Fumarylacetoacetate Hydrolase Research Articles

Renal tubular function in children with tyrosinaemia type I treated with nitisinone

Tyrosinaemia type I (TTI) is an inherited deficiency in the enzyme fumarylacetoacetate hydrolase and is frequently complicated by renal tubular dysfunction which may persist in some patients after hepatic transplantation. Nitisinone has revolutionized the management of TTI but its effect on renal tubular dysfunction has not been described in a large cohort of patients. To document the incidence and progression of renal tubular dysfunction in children with TTI treated with nitisinone at a single centre. Twenty-one patients with TTI from a single centre were treated with nitisinone for at least 12 months. Median age at first treatment was 17 weeks (range 1 week to 27 months). Nine patients (43%) presented in acute liver failure, seven (33%) had a chronic presentation and five (24%) were detected pre-clinically. A retrospective case analysis of plasma phosphate, urinary protein/creatinine ratio and tubular reabsorption of phosphate was performed for all patients as markers of tubular function. Renal ultrasounds were examined for evidence of nephrocalcinosis and where available, skeletal radiographs for rickets. All patients had biochemical evidence of renal tubular dysfunction at presentation. After nitisinone and dietary treatment were started, all three markers normalized within one year. Four children had clinical rickets at presentation (which improved), of whom one had nephrocalcinosis, which did not reverse on nitisinone. No child redeveloped tubular dysfunction after commencing nitisinone. All patients with TTI had evidence of tubular dysfunction at presentation and in all cases this resolved with nitisinone and dietary control. The tubulopathy associated with TTI is reversible.

Activation of nuclear factor E2-related factor 2 in hereditary tyrosinemia type 1 and its role in survival and tumor development

In tyrosinemia type 1 (HT1), accumulation of toxic metabolites results in oxidative stress and DNA damage, leading to a high incidence of hepatocellular carcinomas. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a key transcription factor important for cellular protection against oxidative stress and chemical induced liver damage. To specifically address the role of Nrf2 in HT1, fumarylacetoacetate hydrolase (Fah)/Nrf2(-/-) mice were generated. In acute HT1, loss of Nrf2 elicited a strong inflammatory response and dramatically increased the mortality of mice. Following low grade injury, Fah/Nrf2(-/-) mice develop a more severe hepatitis and liver fibrosis. The glutathione and cellular detoxification system was significantly impaired in Fah/Nrf2(-/-) mice, resulting in increased oxidative stress and DNA damage. Consequently, tumor development was significantly accelerated by loss of Nrf2. Potent pharmacological inducers of Nrf2 such as the triterpenoid analogs 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole have been developed as cancer chemoprevention agents. Pretreatment with 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole dramatically protected Fah(-/-) mice against fumarylacetoacetate (Faa)-induced toxicity. Our data establish a central role for Nrf2 in the protection against Faa-induced liver injury; the Nrf2 regulated cellular defense not only prevents acute Faa-induced liver failure but also delays hepatocarcinogenesis in HT1.

A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.

Current techniques for the alteration of gene expression in the liver have a number of limitations, including the lack of stable somatic gene transfer and the technical challenges of germline transgenesis. Rapid and stable genetic engineering of the liver would allow systematic, in vivo testing of contributions by many genes to disease. After fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in FAH-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable Fah transposon cassette. First, net conversion of Fah(-/-) liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, coexpressed short hairpin RNAs (shRNAs) stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant allele of human alpha1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in transformation of transduced hepatocytes. Coexpression with FAH is an effective technique for lifelong expression of transgenes in adult hepatocytes with applicability to a wide variety of genetic studies in the liver.

Structural Insight into Substrate Binding and Catalysis of a Novel 2-Keto-3- deoxy- d-arabinonate Dehydratase Illustrates Common Mechanistic Features of the FAH Superfamily

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3- deoxy- d-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 Å resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded β-sheet flanked by two α-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons α to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

The Caenorhabditis elegans K10C2.4 Gene Encodes a Member of the Fumarylacetoacetate Hydrolase Family: A CAENORHABDITIS ELEGANS MODEL OF TYPE I TYROSINEMIA

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.

Hepatic stress in hereditary tyrosinemia type 1 (HT1) activates the AKT survival pathway in the fah−/− knockout mice model

The AKT survival pathway is involved in a wide variety of human cancers. We investigated the implication of this pathway in hereditary tyrosinemia type 1 (HT1), a metabolic disease exhibiting hepatocellular carcinoma (HCC), despite treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexadione (NTBC) which prevents liver damage. HT1 is an autosomal recessive disorder caused by accumulation of toxic metabolites due to a deficiency in fumarylacetoacetate hydrolase (FAH), the last enzyme in the catabolism of tyrosine. NTBC withdrawal in the murine fah(-/-) knockout model was used to analyze in vivo the correlation between pathophysiological, biochemical and histological features consistent with hepatocarcinogenesis and activation of the AKT survival pathway. The HT1 stress initiated by NTBC discontinuation causes a progressive increase of liver and kidney pathophysiology. A stable activation of the AKT survival pathway is observed in the liver but not in kidneys of fah(-/-) mice. Hepatic survival is reinforced by inhibition of mitochondrial-mediated apoptosis through inactivation of Bad and induction of BCl-X(L) and BCl-2. The chronic stress induced by liver disease in HT1 activates the AKT survival signal and inhibits intrinsic apoptosis to confer cell death resistance in vivo and favor hepatocarcinogenesis.

Rescue from neonatal death in the murine model of hereditary tyrosinemia by glutathione monoethylester and vitamin C treatment

Hereditary tyrosinemia type 1 (HT1) is a recessive disease caused by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) that catalyzes the conversion of fumarylacetoacetate (FAA) into fumarate and acetoacetate. In mice models of HT1, FAH deficiency causes death within the first 24 h after birth. Administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC) prevents neonatal death in HT1 mice, ameliorates the HT1 phenotype but does not prevent development of hepatocellular carcinoma later on. FAA has been shown to deplete cells of glutathione by forming adducts. We tested whether a combination of a cell membrane permeable derivative of glutathione, glutathione monoethylester (GSH-MEE) and vitamin C could provide an alternative effective treatment for HT1. GSH-MEE (10 mmol/kg/j)/vitamin C (0.5 mmol/kg/j) treatment was given orally to pregnant/nursing female mice. While FAH −/− pups died in absence of treatment, all FAH −/− pups survived the critical first 24 h of life when the mothers were on the GSH-MEE/vitamin C treatment and showed normal growth until postnatal day 10 (P10). However, after P10, pups showed failure to thrive, lethargy and died around P17. Thus, GSH-MEE/vitamin C supplementation could rescue the mice model of HT1 from neonatal death but it did not prevent the appearance of a HT1 phenotype in the second week after birth.

Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice.

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

Messenger RNA as a Source of Transposase for Sleeping Beauty Transposon–mediated Correction of Hereditary Tyrosinemia Type I

The Sleeping Beauty (SB) transposon system mediates chromosomal integration and stable gene expression when an engineered SB transposon is delivered along with transposase. One concern in the therapeutic application of the SB system is that persistent expression of transposase could result in transposon instability and genotoxicity. Here, we tested the use of transposase-encoding RNA plus transposon DNA for correction of murine fumarylacetoacetate hydrolase (FAH) deficiency. A bi-functional transposon containing both mouse FAH and firefly luciferase sequences was used to track the growth of genetically corrected liver tissue by in vivo bioluminescence imaging after delivery of DNA or RNA as a source of transposase. Supplying SB transposase in the form of RNA resulted in selective repopulation of corrected hepatocytes with stable expression of FAH and luciferase. Plasma succinylacetone and amino acid levels were normalized, suggesting normal liver metabolism of catabolized protein products. Secondary FAH-deficient animals transplanted with hepatocytes (250,000) isolated from primary treated animals survived 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) withdrawal, gained weight consistently, and demonstrated stable expression of luciferase. We conclude that transposase-encoding messenger RNA (mRNA) can be used to mediate stable non-viral gene therapy, resulting in complete phenotypic correction, and is thus an effective source of recombinase activity for use in human gene therapy.

Animal Models of Tyrosinemia

Hereditary tyrosinemia I (HT I) is a genetic disorder of tyrosine metabolism characterized by progressive liver damage from infancy and by a high risk for hepatocellular carcinoma. HT I is due to mutations in the fumarylacetoacetate hydrolase (Fah) gene, which encodes the last enzyme in the tyrosine catabolic pathway. Disturbances in tyrosine metabolism lead to increased levels of succinylacetone and succinylacetoacetate. However, the mechanisms causing liver failure, cirrhosis, renal tubular dysfunction, and hepatocarcinoma are still unknown. Lethal albino deletion c14CoS mice and mice with target-disrupted Fah are models for HT I. They die in the perinatal period, although with a different phenotype from that seen in HT I in humans. In addition, 2 mouse strains that carry N-ethyl-N-nitrosourea-induced mutations in the Fah gene have been described. Mice with a splice mutation exhibit the milder features of the clinical phenotype. In mice that carry both Fah and 4-hydroxyphenylpyruvate dioxygenase gene mutations, administration of homogentisate results in rapid apoptosis of hepatocytes. Simultaneously, renal tubular epithelial cells are injured, resulting in Fanconi syndrome. These are central features of visceral injury in patients with HT I. Apoptosis of hepatocyte and renal tubular cells is prevented by the caspase inhibitors acetyl-Tyr-Val-Ala-Asp-CHO or acetyl-Asp-Glu-Val-Asp-CHO. Apoptosis of hepatocytes and renal tubular epithelial cells are central features of this disease. Alterations in gene expression found in the liver of patients with HT I are responsible for the pathogenesis of this disease, for example, acute liver failure. Therefore, gene expression analysis allows a better understanding of the specific pathogenesis. Cell fusion of hematopoietic stem cells with hepatocytes leads to liver regeneration after liver injury. This finding was possible after using the liver injury model of HT I in Fah null mice. Thus, animal models of tyrosinemia are unique and useful tools to reveal mechanisms of interest to both clinical and basic science.

“Silent” Tyrosinemia Presenting as Hepatocellular Carcinoma in a 10‐year‐old Girl

“Silent” Tyrosinemia Presenting as Hepatocellular Carcinoma in a 10‐year‐old Girl

Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284-15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 A; 1 A=0.1 nm) crystal structure of the FAH.CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 mumol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm

When differentiated in the presence of activin A in serum-free conditions, mouse embryonic stem cells efficiently generate an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45-70%) for cells that express the alpha-fetoprotein and albumin proteins. These cells also express transcripts of Afp, Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store glycogen, show ultrastructural characteristics of mature hepatocytes, and are able to integrate into and proliferate in injured livers in vivo and mature into hepatocytes expressing dipeptidyl peptidase IV or fumarylacetoacetate hydrolase. Together, these findings establish a developmental pathway in embryonic stem cell differentiation cultures that leads to efficient generation of cells with an immature hepatocytic phenotype.

The peptide specificities of the autoantibodies elicited by mouse hepatitis virus A59

Synthetic decapeptides ( N = 206) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (FAH) were used to analyze the specificities of the autoantibodies (autoAb) elicited towards this enzyme in mice infected with mouse hepatitis virus (MHV). These autoAb bound mainly to N- and C-terminal FAH peptides, the most reactive sequences being 1–50 and 390–420, respectively. Surprisingly, although FAH sequence 1–50 shares a high degree of homology with various MHV proteins, the C-terminal portion does not. Moreover, whereas the autoAb reacted with homologous peptides surrounding residues 70, 160 and 360, non-similar sequences around residues 130, 210, 240, 250, and 300 were also recognized, indicating that autoAb were not restricted to epitopes with sequence homologies. There was also a lack of correlation between the amount of anti-MHV or anti-FAH antibodies produced and the reactivity towards the peptides. Moreover, the spectrum of peptides recognized by the autoAb of a given mouse did not change significantly with time, which suggests that the MHV-elicited autoimmune response does not induce an epitope recognition spreading. Finally, anti-FAH Ab produced after immunization with rat liver FAH recognized essentially the same mouse FAH regions than autoAb from MHV-infected mice. Results indicated that the induction of the autoAb is not only related to molecular or structural mimicry, but rather supports the Danger model, in which any aggression, in this case the MHV infection, is susceptible to trigger the production of autoAb.

Gene expression profiles of homogentisate-treated Fah−/− Hpd−/−mice using DNA microarrays

Hereditary tyrosinemia I (HT I) is a genetic disorder of tyrosine metabolism caused by abnormalities of fumarylacetoacetate hydrolase. Disturbances in tyrosine metabolism lead to increased levels of succinylacetone and succinylacetoacetate. However, the mechanisms causing liver failure, cirrhosis, renal tubular dysfunction, and hepatocarcinoma are still unknown. Alterations in gene expression found in the livers of patients with HT I are responsible for the pathogenesis of this disease, for example acute liver failure. Therefore, gene expression analysis allows us to better understand its pathogenesis. We analyzed gene expressions in tyrosinemia type I model mice with liver failure using microarrays. The results were confirmed by quantitative PCR to evaluate the pathogenesis of tyrosinemia type I. We found that numerous genes, including amino acid metabolism and apoptosis related genes, were up- or down-regulated at the onset of liver failure. These findings are useful in understanding the pathogenesis of hereditary tyrosinemia.

Enzymes of tyrosine catabolism in Arabidopsis thaliana

Tyrosine catabolism is an essential pathway in animals, but its role in plants is unclear. The first steps of tyrosine degradation lead to the formation of homogentisate. In animals this is then sequentially acted on by homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI) and fumarylacetoacetate hydrolase (FAH) to generate fumarate and acetoacetate. In plants, homogentisate is used to generate the essential redox metabolites tocopherol and plastoquinone, which effectively act as an alternative metabolic fate for tyrosine. Having determined that a zeta class glutathione transferase from Arabidopsis thaliana is a functional MAAI, we have looked for evidence that the mammalian degradation pathway could also operate in plants. Based on array and quantitative PCR experiments, the A. thaliana homologues AtHGO, AtMAAI and AtFAH could be shown to be expressed, with AtHGO and AtMAAI showing evidence of co-regulation. cDNAs encoding AtHGO, AtMAAI and AtFAH were cloned in Escherichia coli and shown to represent a fully functional catabolic pathway when combined in vitro. The significance of this pathway, including increased transcription of the associated enzymes in senescing tissue, compartmentalisation and impact on flux into synthesis of Vitamin E and other tocopherols of biotechnological interest is discussed.

The genetic tyrosinemias

The genetic tyrosinemias are characterized by the accumulation of tyrosine in body fluids and tissues. The most severe form of tyrosinemia, Type I, is a devastating disorder of childhood that causes liver failure, painful neurologic crises, rickets, and hepatocarcinoma. This disorder is caused by a deficiency of fumarylacetoacetate hydrolase (FAH). If untreated, death typically occurs at less than 2 years of age, with some chronic forms allowing longer survival. It has a prevalence of about 1 in 100,000 newborns in the general population. Oculocutaneous tyrosinemia, Type II, is caused by a deficiency of tyrosine aminotransferase (TAT). It clinically presents with hyperkeratotic plaques on the hands and soles of the feet and photophobia due to deposition of tyrosine crystals within the cornea. Tyrosinemia Type III is an extremely rare disorder caused by a deficiency of 4-hydroxyphenylpyruvic dioxygenase. It has been associated with ataxia and mild mental retardation. These disorders are diagnosed by observing elevated tyrosine by plasma amino acid chromatography and characteristic tyrosine metabolites by urine organic acid analysis. In tyrosinemia Type I, methionine is also elevated, reflecting impaired hepatocellular function. Urine organic acids show elevated p-hydroxy-phenyl organic acids in each type of tyrosinemia, and the pathognomic succinylacetone in tyrosinemia Type I. Diagnosis can be confirmed by enzyme or molecular studies in tyrosinemia Type I. Therapy consists of a diet low in phenylalanine and tyrosine for each of the tyrosinemias and 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) for tyrosinemia Type I.

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5 g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5 g/L) and 94% (5 g/L) of untreated wild-type mice. Interestingly, FAH −/− mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10–20% of control. Thus the failure of DCA treatment in FAH −/− mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.

Kidneys of Mice With Hereditary Tyrosinemia Type I Are Extremely Sensitive to Cytotoxicity

Children with hereditary tyrosinemia type 1 (HT1) suffer from liver failure, renal tubular dysfunction, and rickets. The disease is caused by deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of tyrosine catabolism, and leads to accumulation of the toxic substrate fumarylacetoacetate (FAA) in hepatocytes and renal proximal tubular cells. Patients are treated with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC), which prevents accumulation of FAA by blocking an enzyme upstream of FAH. Liver transplantation is performed when patients do not respond to NTBC or develop hepatocellular carcinoma. This reduces the tyrosine load for the kidney but does not abolish renal exposure to locally produced FAA. To investigate the pathogenesis of liver and kidney damage induced by tyrosine metabolites, we challenged FAH-deficient mice with various doses of homogentisic acid (HGA), a precursor of FAA. Injecting NTBC-treated Fah-/- mice with low doses of HGA caused renal damage and death of renal tubular cells, as was shown by histologic analyses and deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay but did not lead to liver damage. In addition, kidney function, but not liver function, was affected after exposure to low doses of HGA. Administration of high doses of HGA led to massive cell death in both the liver and kidneys. Resistance to HGA-induced cell death was seen after withdrawing NTBC from Fah-/- mice. The finding that the kidneys of Fah-/- mice are especially sensitive to damage induced by low doses of HGA underscores the need to perform careful monitoring of the kidney function of tyrosinemia patients undergoing any form of treatment.

Involvement of Endoplasmic Reticulum Stress in Hereditary Tyrosinemia Type I

Hereditary tyrosinemia type I (HTI) is the most severe disease of the tyrosine degradation pathway. HTI is caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the enzyme responsible for the hydrolysis of fumarylacetoacetate (FAA). As a result, there is an accumulation of metabolites such as maleylacetoacetate, succinylacetone, and FAA. The latter was shown to display mutagenic, cytostatic, and apoptogenic activities and to cause chromosomal instability. Herein, we demonstrate that FAA also causes a cellular insult leading to the endoplasmic reticulum (ER) stress signaling. Treatment of V79 Chinese hamster lung cells with an apoptogenic dose of FAA (100 mum) causes an early induction of the ER resident chaperone GRP78/BiP and a simultaneous phosphorylation of the eIF2alpha. FAA treatment also causes a subsequent induction of the proapoptotic CHOP (CEBP homologous protein) transcription factor as well as a late activation of caspase-12. Data obtained from fah(-/-) mice taken off the therapeutic 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione drug are similar. However, in this mouse model, there is also an increase in proteasome activity indicative of ER-associated degradation. This difference observed between the two models may be due to the fact that the murine model measures the effects of all metabolites accumulating in hereditary tyrosinemia type I as opposed to the cellular model that only measures the effects of exogenous FAA.