QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.072785v1 311/3/864    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McCarthy, T. C. Articles by Sinal, C. J. Search for Related Content PubMed PubMed Citation Articles by McCarthy, T. C. Articles by Sinal, C. J.

TOXICOLOGY

Disruption of Hepatic Lipid Homeostasis in Mice after Amiodarone Treatment Is Associated with Peroxisome Proliferator-Activated Receptor-Target Gene Activation

Department of Pharmacology (T.C.M, P.T.P, E.A.H., C.J.S.) and Department of Medicine (P.T.P), Dalhousie University, Halifax, Nova Scotia, Canada

Received for publication June 14, 2004
Accepted July 20, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Amiodarone, an efficacious and widely used antiarrhythmic agent, has been reported to cause hepatotoxicity in some patients. To gain insight into the mechanism of this unwanted effect, mice were administered various doses of amiodarone and examined for changes in hepatic histology and gene regulation. Amiodarone induced hepatomegaly, hepatocyte microvesicular lipid accumulation, and a significant decrease in serum triglycerides and glucose. Northern blot analysis of hepatic RNA revealed a dose-dependent increase in the expression of a number of genes critical for fatty acid oxidation, lipoprotein assembly, and lipid transport. Many of these genes are regulated by the peroxisome proliferator-activated receptor- (PPAR), a ligand-activated nuclear hormone receptor transcription factor. The absence of induction of these genes as well as hepatomegaly in PPAR knockout [PPAR(—/—)] mice indicated that the effects of amiodarone were dependent upon the presence of a functional PPAR gene. Compared to wild-type mice, treatment of PPAR(—/—) mice with amiodarone resulted in an increased rate and extent of total body weight loss. The inability of amiodarone to directly activate either human or mouse PPAR transiently expressed in human HepG2 hepatoma cells indicates that the effects of amiodarone on the function of this receptor were indirect. Based upon these results, we conclude that amiodarone disrupts hepatic lipid homeostasis and that the increased expression of PPAR target genes is secondary to this toxic effect. These results provide important new mechanistic information regarding the hepatotoxic effects of amiodarone and indicate that PPAR protects against amiodarone-induced hepatotoxicity.

Amiodarone has been shown to induce steatosis in both animal models and humans (Fromenty et al., 1990; Card et al., 1998; Bolt et al., 2001), possibly through inhibition of the mitochondrial -oxidation of long-, medium-, and short-chain fatty acids (Fromenty et al., 1990). Steatosis is a common, early histological finding of hepatic injury and is characterized by micro- and/or macrovesicular hepatocellular lipid accumulation. Although steatosis is reversible, it can lead to steatohepatitis involving hepatocellular necrosis and/or apoptosis (Koteish and Diehl, 2001). Although specific mechanisms for the inhibition of mitochondrial -oxidation by amiodarone have not been unequivocally defined, this drug is known to inhibit carnitine palmitoyltransferase-I (CPTI)-dependent transport of long-chain fatty acids across the mitochondrial membrane (Kennedy et al., 1996; Bonnefont et al., 1999). Amiodarone also exerts multiple effects on mitochondrial respiration by inhibiting the normal function of the mitochondrial electron transport chain and thereby the generation of ATP (Bolt et al., 2001; Spaniol et al., 2001). Nonspecific hydrophobic interactions with either mitochondrial membrane phospholipids and/or membrane-associated proteins have also been suggested as a mechanism by which amiodarone disrupts mitochondrial function (Fromenty et al., 1990).

An important regulator of the hepatic -oxidation of fatty acids is the peroxisome proliferator-activated receptor- (PPAR), a member of the gene superfamily of ligand-activated nuclear hormone transcription factors (Francis et al., 2003b). The most important endogenous ligands for PPAR are fatty acids, particularly medium- and long-chain fatty acids, and eicosanoids such as leukotriene B4 and other arachidonate derivatives (Wahli et al., 1999). PPAR binds as a heterodimer with the retinoid X-receptor to specific DNA sequences, known as peroxisome proliferator response elements, that are present within the regulatory regions of target genes. After activation by cognate ligands, PPAR induces the expression of target genes through the recruitment of coactivators that facilitate gene transcription. Thus, by serving as a sensor of intracellular fatty acid concentrations and regulating genes involved in the transport and catabolism of these fatty acids, PPAR plays an important role in normal lipid homeostasis (Staels et al., 1998; Mascaro et al., 1999; Wolfrum et al., 2001).

Although most of the adverse hepatic effects of amiodarone are transient and reversible, deaths resulting from amiodarone-induced hepatotoxicity have been reported (Simon et al., 1984; Richer and Robert, 1995; Stravitz and Sanyal, 2003). Histopathological signs of amiodarone-induced hepatotoxicity in patients resemble nonalcoholic steatohepatitis and include steatosis, cellular degeneration, and cellular necrosis (Rigas et al., 1986; Stravitz and Sanyal, 2003). Previous studies have suggested that PPAR may have a central role in the pathogenesis of microvesicular steatosis and nonalcoholic steatohepatitis induced by feeding a methionineand choline-deficient diet (Rao et al., 2002; Ip et al., 2003). Given the similar pathological consequences of this model and amiodarone-induced hepatotoxicity, PPAR may also be relevant to the toxic effects associated with this drug.

At present, amiodarone is one of the most effective drugs available for the treatment of the increasingly prevalent arrhythmia, atrial fibrillation. However, the clinical use of amiodarone has been tempered by a reputation for causing hepatotoxicity. Consequently, patients are often placed on less effective medications that, although they present a lower risk of hepatotoxicities, may cause potentially fatal proarrhythmia. A greater knowledge of the biochemical and molecular changes that occur in the liver as a result of amiodarone treatment will advance our understanding of the mechanisms of amiodarone-induced liver toxicity and, as such, will be of great benefit to patients. The objective of the present study was to examine the role of PPAR in amiodarone-induced hepatotoxicity through a combination of complementary in vitro and in vivo approaches.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals and Experimental Protocols. Amiodarone was purchased from Sigma-Aldrich (Oakville, ON, Canada). N-Desethylamiodarone (DEA) was a generous gift of Sanofi-Synthelabo Inc. (New York, NY). Wild-type (C57BL/6) and PPAR(—/—) null (C57BL/6 background; obtained originally from Frank Gonzalez, National Cancer Institute, National Institutes of Health, Bethesda, MD) mice were obtained from our breeding colony and were housed under a 12-h light/dark cycle. Mice were allowed water and standard rodent chow ad libitum before and during treatment. All experiments were performed with male mice weighing 25 to 30 g at 9 to 11 weeks of age. Mice were randomly divided into experimental groups and weighed once a day, 4 days before treatment and daily throughout the treatment period. Treatment consisted of daily intraperitoneal injections (0.1 ml) of amiodarone at various doses (10 mg/kg, 40 mg/kg, 80 mg/kg, and 150 mg/kg) or vehicle (0.5% Tween 20, 10% DMSO in sterile H₂O) or oral gavage of clofibrate (200 mg/kg) in corn oil (0.1 ml) daily for a total of 4 or 7 days. Mice were anesthetized with an intraperitoneal injection of 80 mg/kg sodium pentobarbital (MTC Pharmaceuticals, Cambridge, ON, Canada) 24 h after the last treatment. Blood was collected into heparinized tubes by cardiac puncture and centrifuged; plasma was stored at 4°C for a maximum of 1 week before assay or frozen until assay for amiodarone or DEA concentrations. Liver and kidney were snap frozen in liquid nitrogen before RNA extraction or frozen in a Tissue-Tek OCT (Sakura Finetek, Torrance, CA)/20% sucrose (v/v) solution for histological analysis. All protocols and procedures were approved by the Dalhousie University Committee on Laboratory Animals and are in accordance with the Canadian Council on Animal Care guidelines.

Plasma Analysis. Glucose concentrations were determined using whole blood at the time of sacrifice using the FreeStyle blood glucose monitoring system (TheraSense, Alameda, CA). Triglycerides, total cholesterol, and AST levels were determined using Infinity assay kits (Sigma-Aldrich); total bile acids were measured using a kit purchased from Wako Chemicals USA (Richmond, VA). All kits were used per the manufacturer's instructions, and absorbances were read on a PowerWaveX microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT). Serum amiodarone and DEA metabolite concentrations were measured simultaneously using a liquid chromatographic assay with a limit of quantitation of 0.1 mg/l (Pollak, 1996). Amiodarone and DEA were extracted using a solid-phase technique and duplicate 20-µl injections made onto a Zorbax cyano analytical column at 45°C (Chromatographic Specialties, Brockville, ON, Canada). Mobile phase (phosphate buffer/methanol/acetonitrile, 40:37: 23, adjusted to pH 3.5) was pumped at 0.8 ml/min with detection at 241 nm.

Northern Blot Analysis. Total RNA was isolated from frozen liver using TRIzol reagent (Invitrogen, Burlington, ON, Canada), as per the manufacturer's instructions. Northern blot analysis was performed essentially as previously described (Sinal et al., 2000). Briefly, RNA (10 µg/sample) was separated by electrophoresis in a denaturing 1.1% agarose/0.22 M formaldehyde gel followed by transfer to Immobilon-Ny+ nylon membranes (Millipore Corporation, Billerica, MA) in 20x standard saline citrate buffer. After cross-linking with UV light and drying, the blots were hybridized with cDNA probes labeled with [³²P]dCTP by the random primer method. After hybridization overnight at 65°C in PerfectHyb buffer (Sigma-Aldrich), the blots were washed once with 2x standard saline citrate/0.5% SDS for 15 min at 65°C, followed by two identical washes for 30 min each. Blots were exposed to storage phosphor screens, and images were detected using a Storm 840 PhosphorImager (Amersham Biosciences Inc., Baie d'Urfé, QC, Canada); results were quantitated using ImageQuant 5.2 software (Amersham Biosciences Inc.). Plasmids containing murine (unless otherwise indicated) cDNA sequences for straight chain fatty acyl-CoA oxidase (ACOX), peroxisomal D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (BIEN), 3-ketoacyl-CoA thiolase (thiolase), cytochromes P450 CYP4A1 and CYP4A3, and -actin were generous gifts of Frank Gonzalez (National Cancer Institute, National Institutes of Health). Reverse transcription-polymerase chain reaction of mouse liver total RNA was used to generate liver-CPTI and CPTII, microsomal triglyceride transfer protein (MTTP), fatty acid translocase (FAT/CD36), mouse PPAR (mPPAR), and apolipoprotein A-I (ApoA-I) cDNA sequences. The identity of all sequences was verified by restriction mapping and sequencing.

Cell Culture and Toxicity Assays. HepG2 human hepatocellular carcinoma cells were maintained in a complete medium consisting of phenol red-free Dulbecco's modified Eagle's medium (Hyclone Laboratories, Logan, UT) supplemented with 4 mM L-glutamine (Hyclone Laboratories), 10% heat-inactivated charcoal-dextranstripped fetal bovine serum (Gemini Bio-Products, Woodland, CA), 1 mM sodium pyruvate (Sigma-Aldrich), and 100 U/ml penicillin-100 µg/ml streptomycin (Sigma-Aldrich) at 37°C in 95% air, 5% CO₂. Freshly isolated rat (Sprague-Dawley, male) primary hepatocytes were obtained in 96-well type I collagen-coated plates from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD) and were maintained in Hepatocyte Culture Medium (Cambrex Bio Science Walkersville, Inc.) at 37°C in 95% air, 5% CO₂. For assays of cell toxicity, HepG2 cells and primary rat hepatocytes were plated at a density of 13,700 cells/well and 40,000 cells/well, respectively, in 96-well plates and allowed to adhere for 18 to 24 h. Cells were treated by the addition of DMSO (0.05%, v/v, in media), amiodarone, or DEA (both in DMSO) at the indicated final concentrations. After 24 h of exposure to the test compounds, 20 µl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) reagent in phosphate-buffered saline (5 mg/ml) was added to each well. After 2 h at 37°C in 95% air, 5% CO₂, the wells were aspirated, and 100 µl of DMSO was added. The plates were then shaken for 5 min, and the absorption at 490 nm was measured with a PowerwaveX microplate spectrophotometer (Bio-Tek Instruments).

Reporter Gene Assays. The GenBank sequences encoding the nuclear receptor ligand-binding domain (LBD) of mouse (bases 945-1853 of NM_011144 [GenBank] ) and human (bases 711-1619 of NM 005036) PPAR were amplified by reverse transcription-polymerase chain reaction from mouse and human liver total RNA, respectively. The products were cloned into the BamHI/HindIII sites of pCMV-BD (Stratagene, La Jolla, CA) to produce the LBD-GAL fusion expression constructs pCMV-BD-mPPAR and pCMV-BD-hPPAR. The identity of the constructs was verified by restriction mapping and sequencing. HepG2 cells were plated at 200,000 cells/well in 12-well plates and transfected 18 to 24 h later with pFR-luc (0.35 µg), pBSK (0.24 µg), and pSV2--gal (0.35 µg) and either pCMV-BD-hPPAR or pCMV-BD-mPPAR (0.06 µg) using TransIT-LT1 transfection reagent (Mirus Corporation, Madison, WI) per the manufacturer's instructions. Cells were treated 18 to 24 h after transfection with DMSO (control) or the test compounds (clofibrate, amiodarone, or DEA) at the indicated concentrations. All test compounds were solubilized in DMSO and diluted in media to a maximum final concentration of 0.5% (v/v) DMSO. Between 18 and 24 h post-treatment, cells were harvested using Reporter Lysis Buffer (Promega, Madison, WI) as per the manufacturer's instructions. Cell lysates were assayed for luciferase activity using the Luciferase Assay System (Promega) and measured using a Luminoskan Ascent (Thermo Electron, Franklin, MA). All luciferase values were corrected for lysate -galactosidase activity assayed as previously described (Sinal et al., 2001) and measured colorimetrically on a PowerWaveX microplate spectrophotometer (Bio-Tek Instruments).

Histological Analysis. Ten-micrometer sections of liver frozen in Tissue-Tek OCT/20% sucrose (v/v) were fixed in 37% formaldehyde, stained with oil red O, and counterstained with Gills' hematoxylin. Histological analysis, image capture, and processing were done on a Zeiss Axiovert 200 microscope equipped with an AxioCam camera system (Zeiss Canada, Toronto, ON, Canada).

Statistics. All data were analyzed by the unpaired Student's t test (unpaired) using InStat 3 for Macintosh.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Amiodarone Treatment Causes Body Weight Loss and Organomegaly. To investigate the mechanism(s) of amiodarone-induced hepatotoxicity, groups of mice were administered various doses of amiodarone daily for a maximum of 7 days. Animal weights were monitored, daily, 4 days before the first treatment with amiodarone and every day thereafter for the duration of the treatment period. Treatment with the highest dose of amiodarone (150 mg/kg) caused a progressive loss of total body weight when compared with vehicle-treated mice (Fig. 1A). Measurement of liver and kidney weights revealed a statistically significant increase in kidney weight, but not liver weight, in amiodaronetreated (150 mg/kg) versus vehicle-treated mice, after 7 days (Fig. 1B). However, when normalized to total body weight, amiodarone-treated (150 mg/kg) mice exhibited approximately 40 and 75% greater liver/body weight and kidney/body weight ratios, respectively, when compared with vehicle-treated mice (Fig. 1C). The large decrease of total body weight (approximately 10%) for mice treated with the highest dose of amiodarone for 7 days indicated substantial toxicity and wasting in these animals. To reduce confounding secondary factors associated with severe toxicity and to examine the early events in amiodarone-induced hepatotoxicity, subsequent experiments were limited to using mice treated with various doses of amiodarone for 4 days. At this time, overt signs of toxicity were not apparent and loss of body weight was, on average, less than 2%. Administration of lower doses of amiodarone (10, 40, or 80 mg/kg) for up to 7 days did not significantly affect total body weight or organ/body weight ratios (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1. High-dose amiodarone treatment results in a loss of total body mass and organomegaly. A, effect of amiodarone on total body weight. Body weights of mice treated with either vehicle or amiodarone (150 mg/kg) were monitored beginning 4 days before treatment and every day thereafter until sacrifice 24 h after the final injection. The first treatment commenced on day 0. Data points are expressed as the percentage of body weight determined for each animal at the indicated time points compared with the initial value at day minus 4. Values shown are mean ± S.D., n = 8. B, organ weight, and C, organ to body weight ratio. Liver and kidney weights of mice were determined and normalized to total body weight 24 h after receiving either vehicle or amiodarone (150 mg/kg) i.p. once a day for 7 days. Values shown are mean ± S.D., n = 4. *, p < 0.05 versus vehicle; **, p < 0.01 versus vehicle; ***, p < 0.001 versus vehicle.

Disruption of Hepatic Lipid Homeostasis by Amiodarone. To determine the effect of amiodarone treatment on hepatic lipid concentrations, 10-µm liver sections prepared from vehicle- or amiodarone-treated (150 mg/kg) mice were stained with oil red O, a lipophilic red stain that is effectively solubilized by neutral lipids within cells. Histologic examination revealed the presence of numerous red-stained lipidcontaining vacuoles within the hepatocytes of mice treated with amiodarone (Fig. 2). In contrast, oil red O staining was essentially absent in liver sections prepared from vehicle-treated mice. Analysis of plasma lipids revealed that amiodarone (150 mg/kg) treatment resulted in a significant decrease of plasma triglycerides but no significant change in total cholesterol when compared with vehicle-treated mice (Fig. 3). Amiodarone treatment also produced a hypoglycemic response as indicated by a decrease in blood glucose concentration in drug- versus vehicle-treated mice. In contrast, plasma concentrations of total bile acids and AST activity levels (indicators of hepatic function and toxicity, respectively) were not affected by amiodarone treatment. Lower doses of amiodarone (10, 40, and 80 mg/kg) did not have a significant effect on plasma chemistry (data not shown).

View larger version (88K): [in this window] [in a new window]   Fig. 2. Amiodarone treatment results in hepatocellular microvesicular lipid accumulation. Representative photomicrograph of liver sections prepared from mice treated daily with vehicle or amiodarone (150 mg/kg) for 4 days. Sections (10 µm) were fixed in 37% formaldehyde followed by staining with oil red O and counterstaining with hematoxylin. Original magnification, 100x.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Amiodarone treatment reduces plasma triglycerides and glucose. Mice received daily injections of vehicle or amiodarone (150 mg/kg) for 4 days and were sacrificed 24 h after the last treatment. Plasma glucose, triglycerides, total cholesterol, AST, and bile acids were determined as described under Materials and Methods. Values are expressed as mean ± S.D., n = 5. *, p < 0.05 versus vehicle.

Dose-Dependent Toxicity of Amiodarone and DEA. The microvesicular lipid accumulation observed in the livers of amiodarone-treated mice, concomitant with an absence of significant increase in plasma total bile acids or AST activity values, suggested that substantial impairment of hepatic lipid homeostasis could occur in the absence of overt hepatotoxicity. To establish a dose-response relationship for toxicity, HepG2 cells and primary rat hepatocytes were treated with increasing concentrations of amiodarone and assayed for cell viability by the MTT assay. Amiodarone treatment for 24 h resulted in a dose-dependent toxicity with an apparent LD₅₀ of 43 µM for HepG2 cells (Fig. 4A) and 28 µM for primary rat hepatocytes (Fig. 4B). Although the LD₅₀ for primary rat hepatocytes was slightly lower compared with the LD₅₀ for HepG2 cells, both of these values were considerably greater than the mean plasma amiodarone concentration of 0.53 µM determined for mice treated with 150 mg/kg amiodarone for 4 days. The major metabolite of amiodarone in humans is DEA (Latini et al., 1984). Similar to the effects of amiodarone, DEA treatment caused a dose-dependent toxicity, but with a lower apparent LD₅₀ of 9 µM in HepG2 cells and 10 µM in primary rat hepatocytes.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Amiodarone and DEA are toxic to HepG2 cells and primary rat hepatocytes. HepG2 cells (A) and primary rat hepatocytes (B) were plated in 96-well plates and treated with amiodarone or DEA at the indicated concentrations. After 24 h, cells were assayed for cell viability using a standard MTT assay as described under Materials and Methods. The results are normalized to vehicle (DMSO) control and are expressed as mean ± S.D., n = 4.

Increased Expression of Hepatic PPAR Target Genes after Amiodarone Treatment. PPAR is a critical regulator of hepatic fatty acid oxidation, and activation of this receptor mediates the liver enlargement that occurs as a consequence of peroxisome proliferation upon fibrate exposure to rodents (Lee et al., 1995). As such, the hepatomegaly observed in mice treated with 150 mg/kg amiodarone for 7 days (Fig. 1C) and the microvesicular lipid accumulation observed in response to 150 mg/kg amiodarone treatment for 4 days led us to investigate the function of PPAR in these mice. To accomplish this, hepatic mRNA levels for several PPAR target genes encoding proteins critical for the metabolism and transport of hepatic fatty acids were determined. Genes investigated include those encoding ACOX, the initial, and rate-limiting, enzyme in the peroxisomal -oxidation of fatty acids; BIEN and thiolase, enzymes also involved in the peroxisomal -oxidation pathway; CYP4A1 and CYP4A3, microsomal cytochrome P450 enzymes that catalyze the -oxidation of fatty acids; CPTI and CPTII, both involved in mitochondrial uptake of long-chain fatty acids; MTTP, which is essential for the hepatic secretion of apolipoprotein B-containing lipoproteins; FAT/CD36, a scavenger receptor with numerous physiological functions including fatty acid uptake into the liver; and ApoA-I, the major apolipoprotein constituent of high-density lipoprotein (Staels et al., 1998; Mascaro et al., 1999; Wolfrum et al., 2001). Total RNA was isolated from the livers of mice treated with vehicle, 10 to 150 mg/kg amiodarone, or 200 mg/kg clofibrate, a prototypical PPAR ligand activator. Northern blot analysis demonstrated that amiodarone treatment caused a dose-dependent increase in the mRNA levels of a number of PPAR target genes when compared with vehicle-treated mice (Fig. 5, A and B). Although all of the genes exhibited variable levels of response, the greatest increases occurred for hepatic mRNA levels for thiolase, CYP4A1, and CYP4A3. In this respect, the qualitative effects of amiodarone treatment on the mRNA levels for several target genes were very similar in extent to that observed after treatment with the bona fide PPAR activator clofibrate.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Amiodarone treatment increases the hepatic mRNA levels of PPAR target genes. Mice received daily injections of vehicle or the indicated doses of amiodarone for 4 days and were sacrificed 24 h after the last treatment. Clofibrate treatment was used as a positive control for PPAR activation. Total hepatic RNA was prepared and analyzed by Northern blot analysis as described under Materials and Methods. All probes were derived from murine cDNA sequences except where indicated otherwise: A, ACOX, BIEN, thiolase, rat CYP4A1 (detects mouse CYP4a10), and rat CYP4A3 (mouse CYP4a14); B, CPTI, CPTII, MTTP, FAT/CD36, mPPAR, and ApoA-I. A cDNA probe for -actin was used as a loading control.

The similarity of the effects of high-dose amiodarone and clofibrate treatment on the expression of various PPAR target genes suggested that the hepatic responses to amiodarone treatment are mediated by this nuclear receptor. To address this question, PPAR knockout [PPAR(—/—)] and congenic C57BL/6 wild-type mice [PPAR(+/+)] were treated with amiodarone (150 mg/kg) or vehicle and used to isolate total hepatic RNA for Northern blotting. Consistent with the previous data, wild-type mice treated with amiodarone exhibited an increase in the hepatic mRNA levels for the PPAR target genes ACOX, BIEN, thiolase, CYP4A1, and CYP4A3 (Fig. 6). In contrast, PPAR(—/—) mice exhibited lower constitutive expression levels of all of these genes. Furthermore, upon treatment with amiodarone, no induction of the hepatic expression of these genes was observed.

View larger version (69K): [in this window] [in a new window]   Fig. 6. Induction of hepatic PPAR target genes requires a functional PPAR gene. PPAR-null [PPAR(—/—)] or wild-type [PPAR(+/+)] mice received daily injections of vehicle or 150 mg/kg amiodarone for 4 days and were sacrificed 24 h after the last treatment. Total hepatic RNA was prepared and analyzed by Northern blot analysis as described under Materials and Methods. All probes were derived from murine cDNA sequences except where indicated otherwise: ACOX, BIEN, thiolase, rat CYP4A1 (detects mouse CYP4a10), and rat CYP4A3 (mouse CYP4a14). A cDNA probe for -actin was used as a loading control.

Similar to wild-type, PPAR(—/—) mice exhibited a progressive loss of total body weight in response to daily injections of 150 mg/kg amiodarone (Fig. 7A). However, the extent of weight loss for the PPAR(—/—) mice was significantly greater compared with that for wild-type mice as measured at days 5 through 8 of the experimental protocol. Coincident with these time points, PPAR(—/—) mice exhibited increasingly overt signs of toxicity including an obvious drop in body temperature, dehydration, constipation, and lethargy. Although amiodarone-treated wild-type mice exhibited similar signs, they did not appear until the day of sacrifice and were generally much milder in comparison to those exhibited by PPAR(—/—) mice. Furthermore, in contrast to wild-type mice, PPAR(—/—) mice did not exhibit hepatomegaly in response to amiodarone treatment (Fig. 7B). Histological analysis of the livers of these mice indicated that there were no substantive differences in the extent of microvesicular lipid accumulation in the hepatocytes of amiodarone-treated wild-type compared with PPAR(—/—) mice (Fig. 7C). Similarly, analysis of serum lipids and glucose did not indicate any significant differences between amiodarone-treated wild-type and PPAR(—/—) mice (data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 7. Differential effects of amiodarone treatment on wild-type and PPAR(—/—) mice. A, total body weight. Body weights of wild-type mice treated with either vehicle or amiodarone (150 mg/kg) were monitored beginning 3 days before treatment and every day thereafter until sacrifice 24 h after the final injection. The first treatment commenced on day 0. Data points are expressed as the percentage of body weight determined for each animal at the indicated time points compared with the initial value at day minus 3. Values shown are mean ± S.D., n = 3; *, p < 0.05 versus PPAR(+/+). B, liver to body weight ratio. Liver weights of mice were determined and normalized to total body weight 24 h after receiving either vehicle or amiodarone (150 mg/kg) i.p. once a day for 7 days. *, p < 0.05 versus vehicle. C, liver histology. Representative photomicrograph of liver sections prepared from mice treated daily with amiodarone (150 mg/kg) for 4 days. Sections (10-µm) were fixed in 37% formaldehyde followed by staining with oil red O and counterstaining with hematoxylin. Original magnification, 100x.

Amiodarone Is Not a Ligand-Activator of PPARa. The experiments with PPAR(—/—) mice clearly demonstrated that the increase in hepatic mRNA levels for ACOX, BIEN, thiolase, CYP4A1, and CYP4A3 in response to amiodarone treatment was dependent upon the presence of a functional PPAR gene. To address the question of whether the molecular mechanism of action of amiodarone was similar to that of clofibrate, a reporter gene assay that measured the activation of PPAR by cognate ligands was used. The coding regions for the LBDs of the murine or human PPAR were cloned and expressed as fusion proteins with the yeast GAL4 DNA-binding domain by subcloning into pCMV-BD. Transient transfection of these expression constructs into HepG2 cells, along with an upstream activation sequence-driven luciferase reporter construct, allowed assessment of ligand-dependent transcriptional activity of PPAR. As shown in Fig. 8A, clofibrate treatment of HepG2 cells transfected with either the murine or human PPAR-GAL expression construct caused a robust (7- to 10-fold) increase in reporter gene activity. In contrast, no increase of reporter gene activity was observed for treatment with amiodarone at any of the tested concentrations (0.01–50 µM). To consider the possibility that in vivo metabolism of amiodarone may have resulted in the generation of a PPAR ligand, we also tested the response of transfected HepG2 cells to the major metabolite DEA using the reporter gene assay. Similar to the results for the parent compound, no increase in reporter gene activity was observed in response to treatment of transfected cells with DEA (Fig. 8B).

View larger version (18K): [in this window] [in a new window]   Fig. 8. Amiodarone and DEA do not activate PPAR in a ligand activation reporter assay. HepG2 cells were cotransfected with an upstream activation sequence-driven luciferase reporter construct (pFR-luc) and GAL-LBD expression constructs for mPPAR or human (hPPAR) isoforms. Luciferase activities were determined as described under Materials and Methods using cell lysates prepared after 24 h of exposure to vehicle (DMSO) or the indicated concentrations of amiodarone (A) or DEA (B). Clofibrate (CFB) was used as a positive control for both assays. The results were corrected for -galactosidase activity and normalized to vehicle (DMSO) control values. Luciferase values were below background levels for 30 µM (hPPAR) and 50 µM (hPPAR and mPPAR) DEA. Values are expressed as mean ± S.D., n = 4.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Amiodarone has been reported to inhibit the hepatic mitochondrial -oxidation of fatty acids and to produce microvesicular steatosis of the liver in mice (Fromenty et al., 1990). Consistent with these previous findings, the amiodaronetreated (150 mg/kg) mice in the present study exhibited clear and obvious hepatocyte microvesicular lipid accumulation. Given that oil red O is a general histological stain for neutral lipids rather than for specific classes of lipids (e.g., sterols, triglycerides, phospholipids), it is not possible to determine the composition of the lipids that accumulated within hepatocytes in response to amiodarone treatment. However, it is likely that inhibition of mitochondrial -oxidation, the major route of fatty acid catabolism in the liver, contributed to lipid accumulation observed in the livers of amiodarone-treated mice. Such accumulation of fatty acids and subsequent esterification to form triglycerides is believed to be an important early causative event in hepatic steatosis and the eventual development of steatohepatitis (Fromenty and Pessayre, 1995; Koteish and Diehl, 2001).

Analysis of various plasma chemistry parameters revealed additional evidence consistent with impaired lipid homeostasis. For example, amiodarone treatment resulted in a significant decrease of plasma triglycerides and glucose. The absence of effect of amiodarone on plasma total bile acids and AST activity indicates that hepatocyte microvesicular lipid accumulation and decreased plasma triglycerides preceded overt hepatotoxicity. This is consistent with the pattern seen in nonalcoholic liver disease in humans, which can manifest as steatohepatitis with advanced fibrosis in the absence of elevated serum alanine aminotransferase values (Mofrad et al., 2003). Also consistent is the observation that relatively high concentrations of amiodarone or DEA were required to cause substantial toxicity to HepG2 cells (LD₅₀ = 43 and 9 µM, respectively) and primary rat hepatocytes (LD₅₀ = 28 and 10 µM, respectively) in an acute (24-h) treatment model. This is much higher than the mean plasma amiodarone concentration (0.53 µM) determined for mice treated with 150 mg/kg amiodarone for 4 days. However, it is important to note that the toxicity of low-dose amiodarone may be manifest only after longer periods of exposure to the drug.

Recently, amiodarone was reported to inhibit MTTP function in mice (Letteron et al., 2003). MTTP is an essential protein involved in the lipidation of apolipoprotein B in the lumen of the endoplasmic reticulum to form triglyceride-rich very low-density lipoprotein (VLDL) particles. Fully lipidated VLDL particles follow the vesicular flow and are secreted into the plasma, whereas incompletely lipidated apolipoprotein B particles are retained and partially degraded. Thus, it is likely that the decrease in serum triglycerides caused by amiodarone treatment observed in the present study was caused, at least in part, by an inhibition of hepatic VLDL synthesis and secretion. Importantly, these data also indicate that amiodarone can induce hepatocyte microvesicular lipid accumulation through two mechanisms: inhibition of mitochondrial -oxidation of fatty acids, resulting in an increase in hepatic triglycerides, and inhibition of MTTP, causing a decrease in the hepatic secretion of triglycerides into the blood.

Mitochondrial fatty acid oxidation is the primary process in mice by which fatty acids are utilized to produce energy through the -oxidation of short (<C₈)-, medium (C_8–12)-, and long (C_12–20)-chain fatty acids (Reddy and Hashimoto, 2001). Consistent with a critical regulatory role in this process, PPAR expression is highest in tissues that possess a significant capacity for fatty acid catabolism including liver, heart, kidney, and skeletal muscle (Auboeuf et al., 1997; Escher et al., 2001). Endogenous fatty acids and their metabolites, particularly very-long (>C₂₀)-chain and polyunsaturated fatty acids, appear to be the most important endogenous PPAR ligands (Wahli et al., 1999). Hypolipidemic drugs of the fibrate class, including clofibrate, fenofibrate, bezafibrate, ciprofibrate, and gemfibrozil, are exogenous ligands that exert their beneficial actions via PPAR activation (Staels et al., 1998). Fibrates are also known as peroxisome proliferators due to the increase in size and number of hepatic peroxisomes (i.e., peroxisome proliferation) as well as hepatomegaly known to occur in rodents as a result of PPAR activation by these xenobiotics (Lee et al., 1995). Given the hepatomegaly and disruption of lipid homeostasis observed for amiodarone-treated mice, it was hypothesized that changes in the activation state of PPAR would be associated with amiodarone treatment. Consistent with this hypothesis, amiodarone treatment caused a dose-dependent increase in the mRNA levels of a number of known PPAR target genes encoding enzymes involved in the peroxisomal, mitochondrial, and microsomal oxidation of fatty acids. Interestingly, genes involved in fatty acid transport (FAT/CD36) as well as the gene encoding PPAR were also increased to varying degrees by amiodarone treatment.

The clear increase in hepatic expression for various PPAR target genes caused by amiodarone treatment suggested the involvement of this nuclear receptor as the mediator of this effect. To test this possibility, we used the well characterized PPAR(—/—) mouse model. These mice lack a functional PPAR gene and, thus, do not exhibit typical pleiotropic responses (i.e., hepatomegaly, peroxisome proliferation, and target gene induction) after exposure to PPAR ligands (Lee et al., 1995; Aoyama et al., 1998). Our experiments with PPAR(—/—) mice demonstrated that, in contrast to wild-type mice, amiodarone treatment did not increase expression of hepatic PPAR target genes. Consistent with these data, amiodarone-induced hepatomegaly was also abolished in PPAR(—/—) mice. These results indicate that increased hepatic expression of various genes involved in lipid homeostasis as well as the hepatomegaly associated with amiodarone treatment is absolutely dependent upon the presence of a functional PPAR gene. In this qualitative sense, the in vivo effects of amiodarone are similar to that for PPAR agonists such as fibrates. However, the inability of amiodarone or DEA to induce ligand-dependent expression of the reporter gene construct in the PPAR activation assay in a manner similar to that for clofibrate clearly indicates that the mechanisms of action are different. Rather, it is more likely that amiodarone increases the expression of PPAR target genes through an indirect mechanism related to a disruption of hepatic fatty acid metabolism.

Compared with wild-type mice, treatment of PPAR(—/—) mice with amiodarone was associated with a greater rate and extent of total body weight loss as well as more severe overt signs of toxicity. These data indicate that PPAR provides protection against the toxicity associated with high-dose administration of amiodarone. This finding is consistent with previous studies supporting a protective role for this receptor in models of methionine/choline-deficient (MCD) diet-induced steatohepatitis (Rao et al., 2002; Ip et al., 2003, 2004). Interestingly, amiodarone treatment caused a similar degree of microvesicular lipid accumulation in the livers of wild-type and PPAR(—/—) mice. This was somewhat surprising given reports that the potent PPAR agonist Wy-14,643 protects against hepatic lipid accumulation in the MCD diet-induced model of steatohepatitis (Ip et al., 2003, 2004). However, a number of important differences between our study and the previous work are notable. For example, the MCD diet has a markedly elevated fat content (20%) and was administered for approximately 5 weeks, resulting in signs of advanced steatohepatitis (severe macrovesicular steatosis, infiltration of inflammatory cells, fibrosis, and elevated serum AST). Thus, the source of hepatic lipid accumulation in this model is primarily exogenous. In contrast, the mice in our model were fed a normal rodent chow diet (7% fat) and exhibited only early signs of steatohepatitis (moderate microvesicular steatosis without elevated serum AST). Thus, there are a number of qualitative and quantitative differences between the present study and those published previously that confound direct comparison. Also of note, oil red O is a general stain for neutral lipids that provides no information regarding the identity of lipid. Thus, we cannot rule out qualitative differences in the amiodarone-induced microvesicular lipid accumulation in wild-type and PPAR(—/—) mouse liver.

Based upon the results of this study, we conclude that amiodarone causes a profound disruption of normal lipid homeostasis and that the increased expression of PPAR target genes is secondary to perturbation of normal fatty acid catabolism. The combined effects of amiodarone to inhibit mitochondrial -oxidation and decrease the secretion of hepatic triglycerides through MTTP inhibition probably constitute early events leading to microvesicular lipid accumulation within the hepatocyte. It is likely that this accumulated lipid consists, at least in part, of various fatty acids that are capable of ligand activation of PPAR. This leads to increased expression of PPAR target genes, many of which are involved in the oxidation of fatty acids and efflux of lipids from the hepatocyte. Thus, the indirect activation of PPAR by amiodarone most likely represents a homeostatic increase in the consumption of fatty acids in an attempt to decrease the accumulation of triglycerides in hepatocytes.

In contrast to most rodents, activation of human PPAR does not lead to significant increases in the expression of genes encoding hepatic enzymes of peroxisomal fatty acid -oxidation, nor does it cause peroxisome proliferation (Staels et al., 1995). Mitochondrial, rather than peroxisomal, -oxidation appears to be the major target of PPAR regulation of fatty acid metabolism in humans. However, in addition to the dramatic effects on peroxisomal -oxidation, constitutive levels of mitochondrial -oxidation are also reduced in the PPAR(—/—) mouse model (Aoyama et al., 1998), demonstrating the importance of this protein in overall fatty acid homeostasis. Furthermore, many of the beneficial actions of fibrates, including hypolipidemia and anti-inflammatory and antiatherosclerotic effects, are common to both humans and rodents (Xie et al., 2002; Francis et al., 2003a). Determination of whether the present findings are relevant to amiodarone-induced hepatotoxicity in humans requires additional study. However, the similarity between humans and mice with respect to the physiological role of PPAR suggests that this may indeed be the case.

	Acknowledgements

 
We are grateful to Drs. Ken Renton and Kerry Goralski for careful reading of the manuscript and Denise Kidson for excellent technical assistance.

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Nova Scotia.

doi:10.1124/jpet.104.072785.

ABBREVIATIONS: CPTI and CPTII, carnitine palmitoyltransferase I and II; ACOX, straight chain fatty acyl-CoA oxidase; ApoA-I, apolipoprotein A-I; AST, aspartate aminotransferase; BIEN, peroxisomal D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein; DEA, N-desethylamiodarone; DMSO, dimethyl sulfoxide; FAT/CD36, fatty acid translocase; GAL, galactosidase; LBD, ligand-binding domain; MCD, methionine/cholinedeficient; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; MTTP, microsomal triglyceride transfer protein; PPAR, peroxisome proliferator-activated receptor-; thiolase, 3-ketoacyl-CoA thiolase; VLDL, very low-density lipoprotein; m, murine; h, human; Wy-14,643, pirinixic acid; 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid.

Address correspondence to: Christopher J. Sinal, Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. E-mail: csinal{at}dal.ca.

	References

Top Abstract Materials and Methods Results Discussion References

 

Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, and Gonzalez FJ (1998) Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 273: 5678–5684.[Abstract/Free Full Text]
Auboeuf D, Rieusset J, Fajas L, Vallier P, Frering V, Riou JP, Staels B, Auwerx J, Laville M, and Vidal H (1997) Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients. Diabetes 46: 1319–1327.[Abstract]
Bolt MW, Card JW, Racz WJ, Brien JF, and Massey TE (2001) Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity. J Pharmacol Exp Ther 298: 1280–1289.[Abstract/Free Full Text]
Bonnefont JP, Demaugre F, Prip-Buus C, Saudubray JM, Brivet M, Abadi N, and Thuillier L (1999) Carnitine palmitoyltransferase deficiencies. Mol Genet Metab 68: 424–440.[CrossRef][Medline]
Burkart F, Pfisterer M, Kiowski W, Follath F, and Burckhardt D (1990) Effect of antiarrhythmic therapy on mortality in survivors of myocardial infarction with asymptomatic complex ventricular arrhythmias: Basel Antiarrhythmic Study of Infarct Survival (BASIS). J Am Coll Cardiol 16: 1711–1718.[Abstract]
Card JW, Lalonde BR, Rafeiro E, Tam AS, Racz WJ, Brien JF, Bray TM, and Massey TE (1998) Amiodarone-induced disruption of hamster lung and liver mitochondrial function: lack of association with thiobarbituric acid-reactive substance production. Toxicol Lett 98: 41–50.[CrossRef][Medline]
Doval HC, Nul DR, Grancelli HO, Perrone SV, Bortman GR, and Curiel R (1994) Randomised trial of low-dose amiodarone in severe congestive heart failure. Grupo de Estudio de la Sobrevida en la Insuficiencia Cardiaca en Argentina (GESICA). Lancet 344: 493–498.[CrossRef][Medline]
Escher P, Braissant O, Basu-Modak S, Michalik L, Wahli W, and Desvergne B (2001) Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding. Endocrinology 142: 4195–4202.[Abstract/Free Full Text]
Francis GA, Annicotte JS, and Auwerx J (2003a) PPAR agonists in the treatment of atherosclerosis. Curr Opin Pharmacol 3: 186–191.[CrossRef][Medline]
Francis GA, Fayard E, Picard F, and Auwerx J (2003b) Nuclear receptors and the control of metabolism. Annu Rev Physiol 65: 261–311.[CrossRef][Medline]
Fromenty B, Fisch C, Labbe G, Degott C, Deschamps D, Berson A, Letteron P, and Pessayre D (1990) Amiodarone inhibits the mitochondrial beta-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther 255: 1371–1376.[Abstract/Free Full Text]
Fromenty B and Pessayre D (1995) Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol Ther 67: 101–154.[CrossRef][Medline]
Gill J, Heel RC, and Fitton A (1992) Amiodarone. An overview of its pharmacological properties and review of its therapeutic use in cardiac arrhythmias. Drugs 43: 69–110.[Medline]
Ip E, Farrell G, Hall P, Robertson G, and Leclercq I (2004) Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice. Hepatology 39: 1286–1296.[CrossRef][Medline]
Ip E, Farrell GC, Robertson G, Hall P, Kirsch R, and Leclercq I (2003) Central role of PPARalpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice. Hepatology 38: 123–132.[CrossRef][Medline]
Kennedy JA, Unger SA, and Horowitz JD (1996) Inhibition of carnitine palmitoyltransferase-1 in rat heart and liver by perhexiline and amiodarone. Biochem Pharmacol 52: 273–280.[CrossRef][Medline]
Khairy P and Nattel S (2002) New insights into the mechanisms and management of atrial fibrillation. Can Med Assoc J 167: 1012–1020.[Abstract/Free Full Text]
Koteish A and Diehl AM (2001) Animal models of steatosis. Semin Liver Dis 21: 89–104.[CrossRef][Medline]
Latini R, Reginato R, Burlingame AL, and Kates RE (1984) High-performance liquid chromatographic isolation and fast atom bombardment mass spectrometric identification of di-N-desethylamiodarone, a new metabolite of amiodarone in the dog. Biomed Mass Spectrom 11: 466–471.[CrossRef][Medline]
Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H, and Gonzalez FJ (1995) Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15: 3012–3022.[Abstract]
Letteron P, Sutton A, Mansouri A, Fromenty B, and Pessayre D (2003) Inhibition of microsomal triglyceride transfer protein: another mechanism for drug-induced steatosis in mice. Hepatology 38: 133–140.[CrossRef][Medline]
Mascaro C, Acosta E, Ortiz JA, Rodriguez JC, Marrero PF, Hegardt FG, and Haro D (1999) Characterization of a response element for peroxisomal proliferator activated receptor (PPRE) in human muscle-type carnitine palmitoyltransferase I. Adv Exp Med Biol 466: 79–85.[Medline]
Mofrad P, Contos MJ, Haque M, Sargeant C, Fisher RA, Luketic VA, Sterling RK, Shiffman ML, Stravitz RT, and Sanyal AJ (2003) Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 37: 1286–1292.[CrossRef][Medline]
Pollak PT (1996) A systematic review and critical comparison of internal standards for the routine liquid chromatographic assay of amiodarone and desethylamiodarone. Ther Drug Monit 18: 168–178.[CrossRef][Medline]
Rao MS, Papreddy K, Musunuri S, and Okonkwo A (2002) Prevention/reversal of choline deficiency-induced steatohepatitis by a peroxisome proliferator-activated receptor alpha ligand in rats. In Vivo 16: 145–152.[Medline]
Reddy JK and Hashimoto T (2001) Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu Rev Nutr 21: 193–230.[CrossRef][Medline]
Richer M and Robert S (1995) Fatal hepatotoxicity following oral administration of amiodarone. Ann Pharmacother 29: 582–586.[Abstract]
Rigas B, Rosenfeld LE, Barwick KW, Enriquez R, Helzberg J, Batsford WP, Josephson ME, and Riely CA (1986) Amiodarone hepatotoxicity. A clinicopathologic study of five patients. Ann Intern Med 104: 348–351.
Shinagawa K, Shiroshita-Takeshita A, Schram G, and Nattel S (2003) Effects of antiarrhythmic drugs on fibrillation in the remodeled atrium: insights into the mechanism of the superior efficacy of amiodarone. Circulation 107: 1440–1446.[Abstract/Free Full Text]
Simon JB, Manley PN, Brien JF, and Armstrong PW (1984) Amiodarone hepatotoxicity simulating alcoholic liver disease. N Engl J Med 311: 167–172.[Medline]
Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, and Gonzalez FJ (2000) Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 102: 731–744.[CrossRef][Medline]
Sinal CJ, Yoon M, and Gonzalez FJ (2001) Antagonism of the actions of peroxisome proliferator-activated receptor-alpha by bile acids. J Biol Chem 276: 47154–47162.[Abstract/Free Full Text]
Spaniol M, Bracher R, Ha HR, Follath F, and Krahenbuhl S (2001) Toxicity of amiodarone and amiodarone analogues on isolated rat liver mitochondria. J Hepatol 35: 628–636.[CrossRef][Medline]
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, and Fruchart JC (1998) Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 98: 2088–2093.[Abstract/Free Full Text]
Staels B, Vu-Dac N, Kosykh VA, Saladin R, Fruchart JC, Dallongeville J, and Auwerx J (1995) Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Investig 95: 705–712.
Stravitz RT and Sanyal AJ (2003) Drug-induced steatohepatitis. Clin Liver Dis 7: 435–451.[CrossRef][Medline]
Wahli W, Devchand PR, IJpenberg A, and Desvergne B (1999) Fatty acids, eicosanoids and hypolipidemic agents regulate gene expression through direct binding to peroxisome proliferator-activated receptors. Adv Exp Med Biol 447: 199–209.[Medline]
Wolfrum C, Borrmann CM, Borchers T, and Spener F (2001) Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha- and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc Natl Acad Sci USA 98: 2323–2328.[Abstract/Free Full Text]
Xie Y, Yang Q, and DePierre JW (2002) The effects of peroxisome proliferators on global lipid homeostasis and the possible significance of these effects to other responses to these xenobiotics: an hypothesis. Ann NY Acad Sci 973: 17–25.[Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.072785v1 311/3/864    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McCarthy, T. C. Articles by Sinal, C. J. Search for Related Content PubMed PubMed Citation Articles by McCarthy, T. C. Articles by Sinal, C. J.