CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I.

Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

Protein Degradation and the Pathologic Basis of Phenylketonuria and Hereditary Tyrosinemia.

A delicate intracellular balance among protein synthesis, folding, and degradation is essential to maintaining protein homeostasis or proteostasis, and it is challenged by genetic and environmental factors. Molecular chaperones and the ubiquitin proteasome system (UPS) play a vital role in proteostasis for normal cellular function. As part of protein quality control, molecular chaperones recognize misfolded proteins and assist in their refolding. Proteins that are beyond repair or refolding undergo degradation, which is largely mediated by the UPS. The importance of protein quality control is becoming ever clearer, but it can also be a disease-causing mechanism. Diseases such as phenylketonuria (PKU) and hereditary tyrosinemia-I (HT1) are caused due to mutations in PAH and FAH gene, resulting in reduced protein stability, misfolding, accelerated degradation, and deficiency in functional proteins. Misfolded or partially unfolded proteins do not necessarily lose their functional activity completely. Thus, partially functional proteins can be rescued from degradation by molecular chaperones and deubiquitinating enzymes (DUBs). Deubiquitination is an important mechanism of the UPS that can reverse the degradation of a substrate protein by covalently removing its attached ubiquitin molecule. In this review, we discuss the importance of molecular chaperones and DUBs in reducing the severity of PKU and HT1 by stabilizing and rescuing mutant proteins.

The Dynamic Chromatin Architecture of the Regenerating Liver.

BACKGROUND & AIMS: The adult liver is the main detoxification organ and routinely is exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, extensive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood. METHODS: We isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in human beings. We then performed the assay for transposase accessible chromatin with high-throughput sequencing specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. In addition, we used motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing for hepatocyte nuclear factor 4α (HNF4α) and CCCTC-binding factor (CTCF). RESULTS: Chromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with the regulation of gene expression, with enhancers of many liver function genes showing a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4α binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation. CONCLUSIONS: Our analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4α as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4α) to temporarily limit enhancer activity. All sequencing data in this study were deposited to the Gene Expression Omnibus database and can be downloaded with accession number GSE109466.

Mutational spectrum of Mexican patients with tyrosinemia type 1: In silico modeling and predicted pathogenic effect of a novel missense FAH variant.

BACKGROUND: Tyrosinemia type 1 (HT1, MIM#276700) is caused by a deficiency in fumarylacetoacetate hydrolase (FAH) and it is associated with severe liver and renal disfunction. At present, the mutational FAH (15q25.1, MIM*613871) spectrum underlying HT1 in the Mexican population is unknown. The objective of this study was to determine the FAH genotypes in eight nonrelated Mexican patients with HT1, who were diagnosed clinically. METHODS: Sequencing of FAH and their exon-intron boundaries and in silico protein modeling based on the crystallographic structure of mouse FAH. RESULTS: We identified pathogenic variants in 15/16 studied alleles (93.8%). Nine different variants were found. The most commonly detected HT1-causing allele was NM_000137.2(FAH):c.3G > A or p.(?) [rs766882348] (25%, n = 4/16). We also identified a novel missense variant NM_000137.2(FAH):c.36C > A or p.(Phe12Leu) in a homozygous patient with an early and fatal acute form. The latter was classified as a likely pathogenic variant and in silico protein modeling showed that Phe-12 residue substitution for Leu, produces a repulsion in all possible Leu rotamers, which in turn would lead to a destabilization of the protein structure and possible loss-of-function. CONCLUSION: HT1 patients had a heterogeneous mutational and clinical spectrum and no genotype-phenotype correlation could be established.

HPD degradation regulated by the TTC36-STK33-PELI1 signaling axis induces tyrosinemia and neurological damage.

Decreased expression of 4-hydroxyphenylpyruvic acid dioxygenase (HPD), a key enzyme for tyrosine metabolism, is a cause of human tyrosinemia. However, the regulation of HPD expression remains largely unknown. Here, we demonstrate that molecular chaperone TTC36, which is highly expressed in liver, is associated with HPD and reduces the binding of protein kinase STK33 to HPD, thereby inhibiting STK33-mediated HPD T382 phosphorylation. The reduction of HPD T382 phosphorylation results in impaired recruitment of FHA domain-containing PELI1 and PELI1-mediated HPD polyubiquitylation and degradation. Conversely, deficiency or depletion of TTC36 results in enhanced STK33-mediated HPD T382 phosphorylation and binding of PELI1 to HPD and subsequent PELI1-mediated HPD downregulation. Ttc36-/- mice have reduced HPD expression in the liver and exhibit tyrosinemia, damage to hippocampal neurons, and deficits of learning and memory. These findings reveal a previously unknown regulation of HPD expression and highlight the physiological significance of TTC36-STK33-PELI1-regulated HPD expression in tyrosinemia and tyrosinemia-associated neurological disorders.

In utero CRISPR-mediated therapeutic editing of metabolic genes.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models.

BACKGROUND: Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes. METHODS: Pig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography-computed tomography. Fumarylacetoacetate hydrolase-deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals' ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione. RESULTS: Positron emission tomography-computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell-transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase-positive hepatocytes in the liver, correlating with similar weight stabilization curves. CONCLUSION: Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase-deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.

Markers of cognitive function in individuals with metabolic disease: Morquio syndrome and tyrosinemia type III.

We characterized cognitive function in two metabolic diseases. MPS-IVa (mucopolysaccharidosis IVa, Morquio) and tyrosinemia type III individuals were assessed using tasks of attention, language and oculomotor function. MPS-IVa individuals were slower in visual search, but the display size effects were normal, and slowing was not due to long reaction times (ruling out slow item processing or distraction). Maintaining gaze in an oculomotor task was difficult. Results implicated sustained attention and task initiation or response processing. Shifting attention, accumulating evidence and selecting targets were unaffected. Visual search was also slowed in tyrosinemia type III, and patterns in visual search and fixation tasks pointed to sustained attention impairments, although there were differences from MPS-IVa. Language was impaired in tyrosinemia type III but not MPS-IVa. Metabolic diseases produced selective cognitive effects. Our results, incorporating new methods for developmental data and model selection, illustrate how cognitive data can contribute to understanding function in biochemical brain systems.

In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence.

Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah-/- cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah-/- mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury.

Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah-/- mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type-specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.

Abnormal social behavior in mice with tyrosinemia type I is associated with an increase of myelin in the cerebral cortex.

Hereditary tyrosinemia type I (HT1) is caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene, the template for the final enzyme in the tyrosine catabolism pathway. If left untreated this deficiency of functional FAH leads to a buildup of toxic metabolites that can cause liver disease, kidney dysfunction and high mortality. The current treatment with the drug NTBC prevents the production of these metabolites and has consequently increased the survival rate in HT1 children. As a result of this increased survival, long term complications of HT1 are now being observed, including slower learning, impaired cognition and altered social behavior. We studied a mouse model of HT1 to gain insight into the effects of HT1 and treatment with NTBC on social behavior in mice. We showed that mice with HT1 display abnormal social behavior in that they spend more time in the absence of another mouse and do not discriminate between a novel mouse and an already familiar mouse. This altered behavior was due to HT1 and not treatment with NTBC. Quantification of cerebral cortex myelin in mice with HT1 showed a two to threefold increase in myelin expression. Our findings suggest that absence of FAH expression in the brain produces an altered brain biochemistry resulting in increased expression of myelin. This increase in myelination could lead to abnormal action potential velocity and altered neuronal connections that provide a mechanism for the altered learning, social behavior and cognitive issues recently seen in HT1.

Biochemical and Clinical Aspects of Hereditary Tyrosinemia Type 1.

Inborn errors of metabolism (IEMs) are a group of diseases involving a genetic defect that alters a metabolic pathway and that presents usually during infancy. The tyrosine degradation pathway contains five enzymes, four of which being associated with IEMs. The most severe metabolic disorder associated with this catabolic pathway is hereditary tyrosinemia type 1 (HT1; OMIM 276700). HT1 is an autosomal recessive disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. Although a rare disease worldwide, HT1 shows higher incidence in certain populations due to founder effects. The acute form of the disease is characterized by an early onset and severe liver failure while the chronic form appears later and also involves renal dysfunctions. Until 1992 the only treatment for this disease was liver transplantation. Since then, NTBC/Nitisone (a drug blocking the pathway upstream of FAH) is successfully used in combination with a diet low in tyrosine and phenylalanine, but patients are still at risk of developing hepatocellular carcinoma. This chapter summarizes the biochemical and clinical features of HT1.

The Liver in Tyrosinemia Type I: Clinical Management and Course in Quebec.

HT1 is a severe autosomal recessive disorder due to the deficiency of fumarylacetoacetate hydrolase (FAH), the final enzyme in the degradation of tyrosine. Before the era of treatment with 2-(2-N-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), even with newborn screening and optimal diet therapy, HT1 patients often developed liver failure. Death was common in patients who did not undergo liver transplantation. For the last two decades, NTBC has revolutionized the management of HT1 patients. In screened newborns treated within the first month of life, we have not observed hepatocarcinoma. If patients are not detected at birth by neonatal screening, the diagnosis and treatment must be performed on an emergency basis, and patients are at risk for complications. Long term adhesion to treatment and reliable early detection of hepatocellular carcinoma (HCC) are two important challenges. In this chapter, we describe the clinical, biological, histo-pathological and imaging findings of HT1 in Québec before the era of NTBC. We also describe the hepatic status of nontransplanted tyrosinemic patients in Quebec and current management practices in the Quebec NTBC Study.

Liver Cancer in Tyrosinemia Type 1.

Hereditary Tyrosinemia type I (HT1) is clinically mainly characterised by severe liver disease. Most patients present in their first months of life with liver failure, but others can present later with issues of compensated cirrhosis, renal tubulopathy or acute intermittent porphyria. If patients survive the acute phase with liver failure or if they present later with compensated cirrhosis, they often develop hepatocellular carcinoma early but also later in life. The course of the disease changed after the introduction of 2-(2 nitro-4-3 trifluoro-methylbenzoyl)-1, 3-cyclohexanedione (NTBC), which blocks the tyrosine degradation pathway at an earlier step. Therefore, the toxic products did not accumulate anymore and all clinical problems resolved. However, the risk (although clearly decreased) for developing liver cancer remained, especially if NTBC treatment is initiated late, a slow decrease of the tumor marker α-fetoprotein is seen or if the α-fetoprotein concentrations remain just above the normal range. A rise of α-fetoprotein in these HT1 patients is more or less pathognomonic for liver cancer. Although hepatoblastoma development occurs in HT1 patients, most HT1 patients develop hepatocellular carcinoma (HCC) or a mixed type of carcinoma consisting of HCC and hepatoblastoma. Due to the small risk of liver cancer development, screening for liver cancer (especially HCC) is still recommended in HT1 patients using regular measures of α-fetoprotein and imaging. Ultrasound is mostly the modality of choice for surveillance, because it is widely available, it does not use radiation and is noninvasive. When a suspicious lesion is present, the higher sensitivity of MRI could be used for characterization and staging of lesions. At this moment, no HCC development in pre-symptomatically treated patients is reported. These different situations could possibly indicate that NTBC can prevent the start of the development of HCC when initiated early, but can't stop the development of HCC if it is prescribed at a later stage, stressing the importance of early diagnosis.

Neurological and Neuropsychological Problems in Tyrosinemia Type I Patients.

Clinically, Hereditary Tyrosinemia type I (HTI) is especially characterized by severe liver dysfunction in early life. However, recurrent neurological crises are another main finding in these patients when they are treated with a tyrosine and phenylalanine restricted diet only. This is caused by the accumulation of δ-aminolevulinic acid due to the inhibitory effect of succinylacetone on the enzyme that metabolizes δ-aminolevulinic acid. Due to the biochemical and clinical resemblance of these neurological crises and acute intermittent porphyria, this group of symptoms in HTI patients is mostly called porphyria-like-syndrome. The neurological crises in HTI patients disappeared after the introduction of treatment with 2-(2 nitro-4-3 trifluoro-methylbenzoyl)-1, 3-cyclohexanedione (NTBC). However, if NTBC treatment is stopped for a while, severe neurological dysfunction will reappear.If NTBC treatment is started early and given continuously, all clinical problems seem to be solved. However, recent research findings indicate that HTI patients have a non-optimal neurocognitive outcome, showing (among others) a lower IQ and impaired executive functioning and social cognition. Unfortunately the exact neuropsychological profile of these HTI patients is not known yet, neither are the exact pathophysiological mechanisms underlying these impairments. It may be hypothesized that the biochemical changes such as high blood tyrosine or low blood phenylalanine concentrations are important in this respect, but an direct toxic effect of NTBC or production of toxic metabolites (that previously characterized the disease before introduction of NTBC) cannot be excluded either. This chapter discusses the neurological and neuropsychological symptoms associated with HTI in detail. An extended section on possible underlying pathophysiological mechanisms of such symptoms is also included.

Diagnosing Hepatorenal Tyrosinaemia in Europe: Newborn Mass Screening Versus Selective Screening.

Hepatorenal tyrosinaemia (HT1) is a serious condition that used to be fatal before the advent of nitisinone (NTBC, Orfadine®) as a therapeutic option. We have recently shown that selective screening is inadequate as initial symptoms are often uncharacteristic which leads to a considerable delay in diagnosis and treatment. This has a negative impact on morbidity and mortality as well as long-term outcome. For example, the odds ratio to develop hepatocellular carcinoma is 12.7 when treatment is initiated after the first birthday compared to start of treatment in the neonatal period. Timely diagnosis is only possible when neonatal mass screening is operational. HT1 meets all the criteria for neonatal mass screening at a clinical and analytical level. The natural course of the disease is well known, clinically there is a latent phase in most patients when presymptomatic treatment can be initiated. There are no mild phenotypes which do not require treatment. Using succinylacetone as the screening parameter a highly specific and sensitive test is available with acceptable financial burden. Neonatal mass screening for HT1 is acceptable to the target population as it can be performed simultaneously with the already existing screening tests in dried blood, there are no false negative and false positive cases and the financial burden to the health system is moderate. An efficient treatment is available with nitisinone and protein-reduced diet supplemented with special amino acid mixtures. Despite compelling evidence in favour of a neonatal mass screening for HT1 only 57% of European centres taking part in our recent cross-sectional study have included HT1 in their newborn screening programme.

Tyrosinemia Type I in Japan: A Report of Five Cases.

Tyrosinemia type I in Japan was reported for the first time in 1957 by Sakai et al. (Jikei Med J 2:1-10, 1957) and Kitagawa et al. (Proc Jpn Acad Ser B 88:192-200, 1957). Five cases of patients with tyrosinemia type I were reported to be definitively diagnosed in Japan. The first case was reported by Sakai et al. and Kitagawa et al. To the best of our knowledge, this was the first definite report in the world. The second and third cases were those of a brother and a sister who underwent liver transplantation and who were the children of a Japanese-descent migrant worker; the fourth case was that of a girl who underwent liver transplantation after 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) treatment, which was reported by Hata et al.; and the fifth case was that of a patient who was administered NTBC, which was reported by Ito et al. These were of the subacute type, wherein residual activity was considerably present. When combined therapy with a low phenylalanine and tyrosine diet and NTBC administration is started after early diagnosis, patients can survive without liver transplantation. Development of liver cancer is not found in the cases in Japan, but performing liver transplantation without delay is necessary when liver cancer is found.

Role of antioxidant treatment on DNA and lipid damage in the brain of rats subjected to a chemically induced chronic model of tyrosinemia type II.

Tyrosine levels are abnormally elevated in tissues and body fluids of patients with inborn errors of tyrosine metabolism. Tyrosinemia type II, which is caused by tyrosine aminotransferase deficiency, provokes eyes, skin, and central nervous system disturbances in affected patients. However, the mechanisms of brain damage are still poorly known. Considering that studies have demonstrated that oxidative stress may contribute, along with other mechanisms, to the neurological dysfunction characteristic of hypertyrosinemia, in the present study we investigated the effects of antioxidant treatment (NAC and DFX) on DNA damage and oxidative stress markers induced by chronic administration of L-tyrosine in cerebral cortex, hippocampus, and striatum of rats. The results showed elevated levels of DNA migration, and thus DNA damage, after chronic administration of L-tyrosine in all the analyzed brain areas, and that the antioxidant treatment was able to prevent DNA damage in cerebral cortex and hippocampus. However, the co-administration of NAC plus DFX did not prevent the DNA damage in the striatum. Moreover, we found a significant increase in thiobarbituric acid-reactive substances (TBA-RS) and DCFH oxidation in cerebral cortex, as well as an increase in nitrate/nitrite levels in the hippocampus and striatum. Additionally, the antioxidant treatment was able to prevent the increase in TBA-RS levels and in nitrate/nitrite levels, but not the DCFH oxidation. In conclusion, our findings suggest that reactive oxygen and nitrogen species and oxidative stress can play a role in DNA damage in this disorder. Moreover, NAC/DFX supplementation to tyrosinemia type II patients may represent a new therapeutic approach and a possible adjuvant to the current treatment of this disease.