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Characterization of Hepatic Mitochondrial Injury Induced by Fatty Acid Oxidation InhibitorsDrug Safety Evaluation, Allergan, Inc., Irvine, California, USA Correspondence: Alison Vickers, PhD, 2525 Dupont Drive, Irvine, CA 92623, USA; e-mail:vickers_alison{at}allergan.com.
Impairment of liver mitochondrial β-oxidation is an important mechanism of drug-induced liver injury. Four inhibitors of fatty acid oxidation were compared in short-term rat in vivo studies in which the rats were administered one or four doses. The hepatocellular vacuolation represented ultra-structural mitochondrial changes. Urine nuclear magnetic resonance (NMR) spectroscopy revealed that both FOX988 and SDZ51-641 induced a persistent dicarboxylic aciduria, suggesting an inhibition of mitochondrial β-oxidation and incomplete fatty acid metabolism. Etomoxir caused minimal mitochondrial ultrastructural changes and induced only transient dicarboxylic aciduria. CPI975 served as a negative control, in that there were no significant perturbations to the mitochondrial ultrastructural morphology or in the urine NMR composition; however, compound exposure was confirmed by the up-regulation of liver gene expression compared to vehicle control. The liver gene expression changes that were altered by the compounds were indicative of mitochondria, general and oxidative stress, and peroxisomal enzymes involved in β-oxidation, suggestive of a compensatory response to the inhibition in the mitochondria. In addition, both FOX988 and SDZ51-641 up-regulated ribosomal genes associated with apoptosis, as well as p53 pathways linked with apoptosis. In summary, metabonomics and liver gene expression provided mechanistic information on mitochondrial dysfunction and impaired fatty acid oxidation to further define the clinical pathology and histopathology findings of hepatotoxicity.
Key Words: hepatic • in vitro toxicology • mechanisms of toxicity Abbreviations: ALP, alkaline phosphatase • ALT, alanine aminotransferase • AST, aspartate aminotransferase • ATP, adenosine triphosphate • CPT-1, carnitine almitoyltransferase 1 • CPT-2, carnitine palmitoyltransferase 2 • FFA, free fatty acids • FIAU, fialuridine • GSH, glutathione • NIDDM, non-insulin–dependent diabetes mellitus • PPAR, peroxisome proliferator activated receptor • ROS, reactive oxygen species • UCP, uncoupling protein
Integration of Omic Technologies and Organotypic Translational Models into Preclinical Safety Drug-induced organ injury continues to be a significant clinical issue owing to the wide variation of human responses and the limited predictability of various target organ injuries from preclinical animal models (Aldridge et al. 2003). Target organ toxicities are usually identified in studies of one month or less, and the prediction of major organ toxicities or dose-limiting effects (hematological, hepatic, gastrointestinal, cardiovascular, urinary tract) is about 50%–70% (Greaves et al. 2004; Olsen et al. 2000). Furthermore, current biomarkers of organ injury are generally nonspecific and often reflect the extent of organ injury rather than the cause. The cellular pathways leading to organ-specific toxicities are often multifaceted, involving several cell types and biochemical networks of cell–cell and cell–matrix interactions. Developments in organotypic cultures, such as organ slices, as well as the application of gene expression profiling technologies to both in vivo and in vitro models contribute to characterization of key pathways leading to organ dysfunction prior to frank clinical signs and to changes in morphology (Schmeichel and Bissell 2003; Vickers and Fisher 2005). These models and technologies facilitate the molecular characterization as to how chemicals perturb signaling pathways and trigger the activation of cellular pathways of proliferation, apoptosis, oxidative stress, and changes in cell function, which are relevant to increasing our knowledge of drugs and cellular targets (Guengerich 2007; Gunawan and Kaplowitz 2007; Van de Water et al. 2006). In addition, comparisons of animal and human tissue models contribute to the extrapolation of data from animals to humans and aid in decision making about the safety of candidate compounds.
Mitochondrial Injury Several drug failures have been attributed to or associated with mitochondrial toxicity (Chan et al. 2005). Drugs that inhibit complex I or complex III of the respiratory chain (metformin, troglitazone, or fibrates) often lead to the generation of reactive oxygen species and oxidative stress within the mitochondria. Drugs that uncouple oxidative phosphorylation by inhibiting complex IV or activating the uncoupling proteins (nonsteroidal anti-inflammatory drugs [NSAIDs], tolcapone, dinitrophenol) greatly reduce ATP synthesis (Figure 1). Uncoupling agents are associated with weight loss resulting from the increased transport of protons from the mitochondria, producing thermal energy without generating ATP and promoting carbohydrate and fat metabolism, resulting in dramatic weight loss in a short time. Etomoxir stimulates food intake in rats and humans and has been shown to decrease hepatic energy status in the rat (Horn et al. 2004; Kahler et al. 1999). Antiviral agents generally inhibit mitochondrial DNA transcription, translation, or replication, which then affects replication of mitochondria-specific genes (complex I, cytochrome b, COX 1–3) and function.
The cytoplasmic accumulation of long-chain fatty acids, as well as inhibitors of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzyme of mitochondrial β-oxidation, can increase mitochondrial uncoupling protein (UCP) levels. Mitochondrial UCPs contain PPAR response elements, and PPAR  regulates genes that control mitochondrial and peroxisomal fatty acid oxidation (Brandt et al. 1998; Forman et al. 1997; Murray et al. 2005). Etomoxir, an inhibitor of CPT-1, or conditions of an abundance of fatty acid supply to mitochondria, triggers an up-regulation of UCP3 in human muscle (Schrauwen et al. 2002). The fatty acids that are not oxidized become trapped as fatty acid anions inside the mitochondrial matrix, where reactive oxygen species (ROS) are formed (Ho et al. 2002). UCP3 may be involved in the protection of mitochondria against lipid-induced oxidative damage by facilitating the transport of nonmetabolized fatty acids and lipid peroxides from the mitochondrial matrix. A reduced UCP3 content in insulin-resistant subjects suggests that these patients are less protected against lipid-induced damage, which could ultimately lead to mitochondrial damage at lower drug dosages. UCP3 may also prove to be a suitable drug target for diabetes and anti-obesity (Schrauwen and Hesselink 2004; Schrauwen et al. 2002; Schrauwen et al. 2006). Markers of mitochondrial metabolism, cytochrome c oxidase subunit 1 and UCP3, exhibited lower mRNA levels in insulin-resistant obese subjects compared to insulin-sensitive obese subjects. Exercise improved the insulin sensitivity for both groups with an increased correlation in aerobic fitness and the protein content of complex III/V mitochondrial proteins, suggesting that exercise can aid in overcoming insulin resistance (Heilbronn et al. 2007). Pharmaceutical efforts have previously focused on inhibiting the mitochondrial β-oxidation pathway to cause a shift in the substrate use from fatty acids to glucose to reverse the fatty acid–driven gluconeogenesis associated with non-insulin–dependent diabetes mellitus (NIDDM) and to prevent hyperglycemia (Foley 1992). Etomoxir and CPI975 inhibit CPT-1, decreasing the use of FFA as a source of energy (Anderson 1998; Lopaschuk and Stanley 1997; Weis et al. 1994). Etomoxir is an irreversible inhibitor of CPT-1 and has a higher affinity for the liver isoform of CPT-1 than for the muscle form, whereas CPI975 is a reversible inhibitor (Weis et al. 1994). FOX988 and SDZ51-641 reduce the availability of CoA to carnitine palmitoyltransferase 2 (CPT-2) by the metabolic activation of each compound to a carboxylic acid and subsequent formation to a coenzyme A ester metabolite (Anderson 1998; Anderson et al. 1995; Foley 1992; Foley et al. 1997) (Figure 2). These agents, potentially useful in NIDDM, exhibited advantageous effects on lipid metabolism and hypoglycemic effects in rats and monkeys (Deems et al. 1998). The potential for mitochondrial injury, however, and intracellular lipid accumulation in liver cells remained a concern. Etomoxir also induced cardiac and hepatic hypertrophy in animals (Rupp et al. 1992; Yotsumoto et al. 2000). Etomoxir, however, has potential as an antianginal agent for ischemic heart disease to increase the ATP production from the oxygen-efficient conversion of glucose rather than from fatty acid metabolism (Lee et al. 2004; Marazzi et al., in press).
CPT deficiency and genetic defects of fatty acid metabolism were initially recognized in the 1970s. The clinical phenotypes, hypoketotic hypoglycemia, cardiomyopathy, and myopathy are confirmed with whole blood acylcarnitine analysis and the presence of organic acids and dicarboxylic acids in urine. A new test probes for the β-oxidation pathway in fibroblasts (Nada et al. 1995). In this study, liver mitochondrial injury induced by the fatty acid oxidation inhibitors Etomoxir, CPI975, FOX988, and SDZ51-641 was investigated by urinary NMR to evaluate the feasibility of detecting liver injury earlier and to identify bio-markers to monitor the progression or reversibility of the hepatotoxicity. Additionally, liver gene expression provided insight into the mechanism of the mitochondrial injury. To assess the degree of injury of the compounds, both clinical pathology and histopathology parameters were evaluated and compared with the changes in urinary NMR and liver gene expression.
Chemicals Etomoxir was obtained from Research Biochemical Corp., and CPI975, FOX988, and SDZ51-641 were obtained from Novartis (East Hanover, NJ, USA). All other chemicals were research grade and obtained from commercial vendors.
Animals Each dose group consisted of five rats. The compounds were administered by gavage dosing, either as a single dose (one dose) or four doses administered daily (four doses). The compounds included: Etomoxir (125 mg/kg, suspended in Veegum and 1.8% Methocel); CPI975 (250 mg/kg, in 0.05 M sodium phosphate buffer, pH 7); FOX988 (2000 mg/kg, in Labrafil [30%]: corn oil [70%]); or SDZ51-641 (160 mg/kg, in 0.5% CMC Type 7HF/0.2% Tween 80). A control group existed for each vehicle. Following the initiation of dosing, urine was collected at 0–8 hours, 8–24 hours, 24–32 hours, 32–48 hours, 48–72 hours, and 72–96 hours. To minimize bacterial contamination, ice packs were used to chill the collection vials during the urine collection period, and the inside of the metabolism cage funnel was rinsed daily with distilled water to remove any contaminants and residual urine. The collected urine was centrifuged (3000 x g at 4°C), and aliquots were stored at –76°C until NMR analysis. Blood samples (approximately 1.5 mL) were collected from all rats via the retro-orbital venous plexus using light CO2 (60%)/O2 (40%) anesthesia prior to dosing. Serum was prepared for clinical pathology analyses. Following the final urine collection, the animals were euthanized with CO2 (60%)/O2 (40%) anesthesia and exsanguination The liver was weighed, and approximately 200 mg of the median lobe was collected for gene expression analysis, snap-frozen in liquid nitrogen, and stored at –76°C until use. Additional liver from the median lobe was collected and fixed in 10% neutral-buffered formalin for hematoxylin and eosin staining, and a portion was fixed in modified Karnovsky’s fixative for ultrastructural evaluation.
Light and Electron Microscopy
Preparation of Urine Samples for NMR Analysis
1H NMR
RNA Isolation
cDNA Microarray Hybridization and Analysis
Branched DNA (bDNA)
Statistical Evaluation
Rat In Vivo Clinical and Histopathology Etomoxir and SDZ51-641 administered to rats caused significant increases in serum transaminase levels (AST and ALT), suggestive of increased hepatocellular permeability. However, the time course of these changes varied. Etomoxir caused significant increases following one dose, and values returned to predose levels, and effects with SDZ51-641 were seen after four doses. Significant increases of ALP, suggestive of biliary cell injury, were induced by SDZ51-641 and FOX988 treatment, and FOX988 significantly affected bilirubin levels. No compound-related changes in serum clinical pathology were evident with CPI975 treatment (Table 1). Additionally, each vehicle had no effect when compared to predose control values of the respective group.
Evaluation of the liver by light microscopy revealed cytoplasmic vacuolation of periportal and centrilobular hepatocytes. Electron microscopy revealed mild to severely enlarged mitochondria in the majority of hepatocytes following SDZ51-641 and FOX988 treatment. Most of the cristae of the megamitochondria remained connected to the internal mitochondrial membrane, were not markedly distended, and had relatively normal architecture. The electron-lucent matrices contained evenly dispersed, moderately electron-dense flocculent material and occasional myelin figures or membranous structures, and the cristae did not seem to be centralized. There was no clear evidence of mitochondrial fusion or division (Figure 3). No compound-related changes were observable for CPI975 however, after multiple dosing with Etomoxir, one animal exhibited mitochondrial ultrastructural changes. Subtle changes in some hepatocytes were characterized by the presence of tightly whorled myelin figures that were closely apposed to mitochondria (Table 1).
Metabonomics and Gene Expression Etomoxir induced dicarboxylic aciduria, detected in the urine, within eight to twenty-four hours (Figure 4). Following multiple doses of Etoxomir, the dicarboxylic aciduria subsided following the third dose, suggesting that adaptation had occurred. Single and daily doses of FOX988 and SDZ51-641 induced dicarboxylic aciduria, which was sustained for up to ninety-six hours with FOX988 and up to forty-eight hours with SDZ51-641 treatment. Urinary glucose was elevated with the SDZ51-641 animals at twenty-four to forty-eight hours post-dose. Following CPI975 treatment, no detectable perturbation in urine composition was found following either a single or multiple dose.
Liver gene expression profiles were determined following one or four doses of Etomoxir, CPI975, FOX988, or SDZ51-641. The greatest number of gene expression changes was exhibited by FOX988, followed by SDZ51-641, Etomoxir, and CPI975, which paralleled the extent of injury detected by clinical pathology and histopathology. The liver gene expression changes that were striking across the compounds included an up-regulation of genes indicative of mitochondrial function, fatty acid oxidation peroxisomal enzymes, ribotoxic stress, and apoptosis. Other cellular processes that may be affected based on the gene expression profiles included general stress, cell matrix and signaling, and transporters (Table 2).
The gene expression changes following CPI975 treatment were limited. The data revealed a potential effect on mitochondrial function, which is characteristic of these compounds, and confirmed that the rats had been exposed to the compound. Additionally, the limited gene expression changes were in agreement with the lack of change in clinical chemistry, metabonomics, and histopathology for CPI975 (Table 3).
The gene chip data were confirmed by direct hybridization (bDNA) of oligonucleotide probe sets specific for rat genes, which represented some of the functional categories altered: cytochrome c oxidase IV (mitochondrial), clusterin and metallothionein 1 (general stress), and p53 (cell cycle) (Figure 5). In general, this method both confirmed the microarray data and revealed induction in treatment groups not previously detected by the chip. This difference can be explained in part by the analysis of RNA from individual animals rather than RNA pools as done for the microarray analysis, as well as an increased sensitivity of the bDNA method, which is based on direct hybridization and signal amplification.
To further our understanding of mitochondrial injury as a consequence of impairment of mitochondrial β-oxidation, four inhibitors of mitochondrial β-oxidation were investigated in short-term rat in vivo studies (one dose and four doses). Etomoxir and CPI975 are direct inhibitors of the rate-limiting enzyme CPT-1, regulating mitochondrial β-oxidation, whereas FOX988 and SDZ51-641 sequester and deplete the intramitochondrial pool of CoA to effect mitochondrial β-oxidation. The doses for this study were selected from previous two- or four-week rat toxicology studies in which mitochondrial injury had occurred. A short-term study with one and four doses was designed to use both urinary NMR analysis and liver gene expression data to gain insight into the mechanism of organ injury and to identify a biomarker profile of mitochondrial β-oxidation in conjunction with clinical pathology and histopathology. The integration of these technologies will improve the evaluation of compound safety for both compound selection and development. Pharmaceutical agents that affect mitochondrial function possess the propensity to cause mitochondrial injury, and the lesions are diverse, including steatosis, inflammation, apoptosis, necrosis, and fibrosis. Liver injury in humans, manifested as enlarged and giant mitochondria, as seen with the nucleoside analog fialuridine (FIAU), led to lactic acidosis and hepatic failure (McKenzie et al. 1995). Compounds which inhibit β-oxidation directly (e.g., tetracyclines) or sequester CoA (e.g., valproic acid) can result in dicarboxylic aciduria, an increased synthesis of triglycerides, and undesirable fat deposition (Fromenty and Pessayre 1995; Pessayre et al. 1999; Szewczyk and Wojtczak 2002). Patients with defects in the mitochondrial β-oxidation of fatty acids have been reported to exhibit dicarboxylic aciduria. Urinary excretion of dicarboxylic acids can reflect the liver and kidney activity of lipid metabolism and concentration of short-chain fatty acids in the tissues, which can serve as a marker of this effect (Mortensen 1990). In this study, liver dysfunction and mitochondrial damage occurred with the doses selected for Etomoxir, FOX988, and SDZ51-641. The increased hepatocyte permeability following a single dose of Etomoxir was transient, whereas FOX988 and SDZ51-641 induced both hepatocellular and biliary cell dysfunction over the course of the study. The hepatocellular vacuolation observable by light microscopy was verified to be mitochondrial damage by electron microscopy. Evidence of lipid accumulation was minimal in this study. Some animals treated with SDZ51-641 exhibited lipid droplets, whereas triglyceride levels were not altered by any of the compounds.
Metabonomic evaluation of the urine revealed increased levels of dicarboxylic acids, representing incomplete β-oxidation of fatty acids. When mitochondrial β-oxidation is impaired, dicarboxylic acids can be formed from CYP4A
In this study, rat liver gene expression specific to the peroxisome β-oxidation system and the microsomal
The 3-ketoacyl CoA thiolase is the last enzyme in the peroxisome β-oxidation pathway, and the gene expression of both rat forms, thiolase 1 and 2, were increased by Etomoxir. The mitochondrial inhibition of fatty acid oxidation, resulting in an increase of free fatty acids, could activate the expression of the peroxisomal β-oxidation pathway and liver fatty acid binding protein, which transport acyl CoA esters (Faergeman and Knudsen 1997). It has been postulated that inhibition of CPT-1 by Etomoxir leads to a PPAR Inhibitors of mitochondrial β-oxidation can disrupt cell respiration by perturbing the energy-coupling properties and migration of electrons along the respiratory chain to cytochrome c oxidase. In this study each compound increased the gene expression of cytochrome c oxidase subunit IV, a subunit of the terminal enzyme cytochrome c oxidase in the mitochondrial electron transport chain. Additionally, Etomoxir caused an up-regulation of enoyl CoA hydratase gene expression, a step in the mitochondrial β-oxidation of fatty acids. Mitochondrial dysfunction is an important mechanism of drug-induced liver injury. A decline of ATP synthesis by uncoupling respiration from ATP synthesis will lead to a loss of energy-requiring processes, like apoptosis and protein synthesis, in favor of necrosis, and an inflammatory response (Pessayre et al. 1999). Additionally, a critical defense system within mitochondria is GSH, which aids in controlling the levels of reactive oxygen species like hydrogen peroxide, since unlike peroxisomes, mitochondria lack catalase. Depletion of GSH and ATP levels by Etomoxir and CPI975 has been demonstrated in human liver slices (Vickers et al. 2006) and has been reported for Etomoxir in HepG2 cells (Merrill et al. 2002). GSH depletion can trigger cell death and inflammation through the release of inflammatory cytokines, which in turn can activate nonparenchymal cells, and contribute to the overall development of liver injury (Fernandez-Checa et al. 1998; Maher 1999). In human liver slices, both Etomoxir and CPI975 caused an up-regulation of IL-8 gene expression, the proinflammatory cytokine produced by endothelial cells, as well as IL-8 protein levels (Vickers et al. 2006). Toxic effects of CPT-1 inhibitors like Etomoxir demonstrate that oxidative stress is a key mediator. Accumulation of fatty acids in the mitochondrial matrix, where oxidative processes and formation of ROS takes place, could lead to increased lipid peroxidation and damage to mitochondrial proteins. Gene expression data, consistent with oxidative stress, was evident in both HepG2 cells and mice hearts exposed to Etomoxir (Merrill et al. 2002; Cabrero et al. 2003). Gene expression data also revealed changes in cell cycle and apoptosis, protein, and DNA damage (Merrill et al. 2002). In this study, cellular stress was implicated by the up-regulation of clusterin, a protein-processing chaperone (Wilson and Easterbrook-Smith 2000), and metallothionein, which protects the cell from reactive oxygen species. The metallothioneins are specific metal-binding proteins high in cysteine residues (30%) that may function in a manner similar to GSH as an antioxidant to protect the cell from reactive oxygen species. Metallothioneins are readily inducible and transcriptionally regulated by metals, glucocorticoids, and cytokines (Klaassen et al. 1999). Following one or four doses, both FOX988 and SDZ51-641 triggered an up-regulation of ribosomal protein gene expression. Ribosomal proteins can function as regulators of cell proliferation, as inducers of apoptosis and as a stress response in xenobiotic toxicity (Chen and Ioannou 1999; Laskin et al. 2002). Ribosomal protein L13a is a subunit of 60s ribosomal protein, which exhibits a regulated release to control translation, and is postulated to be a regulatory protein (Mazumder et al. 2003). The ribosomal protein S9 encodes a protein with an N-terminal mitochondrial targeting sequence, and overexpression or inhibition of this protein can affect mitochondrial function (Wiltshire et al. 1999). Both FOX988 and SDZ51-641 additionally up-regulated p53 gene expression, which can trigger pathways linked with apoptosis. Cathepsin L gene expression was also up-regulated by both FOX988 and SDZ51-641 following one or four doses. Cathepsin L, a lysosomal protease, has been suggested in some cell types to play a role in apoptosis because of its role in the degradation of collagen and elastin (Wille et al. 2004). The mechanistic information gained about the consequences of mitochondrial fatty acid inhibition from the in vivo studies reported here and from the in vitro studies reported previously (Vickers et al. 2006) are summarized in Figure 6. As a result of incomplete metabolism of the fatty acids, inhibition of mitochondrial fatty acid oxidation by Etomoxir, CPI975, FOX988, and SDZ51-641 led to increased amounts of dicarboxylic acids in the urine. In vivo, liver dysfunction was evident by increases in blood transaminase and ALP levels. Gene expression profiling of liver in vivo samples and in vitro liver slices revealed that similar cellular targets or pathways were affected, including mitochondrial function, general and oxidative stress, fatty acid metabolism, peroxisomal enzymes, and apoptosis. The up-regulation of liver gene expression preceded and persisted with mitochondrial enlargement and megamitochondria histopathology. Mitochondrial dysfunction manifested as decreased liver ATP and GSH levels would result in an overall decline of cellular energy and an increase of oxidative stress. Concurrent with mitochondrial dysfunction is an increased apoptosis, evident by increased caspase activity, and the involvement of a tissue inflammatory response, verified by increased IL-8 protein levels (Vickers et al. 2006). Because of the loss of energy by the mitochondria, the further metabolism of pyruvate from glucose in the mitochondria would be hindered, and enzymes in the cytoplasm would convert the pyruvate to lactic acid, which could progress to lactic acidosis and in a rare event, to liver failure.
These studies demonstrate the value of integrating gene expression, cell function, and histopathology with in vivo and in vitro models for the purpose of defining compound-induced effects on cellular pathways, identifying biomarkers, and for demonstrating the biological relevance in human tissue.
The author thanks Laurie O’Rourke, DVM, PhD for reviewing the Clinical Pathology data; Judit Markovits, DVM, PhD for histopathology review; Sue Irwin, PhD for providing animal treatment support; John Gounarides for NMR analysis; Kristine Rose, PhD for RNA isolation and bDNA analysis; and Maria Rivero for graphics.
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Toxicologic Pathology, Vol. 37, No. 1,
78-88 (2009)
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Abstract
Introduction
1H 0.0), with a final concentration of 1 mM. Following centrifugation to remove precipitates, a 700 µL aliquot of the buffered urine was then transferred into vials for NMR spectroscopic analysis. 
1.75-fold change, 30% cov), which were averaged and normalized to the total fluorescence across the slide using the Phase-1 MATRIXexpress program. Expression profiles were compared from replicate analyses as the chip evolved, for reproducibility, and for similarity within gene categories as new gene probes were added to the chips. 
-oxidation. Typically, dicarboxylic acids are converted to a CoA derivative and further metabolized. In humans the peroxisome β-oxidation system is responsible for the further metabolism of dicarboxylyl-CoAs, whereas in rats a significant portion of dicarboxylyl-CoAs is metabolized via the noninducible branched-chain oxidation system. Even though microsomal 