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Mitochondria and Childhood Liver Diseases

1999; Lippincott Williams & Wilkins; Volume: 28; Issue: 1 Linguagem: Inglês

10.1097/00005176-199901000-00005

1536-4801

Ronald J. Sokol, William R. Treem,

Infectious Encephalopathies and Encephalitis

In recent years, several reports have appeared that describe infants and older children with liver failure caused by defects in mitochondrial function. Because of the growing frequency of these reports, assessment of mitochondrial function has become important in evaluating children with liver disease of undetermined origin. These mitochondrial hepatopathies are now viewed as a subset of disorders of mitochondria that may affect many organ systems (1,2), including the nervous system (1), the cardiac and skeletal muscle (1), the bone marrow (3,4), the endocrine and exocrine pancreas (3-5), the kidney, the inner ear, and the colon (6). These disorders are caused by inherited and acquired defects in specific mitochondrial functions, such as the respiratory chain and fatty acid oxidation, and may be inherited through the mitochondrial and nuclear genomes. In addition, investigators have recently suggested that the accumulation in specific tissues over time of new somatic (noninherited) mutations of mitochondrial genes, caused at least in part by the effect of reactive oxygen species generated by the nearby respiratory chain, may play an important role in the pathogenesis of several neurodegenerative diseases (7) and the process of aging itself (8). In this review, we will summarize recent advances in our understanding of mitochondrial biology and genetics and propose a classification for the primary and secondary mitochondrial hepatopathies, emphasizing those that involve impaired respiration and that appear during infancy and childhood. MITOCHONDRIAL BIOLOGY AND GENETICS The structure of the mitochondrion includes a dual-membrane framework containing a soluble matrix and genome. The outer membrane holds the highly folded inner membrane in place; regulates efflux of mitochondrial enzymes, cations, and substrates into cytosol; and is the specific transport site for various substrates that must be taken up from cytosol into the mitochondrion. The inner mitochondrial membrane contains the electron transport chain that accepts electrons generated from the citric acid cycle and fatty acid oxidation; numerous specialized transporters for small molecules; and the enzyme, adenosine triphosphate (ATP) synthase, that carries out oxidative phosphorylation (OXPHOS) and ATP synthesis (Fig. 1). The mitochondrial matrix contains the enzymes of the tricarboxylic acid cycle, fatty acid β-oxidation, urea synthesis, and other metabolic pathways. Specific enzyme defects have been described for many of these matricial enzymes, causing classic, although rare, disorders of fatty acid oxidation, urea synthesis, gluconeogenesis, and others. These disorders are caused almost entirely by mutations of nuclear DNA. Mitochondria may also play a pivotal role in the induction of cellular necrosis and apoptosis through the generation of oxidant stress, opening of the permeability transition pore and release of cytochrome c and apoptosis-inducing factor, and the activation of intracellular caspases and proteases. These cellular processes are not only important during cell injury but also play a critical role during development, tissue remodeling, and carcinogenesis.FIG. 1: Electron transport chain present in mitochondria. Electrons are donated from various respiratory substrates to the respiratory chain and transferred down the electrochemical gradient of the complexes, thus generating the proton-motive force that drives adenosine triphosphate synthesis by complex V (not shown). Superoxide is also generated during mitochondrial respiration at complex I and at the interaction of coenzyme Q (CoQ) with complex III. ETF, electron transferring flavoprotein; Cyt c, cytochrome c.A fascinating feature of mitochondria in mammalian cells is the presence of a separate genome, distinct from that of the nucleus, and the gamut of enzymes needed to replicate and express nucleic acids (1). In distinction from that of nuclear DNA, virtually all mitochondrial DNA (mtDNA) in a cell is derived from the unfertilized ovum, and therefore all characteristics encoded by the mtDNA are maternally inherited. Sperm contain almost no mitochondria and participate little, if at all, in mitochondrial genetics. The human mtDNA is a 16,569-bp, double-stranded, circular molecule that codes for 37 genes, including the 2 ribosomal RNAs and 22 transfer RNAs of mitochondrial protein synthesis and 13 polypeptides of complex I, III, and IV of the respiratory chain. Genetic information necessary for the replication and transcription of mtDNA is also contained in its genome, and there are few noncoding sequences (introns). Because many other subunits of the respiratory chain proteins are encoded by nuclear DNA and transported into the mitochondria after assembly elsewhere in the cell, mutations in nuclear DNA or mtDNA can result in abnormalities in OXPHOS. Each hepatocyte may contain thousands of copies of mtDNA, because each mitochondrion contains 2 to 10 copies of mtDNA, and cells can contain hundreds or thousands of mitochondria. Thus, normal and mutant mtDNA are present in various proportions in each single cell. This condition of a mixture of differing mitochondrial genomes within the same cell is known as heteroplasmy. When a cell contains uniformly normal mtDNA, homoplasmy is said to be present. The relative proportions of normal and mutated mitochondrial genomes determines the phenotype of the cell. The threshold of mutated genome needed to produce a deleterious phenotype varies among persons, among organ systems, and within individual tissues. Cell damage results from an inadequate supply of energy in metabolically active cells and tissues; increased generation of injurious reactive oxygen species as a consequence of perturbed flow of electrons down the respiratory chain; release of mitochondrial calcium, cytochrome c, or apoptosis-inducing factor into cytosol; the opening of the mitochondrial membrane permeability pore; or other undefined mechanisms. Because the mtDNA has no protective histones and an adequate excision and recombination repair system, the mitochondrial genome is considerably more prone to oxidative injury and a higher somatic mutation rate than is nuclear DNA. This process of gradual accumulation of mtDNA mutations as a person ages may participate in the loss of mitochondrial respiratory function that appears to accompany aging and a number of degenerative neurologic disorders. Further favoring the development of human diseases caused by mtDNA mutations is the absence of redundancy and the high information density in mtDNA. Thus, loss of function of a critical protein by a mutation in a mitochondrial gene leaves little potential for replacement of this function by another "back-up" protein. NEUROMUSCULAR AND INTESTINAL MITOCHONDRIAL DISORDERS Most of the diseases initially associated with maternal inheritance and later with mitochondrial gene mutations involve the nervous system, skeletal muscle, and cardiac muscle (Table 1). Molecular genetic studies have shown mutations that cause many of these disorders, including large deletions or missense mutations of mtDNA involving transfer RNA genes or subunits of the electron transport chain complexes. Other disorders are attributed to mutations of nuclear genes encoding subunits of these complexes. These disorders include mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF) syndrome, Kearns-Sayre syndrome, Leigh disease, and others (1). The onset of mitochondrial encephalomyopathy may be in childhood and must be kept in mind by the pediatrician.TABLE 1: Mitochondrial encephalomyopathiesThe identification of disorders that can be caused by defects of specific mitochondrial enzymes, abnormal OXPHOS, or other mtDNA mutations has expanded at a rapid pace and now includes several gastrointestinal diseases. Pearson's marrow-pancreas syndrome is a progressive, usually fatal childhood disease with refractory sideroblastic anemia, exocrine pancreatic insufficiency, and hepatic dysfunction, that has been associated with a 4977-bp deletion in the mitochondrial genome (4). This deletion encompasses multiple subunits for respiratory complexes I and III (8). It is unclear how this deletion affects the target organs of this disorder, inasmuch as a similar deletion causes the neurologic manifestations of Kearns-Sayre syndrome in other families or even in members of the same family (1). An additional disorder has recently been described. Cormier-Daire et al. (8) have reported respiratory chain enzyme deficiencies and heteroplasmic rearrangements of mtDNA in two unrelated children whose initial symptoms included chronic diarrhea and intestinal villous atrophy. Eventually, hepatic failure, renal tubulopathy, central nervous system and cranial nerve findings, and persistent lactic acidemia prompted an investigation of mitochondrial function. In yet another mitochondrial disorder, recently labeled mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), virtually all patients have intestinal dysmotility, chronic diarrhea, and symptoms of intestinal pseudoobstruction (5). These cases have further extended the target organs in OXPHOS disorders to include the intestine, with visceral smooth muscle and possibly epithelial cell involvement. PRIMARY MITOCHONDRIAL HEPATOPATHIES A growing list of disorders that cause acute or chronic liver disease are now attributed to abnormalities of mitochondrial structure or function. These can be divided into those in which the mitochondrial defect is the primary cause of the liver disorder or those in which secondary or acquired mitochondrial injury or dysfunction is an important component of the pathogenesis of the underlying disease. Categories have been established for these disorders (Table 2).TABLE 2: Classification of mitochondrial hepatopathiesIt is now recognized that the liver is a major target organ in inherited defects of mitochondrial OXPHOS. These disorders of electron transport are an important subgroup and perhaps are the most severe of the primary mitochondrial hepatopathies (Table 2). Reduced activity of respiratory chain complexes and OXPHOS has been associated with liver disease of varying severity and at different ages; however, neonatal and early childhood occurrences predominate. Neonatal liver failure has been reported in association with deficiency of complex IV (cytochrome c oxidase) in several infants (10-12) and of complexes I and III in several others (10,11). Biochemical evidence of liver synthetic failure (hypoglycemia, hypoalbuminemia, hyperbilirubinemia, hyperammonemia, and coagulopathy) accompanies lethargy, hypotonia, vomiting, and poor breast-feeding capability and may be present from birth (13). Fetal hydrops and congenital ascites have been described and may indicate severe prenatal involvement. The key feature to be noted is the presence of significant lactic acidemia and an elevated molar ratio of plasma lactate to pyruvate (normally <20:1). If examined, β-hydroxybutyrate levels are generally elevated with an increased ketone body molar ratio of β-hydroxybutyrate to acetoacetate (normally, <2:1). Liver biopsy specimens typically show microvesicular and macrovesicular steatosis, with increased mitochondrial density and occasional swelling on electron microscopy. Hepatocellular and canalicular cholestasis, bile ductular proliferation, and periportal fibrosis and cirrhosis are other features present in varying proportions depending on the stage of the disease. These biochemical and histologic features should be red flags signaling the possibility of a respiratory chain defect. Despite standard metabolic support, hepatic coma and other complications of liver failure in these very ill neonates eventually cause death. Generally, there is obvious and severe neuromuscular involvement in infancy (seizures, hypotonia) precluding any consideration for liver transplantation. However, because the heteroplasmy for mtDNA mutations is not uniform in all tissues, in the absence of detectable extrahepatic manifestations, affected infants have undergone liver transplantation with mixed results. We have performed a liver transplant in one such infant with no apparent development of neuromuscular involvement during follow-up of more than 3 years. Others have performed transplants in infants with no clinically apparent neuromuscular involvement, only to have neurologic deterioration appear after liver transplantation. The long-term outcome in these infants will not be known for some time, because neuromuscular symptoms may not develop until adulthood in many of the mitochondrial disorders. More recently, a European group has suggested that the spectrum of these respiratory chain disorders is much broader, with many mildly affected patients having a more chronic course, some with delayed onset and some with occurrence in infancy. These findings need confirmation at other centers. The infants with complex I, III, or IV deficiencies have generally had normal mitochondrial genome when it was examined, implying that yet to be defined nuclear genomic mutations are involved in the cause. A similar disorder in older infants has been recognized for some time by the neurology community, but only more recently has liver involvement been appreciated. Infants with Alpers' disease (progressive neuronal degeneration in childhood with liver disease; familial progressive poliodystrophy with cirrhosis of the liver) have symptoms of vomiting, hypotonia, seizures, and liver failure between 6 months and 8 years of age (11,14,15). In Alpers' disease, the infant is initially normal with onset of development delay generally between 6 and 24 months of age, although sometimes development remains normal for a longer period. Failure to thrive, hypotonia, and unexplained vomiting (labeled severe gastroesophageal reflux in some patients) accompany the delayed development. A severe seizure disorder, frequently myotonic in nature, necessitates the use of multiple anticonvulsants because of its intractable nature. Valproic acid is commonly prescribed and then may be implicated in the liver dysfunction that follows. To monitor the use of anticonvulsants, liver blood tests may be obtained for the first time, showing mild elevations of aspartate aminotransferase and alanine aminotransferase. However, evidence of hepatic synthetic failure (low serum albumin, elevated prothrombin time, and ammonia and depressed clotting factor V or VII levels) may also be present, although liver disease is clinically unsuspected. Neurologic deterioration ensues with death generally by age 4 or 5 years if not sooner. Hepatic involvement is usually evident only late in the course, usually when neurologic function is poor. Once identified, the hepatic dysfunction portended death within 6 weeks in our five patients with this disorder (14). A liver tissue specimen shows microvesicular steatosis, mild inflammation, and focal hepatocyte necrosis (14). There is rapid loss of viable hepatocytes without extreme elevations of aminotransferase concentrations as the disease progresses and as liver failure ensues. As in other metabolic liver diseases, this suggests that apoptosis, not cellular necrosis, is the predominant pathway of hepatocyte demise and loss of functional hepatic mass. The typical findings at autopsy are micronodular cirrhosis with marked loss of the hepatocyte mass, hepatic steatosis, and bile ductular proliferation. Electron microscopy may show a variety of mitochondrial changes (including swelling) and increased mitochondrial density, or findings may be normal. Decreased complex I activity has been reported in several of these patients from examination of liver biopsy material (11,15). Although the reported cases involve patients with severe, usually fatal, disease, it is possible that milder cases may exist with less severe neurologic disease and more gradually progressive liver disease. No successful therapy has been described for Alpers' disease. Another fascinating mitochondrial hepatopathy is the mitochondrial DNA depletion syndrome. This disorder appears to be caused by mutation of a nuclear gene (probably controlling mtDNA replication) that results in a generalized reduction of otherwise normal mtDNA molecules in the affected tissues or organs (16-18). The consequence of this defect is a general reduction in the activity of the respiratory chain complexes encoded for by the mitochondrial genome-that is, complexes I, III, and IV. Prior reports have focused on the myopathic presentation of this disorder in infancy or later in childhood, although liver involvement in infancy was fatal in one case (16). Recently, three new cases have been described by Bakker et al. (17), all involving patients with liver failure soon after birth and death in the first 3 months of life. Lactic acidemia and severe hypoglycemia were present in two of the three reported siblings and, similar to children with other metabolic liver diseases, serum aminotransferase levels were only modestly elevated despite hepatic failure. Maaswinkel-Mooij et al. (18) also reported a similar infant with mtDNA depletion who had vomiting, hypotonia, lactic acidemia, and liver dysfunction and died by 7 months of age. Curiously, this patient had hypoketotic hypoglycemia. Neurologic abnormalities developed in these children before death; however, the predominant clinical symptoms were hypoglycemia, acidosis, and liver failure. The full spectrum of this disorder is unknown. Two other pediatric disorders in which depressed respiratory chain enzyme activity leads to liver dysfunction have already been mentioned. Pearson's marrow-pancreas syndrome was originally described in 1979 in four children with severe macrocytic anemia, variable neutropenia and thrombocytopenia, vacuolization of erythroid and myeloid precursors, and ringed sideroblasts in the bone marrow (3). Diarrhea and malabsorption developed later in early childhood. Each child was found to have pancreatic insufficiency associated with extensive pancreatic fibrosis and acinar atrophy. Partial intestinal villous atrophy was noted in several patients. Marked enlargement of the liver, hepatic steatosis, and cirrhosis have been associated with liver failure and death in some patients before the age of 4 years. Recent investigations have shown that mtDNA rearrangements are consistent features in Pearson's syndrome; large (4000-5000 bp) deletions predominate in 75% of reported cases (19-21). Of the respiratory chain enzymes encoded by mtDNA, complex I is the most severely affected by this deletion; however, the deletion also encompasses genes that encode two subunits of complex V, one subunit of complex IV, and five transfer RNA genes. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) is abnormal in lymphocytes from these patients; however, oxygen consumption and respiratory chain enzyme activities are normal in muscle mitochondria. Southern blot analysis reveals a mixed population of normal and deleted mitochondrial genomes in all tissues tested (heteroplasmy). Different proportions of deleted mtDNA molecules are noted in different tissues. In more severely affected tissues, such as bone marrow, polymorphonuclear neutrophils, lymphocytes, pancreas, and gut, mtDNA deletions are found in 80% and 90% of cells, but in only 50% of muscle cells. Thus, it appears that the phenotypic expression of Pearson's syndrome in a given tissue requires a minimum threshold number of mutated mtDNA molecules. As opposed to other mtDNA disorders, the absence of maternal inheritance or positive family histories and the absence of mtDNA rearrangements in the lymphocytes of parents or siblings of detected cases suggest that de novo mutations occurred during oogenesis or early development of fertilized eggs. Other clinical manifestations of Pearson's syndrome include renal tubular disease (Fanconi's syndrome), patchy erythematous skin lesions and photosensitivity, diabetes mellitus, hydrops fetalis, and the late development of a pigmentary retinopathy, visual impairment, tremor, ataxia, proximal muscle weakness, and external ophthalmoplegia. It is of interest that these symptoms are also found in Kearns-Sayre syndrome, a mitochondrial disease also characterized by a large (5-kb) mtDNA deletion (22). The occurrence of Kearns-Sayre syndrome in patients with Pearson's syndrome who survive into childhood is another example of the dependence of phenotypic expression on random partitioning of mutated mtDNA during cell division and possible alterations in the proportion of rearranged mtDNA in various tissues over time (23). Supporting this hypothesis is the clinical observation that the anemia of Pearson's syndrome may improve as patients age, eliminating the need for red blood cell transfusions after the age of 2 years. Thus, the number of hematopoietic cells containing a high proportion of deleted mtDNA appears to decrease with time as a result of apparent selection of cells with normal mtDNA. The new syndrome of chronic diarrhea and intestinal pseudo-obstruction with liver involvement has also been attributed to an mtDNA rearrangement (9). Severe anorexia, vomiting, chronic diarrhea, and villous atrophy are the initial manifestations that appear late in the first year or during the second year of life and are associated with mild elevations of liver enzymes, hepatomegaly, and steatosis. Diarrhea, vomiting, and lactic acidosis worsens in these patients when they are fed high dextrose-containing parenteral or enteral nutrition. Diarrhea improves and even resolves completely by 5 years of age and results of examinations of intestinal biopsy specimens normalize; but neurologic findings such as retinitis pigmentosa, cerebellar ataxia, sensorineural deafness, and proximal muscle weakness appear late in the first decade and herald a fatal outcome. Respiratory chain enzyme assays reveal a complex III deficiency that is present in muscle tissue, but absent in circulating lymphocytes. The term mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) has recently been applied to a syndrome of myopathy with ragged red fibers, prominent peripheral neuropathy, ophthalmoplegia, leukoencephalopathy, and chronic intestinal pseudo-obstruction (6). Nausea, vomiting, borborygmi, and abdominal distention often begin in late childhood or adolescence, eventually leading to a diagnosis of pseudo-obstruction. These symptoms often precede the development of external ophthalmoplegia, ptosis, limb weakness, sensorimotor neuropathy, and hearing loss. Many of these patients show signs of chronic malnutrition, short stature, and diverticulosis (that may involve the small intestine) in their early adult years. Delayed gastric emptying and antroduodenal dysmotility may be revealed by appropriate diagnostic studies. The severity of the chronic intestinal pseudo-obstruction is variable and is attributed to visceral myopathy with atrophic fibrotic longitudinal smooth muscle in the intestinal wall with normal ganglion cells in some patients, whereas fibrosis and vacuolization of autonomic ganglia in the myenteric plexus and decreased nerve fibers innervating intestinal smooth muscle has been described in other patients. Decreased activity of muscle respiratory chain enzymes, including complex IV, complex I, or combinations of these, have been identified in several patients who have been evaluated. Autosomal recessive inheritance appears to underlie the transmission of the disease, because most patients have affected siblings and an equal number of males and females are affected. Several patients have multiple mtDNA deletions similar to those found in other mitochondrial myopathies. Navajo neuropathy is a sensorimotor neuropathy confined to Navajo children that is potentially caused by a mitochondrial defect, although this has not been proved. This disorder is manifested by the development of weakness, hypotonia, areflexia, loss of sensation in the extremities, acral mutilation, corneal ulceration, poor growth, short stature, and serious systemic infections (24,25). In 1990, Singleton et al. (24) reported an association of Reye's-like syndrome episodes, hepatic dysfunction, and death caused by liver failure at a young age in three Navajo children. Progressive white-matter lesions were demonstrated by cerebral magnetic resonance scanning, and analysis of specimens obtained in peripheral nerve biopsy specimens showed severe loss of myelinated fibers. Infectious, biochemical, and known metabolic causes have been excluded in this multisystem, autosomal recessive disorder. The hepatic findings were initially summarized by Walker et al. (26) in 1992, and in more detail recently by Holve et al. (27). Liver disease appeared at a mean age of 30 months, although neonatal appearance with giant-cell hepatitis has subsequently been observed. Patients showed failure to thrive, hepatomegaly, and jaundice, with the development of fatal liver failure over a period of months or years. Elevation of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase was uniform. Histologic analysis of liver tissue showed macrovesicular and microvesicular steatosis, portal fibrosis or micronodular cirrhosis, pseudoacinar formation, syncytial mutinucleated giant cells, cholestasis, and periportal inflammation. Nonspecific mitochondrial changes, such as swollen mitochondria and ringed cristae, were observed in liver tissue specimens from several patients. Blood lactate and pyruvate levels were normal, and skin fibroblasts had normal respiration from one patient. The liver involvement in this disorder is progressive with liver failure developing within months to years. Neurologic symptoms progressed after liver transplant in one patient, illustrating multisystem involvement. There has been no effective treatment to date for affected children. The cause of Navajo neuropathy remains unknown; however, it shares many clinical and histologic features with other mitochondrial hepatopathies. Recently, Zhang and Arias (28) presented preliminary findings of very low expression of mRNA for the MDR3 protein in liver from several Navajo neuropathy patients. Because deficiency of this protein has been associated with progressive intrahepatic cholestasis with elevated γ-glutamyl transpeptidase levels in non-Navajo children, this finding is of interest. However, the non-Navajo children with MDR3 deficiency have neither hepatic steatosis nor neurologic abnormalities. Further investigations into the cause of this fascinating disorder are in progress. Other primary mitochondrial hepatopathies include defects in specific enzymatic pathways, such as the fatty acid oxidation defects, urea cycle enzyme deficiencies, phosphoenolpyruvate carboxy-kinases (PEPCK) deficiency, carnitine palmitoyl-transferase-I and -II deficiency, and others (Table 1). The manifestations of these disorders are dependent on the pathophysiology of the specific biochemical pathways that are interrupted. The reader is referred to pediatric hepatology and metabolic disease textbooks for detailed review of these conditions. SECONDARY MITOCHONDRIAL HEPATOPATHIES A second major group of mitochondrial hepatopathies includes those secondary to an injurious toxin, metal, xenobiotic compound, or endogenous compound (Table 2). Acquired abnormalities of mitochondrial respiration may occur because of toxins and drugs (e.g., fialuridine) (29), accumulation of metals (e.g., Wilson's disease) (30), Reye's syndrome (31), and possibly several other disease states (e.g., alcoholic liver disease and chronic cholestasis) (2,13). The toxicity observed during a trial using fialuridine for treatment of hepatitis B virus infection in adults produced a disorder that mimicked genetic defects of OXPHOS (29). The incorporation of fialuridine into mtDNA led to dysfunction of the mitochondrial genome (32). More recently, the emetic toxin of Bacillus cereus, cereulide, has been shown to be a clinically relevant mitochondrial toxin that causes liver failure (33). Hydrophobic bile acids (34), which accumulate in the liver in cholestasis, and metals (35) appear to exert their effect on mitochondria by oxidant-mediated mechanisms, possibly through the induction of the mitochondrial membrane permeability transition. During experimental cholestasis induced by bile duct ligation in the rat, Krahenbuhl et al. (36) have demonstrated reduced activity of the electron transport chain in hepatic mitochondria with an increased density of mitochondria per hepatocyte. This group further showed that hydrophobic bile acids reduce complex I and complex III activity in isolated hepatic mitochondria (37). Recently, it has been shown that physiologic concentrations of hydrophobic bile acids induce mitochondrial membrane permeability transition and hydroperoxide generation (38). Thus, it has been proposed that during cholestasis and in patients with bile acid synthesis defects increased concentrations of hydrophobic bile acids induce generation of reactive oxygen species from the altered electron transport chain of hepatocyte mitochondria, with the resultant opening of the permeability pore and the onset of cellular necrosis or apoptosis (39). The antioxidant vitamin E has provided significant protection against bile acid toxicity in an in vivo rat model (39). The role of mitochondrial dysfunction in human cholestatic liver disease has not been fully elucidated and awaits further investigation. Other drugs and compounds (salicylates and valproic acid, for example) have well-defined effects on mitochondrial respiration and may cause cellular injury. The contribution of mitochondrial dysfunction in a variety of pediatric disorders in which hepatic iron