cells are means and ranges. The histologic grading is described in Materials and Methods. Alcohol consumption is the most common etiologic agent in both sporadic and familial PCT. 1-3,5 Investigations into the mechanism by which alcohol causes PCT have been impeded by the lack of a suitable animal model. In this report, a new model of ethanol-mediated uroporphyria has been presented. The design of these experiments was based on previous work showing that many, although not all, patients with PCT are homozygous for the C282Y mutation in the HFE gene and usually have elevated hepatic iron levels. 4,8-10 In addition, Hfe( / ) mice with enhanced hepatic parenchymal cell iron are sensitized for the development of uroporphyria. 16,18 It is recognized that Hfe( / ) mice are likely to present a more severe phenotype than mice homozygous for the C282Y mutation because of the higher hepatic iron levels in the former. 17 In this study, when Hfe( / ) mice were given ethanol in their drinking water for 6 to 7 months, they accumulated hepatic URO (Fig. 1A); this correlated with de-

6 356 SINCLAIR ET AL. HEPATOLOGY, February 2003 Table 2. Effect of Ethanol on Hepatic CYPs in Hfe( / ) Mice Hepatic Microsomal Activities Immunodetected CYPs (%) Treatment No. of Mice UROX (pmol URO/ min/mg protein) MROD (pmol resorufin/ min/mg protein) 1A2 2E 3A Untreated EtOH * * NOTE. Where indicated, mice received 10% ethanol in drinking water for 6 to 7 months. Values are means and SDs. Microsomal activities and immunodetection of CYPs are described in Materials and Methods. *Significantly different from untreated mice (P.05). creases in hepatic UROD activity (Fig. 1B) and increases in total hepatic nonheme iron (Fig. 2A), with a marked increase of stainable iron in parenchymal cells (Fig. 3). The results indicate that ethanol consumption alone does not cause uroporphyria, at least in wild-type mice of this 129 subline. Previous studies have not detected uroporphyria in ethanol-treated rodents. 22,23 Whether the effect of the disruption in the Hfe gene on uroporphyria is solely due to increased hepatic iron level or to some other effects of the Hfe gene deletion is not known. Uroporphyria was achieved in this study using 10% ethanol in the drinking water without evidence of significant hepatic injury. Although this route of ethanol administration is not optimal for studies of hepatic steatosis and other forms of alcohol-induced liver injury, 36 its simplicity is a distinct advantage for future studies of the mechanism of the specific effect of ethanol on uroporphyria. Furthermore, the 129 mouse subline used here readily consumes ethanol ad libitum. 37 The mice administered 10% ethanol in drinking water gained weight at the same rate as untreated mice, indicating that caloric intakes were similar. Hepatic ALAS activity is rate-limiting in heme and porphyrin synthesis. ALAS activities in untreated Hfe( / ) mice were greater than in untreated wild-type mice (Fig. 2B), consistent with the known induction of hepatic ALAS in rats by administration of iron. 20 Treatment of Hfe( / ) mice but not wild-type mice with ethanol resulted in further increases in ALAS activity. The effect of ethanol on ALAS in Hfe( / ) mice may be due to the synergistic induction of ALAS by the combination of iron and ethanol. It was previously observed that hepatic ALAS is induced when patients with PCT are administered ethanol acutely. 38 Our present findings suggest that this ethanol induction of ALAS may occur because the patients livers are already iron loaded. Although the levels of hepatic URO in this study are relatively low compared with those achieved in other protocols, they are typical of the early stages of URO accumulation in rodent uroporphyria models. 30,31 To accumulate higher amounts of URO, further decreases in UROD activity may be required as well as further increases in ALAS, which can be replaced by feeding with ALA. 16,31 Decreases in UROD may be associated with production of an as-yet unidentified UROD inhibitor that has been detected in uroporphyric livers from mice and rats (see reviews 3,39 ). Whether this putative UROD inhibitor causes UROD inactivation in vivo has yet to be determined. There is evidence that UROD inhibition results from the increased uroporphyrinogen accumulating when porphyrin synthesis is stimulated by administration of ALA. 40 With the development of a model for alcoholinduced uroporphyria, whether the inhibitor is formed in this uroporphyria can now be examined. Ethanol consumption accelerated development of uroporphyria and caused preferential accumulation of iron in parenchymal cells of Hfe( / ) mice. The mechanism of the ethanol effect on iron localization is unknown. Although transferrin saturation in the wild-type 129 mouse subline used here is 85%, 17 an increase in intestinal absorption could increase non transferrin-bound iron, which is taken up by hepatocytes. 41 Iron is absorbed by duodenal enterocytes from the diet through the protein DMT1 in conjunction with ferric reductase. 42 Whether ethanol alters the kinetics of absorption via this route is not known. Similarly, iron is exported from the basolateral surface of the enterocyte via IREG1 and hephaestin. 43 No data are available on the effect of ethanol on these proteins. Ethanol may also exert its effects within the liver on parenchymal and/or Kupffer cells. The interaction of ethanol, HFE, and other proteins involved in iron metabolism is not yet known. Our findings suggest that one mechanism by which ethanol consumption precipitates PCT is related to its effects on iron metabolism. Increases in hepatic iron levels have previously been reported to enhance the development of uroporphyria. 15,39 In some mouse strains, iron dextran alone, administered as a single large dose, can produce high levels of hepatic URO over a period similar to that used here. 15 In addition, we recently noted that with administration of ALA, very large doses of iron dextran were required to produce hepatic URO accumula-

7 HEPATOLOGY, Vol. 37, No. 2, 2003 SINCLAIR ET AL. 357 tion in C57BL/6 mice. 34 By using Hfe( / ) mice, we have avoided the requirement for administration of iron. 16 As in PCT, the effect of ethanol on hepatic URO accumulation in Hfe( / ) mice was chronic rather than acute. Both ethanol and the loss of HFE protein expression are required for development of the uroporphyria, because wild-type mice did not respond to treatment with ethanol. These results suggest that the importance of ethanol, the most common etiologic agent in human PCT, is due to its effects on both iron metabolism and iron-stimulated increases in ALAS activity. The central effect of ethanol on iron in this mouse model is supported by the clinical finding that all patients are improved with phlebotomy and that chloroquine, the major alternative PCT therapy, may also be effective by altering iron metabolism. 6 There is considerable interest in ethanol-induced oxidative stress and its role in various symptoms of alcohol toxicity such as PCT. Several mechanisms may contribute to ethanol-induced oxidative stress, including production of reactive oxygen species by CYPs, mitochondrial injury, depletion of reduced glutathione and other antioxidant defense mechanisms, increased endotoxin production, and Kupffer cell activation. 44 The development of oxidative stress and its effects on uroporphyria in the model presented in this report will be the subject of future studies. In other protocols, using Cyp1a2( / ) mice, CYP1A2 has been shown to be absolutely required for producing uroporphyria. 26,32,33 This was the case even in uroporphyria produced by acetone, in which there were no increases in CYP1A2 above constitutively expressed levels. 45 In the present study, uroporphyria was produced by ethanol treatment of Hfe( / ) mice without a detectable increase in CYP1A2 as measured enzymatically and immunochemically. Likewise, recent studies in patients with PCT have not produced evidence of increases in hepatic CYP1A2 compared with controls. 46 It has been suggested that 2 other ethanol-induced CYPs (CYP2E1 and CYP3A) that were increased by ethanol might contribute to the development of human PCT. 47 Investigation of whether the ethanol-mediated uroporphyria in mice requires CYP1A2 or other forms of CYP will be the subject of future studies. Our findings that ethanol accelerated hepatic iron accumulation in the Hfe( / ) mice may also be relevant in the clinical penetrance and severity of liver disease in hereditary hemochromatosis. 48,49 There are several other hepatic diseases associated with increased hepatic iron levels, 50 and alcohol may play an important role. In conclusion, this report presents a new mouse model that recapitulates the effect of the 2 primary etiologic agents in PCT and focuses attention on the major effect of ethanol on hepatic iron metabolism in Hfe( / ) mice. Further investigations into the molecular mechanisms underlying this uroporphyria will now be possible with this model. Acknowledgment: The authors thank Drs. N. Jacobs, J. Jacobs, and A. Cederbaum for advice on the manuscript as well as Terry Mattoon and Linda Wilmott for histologic preparation of samples. References 1. Elder GH. Porphyria cutanea tarda. Semin Liver Dis 1998;18: Elder GH. Alcohol intake and porphyria cutanea tarda. HEPATOLOGY 1998;27: Elder GH. Porphyria cutanea tarda and related disorders. In: Kadish KM, Smith KM, Guilard R, eds. The Porphyrin Handbook II. Volume 14. Boston: Academic Press, 2003: Bulaj ZJ, Phillips JD, Ajioka RS, Franklin MR, Griffen LM, Guinee DJ, Edwards CQ, et al. Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda. Blood 2000;95: Egger NG, Goeger DE, Payne DA, Miskovsky EP, Weinman SA, Anderson KE. Porphyria cutanea tarda. Multiplicity of risk factors including HFE mutations, hepatitis C, and inherited uroporphyrinogen decarboxylase deficiency. Dig Dis Sci 2002;47: Sarkany RP. The management of porphyria cutanea tarda. Clin Exp Dermatol 2001;26: Roberts AG, Whatley SD, Morgan RR, Worwood M, Elder GH. Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda. Lancet 1997;349: Tannapfel A, Stolzel U, Kostler E, Melz S, Richter M, Keim V, Schuppan D, et al. C282Y and H63D mutation of the hemochromatosis gene in German porphyria cutanea tarda patients. Virchows Arch 2001;439: Brady JJ, Jackson HA, Roberts AG, Morgan RR, Whatley SD, Rowlands GL, Darby C, et al. Co-inheritance of mutations in the uroporphyrinogen decarboxylase and hemochromatosis genes accelerates the onset of porphyria cutanea tarda. J Invest Dermatol 2000;115: Stuart KA, Busfield F, Jazwinska EC, Gibson P, Butterworth LA, Cooksley WG, Powell LW, et al. The C282Y mutation in the haemochromatosis gene (HFE) and hepatitis C virus infection are independent cofactors for porphyria cutanea tarda in Australian patients. J Hepatol 1998;28: Sampietro M, Piperno A, Lupica L, Arosio C, Vergani A, Corbetta N, Malosio I, et al. High prevalence of the His63Asp HFE mutation in Italian patients with porphyria cutanea tarda. HEPATOLOGY 1998;27: Brunt EM, Olynyk JK, Britton RS, Janney CG, Di Bisceglie AM, Bacon BR. Histological evaluation of iron in liver biopsies: relationship to HFE mutations. Am J Gastroenterol 2001;95: Smith A, Francis J. Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice. Biochem J 1983; 214: Sweeney GD, Jones KG, Cole FM, Basford D, Krestynski F. Iron deficiency prevents liver toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Science 1979;204: Smith AG, Francis JE. Genetic variation of iron-induced uroporphyria in mice. Biochem J 1993;291: Sinclair PR, Gorman N, Walton HS, Bement WJ, Sinclair JF, Gerhard GS, Szakacs JG, et al. Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda. HEPATOL- OGY 2001;33:

8 358 SINCLAIR ET AL. HEPATOLOGY, February Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC. The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999;94: Phillips JD, Jackson LK, Bunting M, Franklin MR, Thomas KR, Levy JE, Andrews NC, et al. A mouse model of familial porphyria cutanea tarda. Proc Natl Acad Sci USA2001;98: Shanley BC, Zail SS, Joubert SM. Effect of ethanol on liver -aminolevulinate synthetase in rats. Lancet 1968;1: Bonkowsky HL, Healey JF, Sinclair PR, Sinclair JF, Pomeroy JS. Iron and the liver. Acute and long-term effects of iron-loading on hepatic haem metabolism. Biochem J 1981;196: Whitfield JB, Zhu G, Heath AC, Powell LW, Martin NG. Effects of alcohol consumption on indices of iron stores and of iron stores on alcohol intake markers. Alcohol Clin Exp Res 2001;25: Shanley BC, Zail SS, Joubert SM. Porphyrin metabolism in experimental hepatic siderosis in the rat. Br J Haematol 1970;18: Teschke R, Gorys-Konemann C, Daldrup T, Goerz G. Influence of chronic alcohol consumption on hepatic heme and porphyrin metabolism. Biochem Pharmacol 1987;36: Lambrecht RW, Jacobs JM, Sinclair PR, Sinclair JF. Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P450-mediated uroporphyrinogen oxidation. Biochem J 1990;269: Sinclair PR, Gorman N, Jacobs JM. Measurement of heme concentration. In: Maines MD, Costa LG, Reed DJ, Sassa S, Sipes IG, eds. Current Protocols in Toxicology. Volume 1. New York: Wiley, 2000: Sinclair PR, Gorman N, Walton HS, Bement WJ, Dalton TP, Sinclair JF, Smith AG, et al. CYP1A2 is essential in murine uroporphyria caused by hexachlorobenzene and iron. Toxicol Appl Pharmacol 2000;162: Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193: Sinclair PR, Gorman N, Walton HS, Sinclair JF, Lee CA, Rifkind AB. Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by Ah receptor ligands in chicken liver and kidney. Drug Metab Dispos 1997;25: Sinclair PR, Gorman N, Cornell NW. Measurement of ALA synthase activity. In: Maines MD, Costa LG, Reed DJ, Sassa S, Sipes LG, eds. Current Protocols in Toxicology. Volume 1. New York: Wiley, 1999: Constantin D, Francis JE, Akhtar RA, Clothier B, Smith AG. Uroporphyria induced by 5-aminolaevulinic acid alone in Ahr d SWR mice. Biochem Pharmacol 1983;52: Gorman N, Walton HS, Bement WJ, Honsinger CP, Szakacs JG, Sinclair JF, Sinclair PR. Role of small differences in CYP1A2 in the development of uroporphyria produced by iron and 5-aminolevulinate in C57BL/6 and SWR strains of mice. Biochem Pharmacol 1999;58: Sinclair PR, Gorman N, Dalton T, Walton HS, Bement WJ, Sinclair JF, Smith AG, et al. Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice. Biochem J 1998;330: Smith AG, Clothier B, Carthew P, Childs NL, Sinclair PR, Nebert DW, Dalton TP. Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin. Toxicol Appl Pharmacol 2001;173: Gorman N, Ross KL, Walton HS, Bement WJ, Szakacs JG, Gerhard GS, Dalton TP, et al. Uroporphyria in mice: thresholds for hepatic CYP1A2 and iron. HEPATOLOGY 2002;35: Louis CA, Wood SG, Kostrubsky V, Sinclair PR, Sinclair JF. Synergistic increases in rat hepatic cytochrome P450s by ethanol and isopentanol. J Pharmacol Exp Ther 1994;269: Hall P de la M, Lieber CS, DeCarli LM, French SW, Lindros KO, Jarvelainen H, Bode C, et al. Models of alcoholic liver disease in rodents: a critical evaluation. Alcohol Clin Exp Res 2001;25:254S-261S. 37. Bowers BJ, Owen EH, Collins AC, Abeliovich A, Tonegawa S, Wehner JM. Decreased ethanol sensitivity and tolerance development in gammaprotein kinase C null mutant mice is dependent on genetic background. Alcohol Clin Exp Res 1999;23: Shanley BC, Zail SS, Joubert SM. Effect of ethanol on liver deltaaminolaevulinate synthetase activity and urinary porphyrin excretion in symptomatic porphyria. Br J Haematol 1969;17: Smith AG. Porphyria caused by chlorinated AH receptor ligands and associated mechanisms of liver injury and cancer. In: Kadish KM, Smith KM, Guilard R, eds. The Porphyrin Handbook II. Volume 14. Boston: Academic Press, 2003: Urquhart J, Elder GH, Roberts AG, Lambrecht RW, Sinclair PR, Bement WJ, Gorman N, et al. Uroporphyria produced in mice by 20-methylcholanthrene and 5-aminolaevulinic acid. Biochem J 1988;252: Brissot P, Wright TL, Ma WL, Weisiger RA. Efficient clearance of nontransferrin-bound iron by rat liver. Implications for hepatic iron loading in iron overload states. J Clin Invest 1985;76: McKie AT, Barow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, et al. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 2001;291: Fleming RE, Sly WS. Mechanisms of iron accumulation in hereditary hemochromatosis. Annu Rev Physiol 2002;64: Cederbaum A. Introduction-serial review: alcohol, oxidative stress and cell injury. Free Radic Biol Med 2001;31: Sinclair PR, Gorman N, Walton HS, Bement WJ, Szakacs J, Gonzalez FJ, Dalton TP, et al. Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone. Arch Biochem Biophys 2000;384: Bulaj ZJ, Franklin MR, Phillips JD, Miller KL, Bergonia HA, Ajioka RS, Griffen LM, et al. Transdermal estrogen replacement therapy in postmenopausal women previously treated for porphyria cutanea tarda. J Lab Clin Med 2000;136: Sinclair PR, Gorman N, Tsyrlov IB, Fuhr U, Walton HS, Sinclair JF. Uroporphyrinogen oxidation catalyzed by human cytochromes P450. Drug Metab Dispos 1998;26: Jackson HA, Carter K, Darke C, Guttridge MG, Ravine D, Hutton RD, Napier JA, et al. HFE mutations, iron deficiency and overload in 10,500 blood donors. Br J Haematol 2001;114: Fletcher LM, Dixon JL, Purdie DM, Powell LW, Crawford DH. Excess alcohol greatly increases the prevalence of cirrhosis in hereditary hemochromatosis. Gastroenterology 2002;122: Bonkovsky HL, Lambrecht RW. Iron-induced liver injury. Clin Liver Dis 2000;4: