Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 Jul 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2007 May 3;222(2):169–179. doi: 10.1016/j.taap.2007.04.008

DIFFERENTIAL GENE EXPRESSION IN MOUSE LIVER ASSOCIATED WITH THE HEPATOPROTECTIVE EFFECT OF CLOFIBRATE1

Jeffrey S Moffit a, Petra H Koza-Taylor b, Ricky D Holland c, Michael S Thibodeau a, Richard D Beger c, Michael P Lawton b, José E Manautou a,2
PMCID: PMC1989769  NIHMSID: NIHMS28093  PMID: 17585979

Abstract

Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor α (PPARα) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARα-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARα-null mice pretreated with vehicle or CFB (500mg/kg, i.p., daily for 10 days) and then challenged with APAP (400mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring ® software. Our analysis identified 53 genes of interest including vanin-1, cell-cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

Keywords: peroxisome proliferators, clofibrate, acetaminophen, vanin-1, hepatoprotection, cysteamine, cystamine, pantothenic acid, pantetheine

Introduction

Peroxisome proliferators mediate their actions in the liver through binding and activating the peroxisome proliferator activated receptors (PPARs). Of the three identified PPAR isoforms, the α receptor (PPARα) is primarily expressed in liver and is responsible for induction of enzymes involved in uptake, metabolism, and β-oxidation of fatty acids. Clofibrate (CFB) and other peroxisome proliferators are known to protect the liver against the toxic actions of chemicals like acetaminophen (APAP) (Nicholls-Grzemski et al., 1992; Manautou et al., 1994). The precise mechanism of this hepatoprotective effect is not known.

Overexposure to APAP results in fulminant centrilobular hepatic necrosis, which can extend to the periportal regions of the liver lobule (Mitchell et al., 1973a). This drug is primarily metabolized by conjugation with sulfate and glucuronic acid, with a small percentage of the dose undergoing bioactivation by cytochrome P450 enzymes to the reactive intermediate N-acetylp-benzoquinoneimine (NAPQI) (Mitchell et al., 1973a). At non-toxic doses, NAPQI is eliminated from the liver after conjugation with reduced glutathione (GSH) (Mitchell et al., 1973). However, with toxic doses, the main conjugation pathways for APAP become saturated, resulting in increased formation of NAPQI. Consequently, detoxification of NAPQI is compromised when existing stores of GSH are depleted and NAPQI then binds to cellular macromolecules, initiating cell death pathways (Jollow et al., 1973). Administration of CFB does not alterAPAP bioactivation or conjugative pathways in mouse liver, and although GSH levels are increased following repeated CFB treatment, this is not the mechanism of resistance (Manautou et al., 1994; Nicholls-Grzemski et al., 2000b). Furthermore, overall biliary and urinary elimination of APAP metabolites is not changed by CFB, thus ruling out changes in hepatobiliary disposition as an explanation for the hepatoprotective effect of CFB (Manautou et al., 1996; Faig et al., 2000).

APAP toxicity is associated with increased production of reactive oxygen species and is often accompanied by lipid peroxidation (Wendel et al., 1979; Gerson et al., 1985). Therefore, the role of other detoxification and antioxidant enzymes has also been investigated. Changes in the activity of enzymes such as glutathione-s-transferase pi and catalase have been excluded as contributors to CFB-mediated hepatoprotection (Manautou et al., 1994; Chen et al., 2002). Previous studies from our laboratory demonstrated that mice lacking the PPAR α nuclear receptor are not protected by CFB treatment from APAP toxicity (Chen et al., 2000). This suggests that the gene(s) contributing to the hepatoprotective effect of CFB are controlled by PPAR αeither directly or indirectly.

To further investigate the mechanism of CFB-mediated hepatoprotection, we identified differentially expressed genes between wild type and PPAR α-null mice treated with vehicle or CFB (500mg/kg, i.p.), daily for 10 days, followed by a toxic challenge of APAP (400mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment, labeled, and hybridized to Affymetrix Murine Genome Array MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring ® software to determine differentially expressed genes (fold change≥1.5, p<0.05). The first comparison was between CFB and vehicle (VEH) pretreated wild type mice, challenged with APAP (WT_CFB_APAP – WT_VEH_APAP). The second comparison was between wild type and PPARα-null mice pretreated with CFB and also challenged with APAP (WT_CFB_APAP – KO_CFB_APAP). Differentially expressed genes that were common to both comparisons were further investigated for their role in CFB-mediated hepatoprotection. Genes linked to peroxisome proliferation, immune function, proteosomal degradation, and transcriptional regulation were most significantly modulated. Of interest, vanin-1 was induced 7-fold in CFB pretreated wild type mice compared to vehicle (WT_CFB_APAP – WT_VEH_APAP) and 52-fold in CFB pretreated wild type mice compared to PPARα-null mice receiving CFB (WT_CFB_APAP – KO_CFB_APAP). All groups of mice in these comparisons received APAP. Vanin-1 may be of importance to CFB-mediated hepatoprotection because it is responsible for cysteamine synthesis, a potent antioxidant capable of ameliorating APAP toxicity in rodents and humans.

Methods

Chemicals

All chemicals used were purchased from Sigma (St. Louis, MO) and were of reagent grade or better.

Animals

Wild type (+/+) and PPARα-null (−/ −) male sv/129 background mice aged 10–12 weeks were provided by Frank Gonzalez (National Institutes of Health, Bethesda, MD). Mice were housed in community cages with free access to water and feed (rodent diet No. 5001, PMI Feeds, St. Louis, MO) and maintained a 12-h dark/light cycle. Groups of mice (n = 3–4) received daily dosing of CFB (500mg/kg) or corn oil vehicle (5ml/kg), i.p. for 10 days. Mice were challenged with APAP (400mg/kg, p.o.) or 50% propylene glycol vehicle (5ml/kg). This dosing paradigm results in peroxisome proliferation and protection from APAP in wild type, but not PPAR α-null mice (Chen et al., 2000). Livers were removed at 4 hr after APAP dosing, snap frozen in liquid nitrogen, and stored at −80°C until assayed. The University of Connecticut Institutional Animal Care and Use Committee (IACUC) approved all experimental animal protocols.

Plasma Alanine Aminotransferase (ALT) Activity

Blood collected at 4 hr afterAPAP was centrifuged at 8,800xg for 5 min. Plasma was then analyzed for ALT activity using Infinity ALT Liquid Stable Reagent (Thermotrace, Melbourne, Australia) as directed by the manufacturer.

Histopathology

A portion of the liver was fixed in 10% phosphate-buffered formalin. Paraffin embedded sections (5μm) were stained with hematoxylin and eosin. Livers were evaluated by light microscopy and graded as previously reported (Bartolone et al., 1989) for the presence and severity of lesions. Grades range from 0–5 (0, no lesions; 1, single or few necrotic cells; 2, 10–25% necrotic cells or mild diffuse degenerative changes; 3, 25–40% necrotic or degenerative cells; 4, 40–50% necrotic or degenerative cells; 5, more than 50% degenerative cells). Histopathology scores greater than 2 indicate significant pathology (Chen et al., 2000).

RNA Isolation

Total tissue RNA was extracted using RNAzol B reagent (Tel-test Inc., Friendswood, TX) according to the manufacturer’s protocol. RNA integrity was confirmed by agarose gel electrophoresis through visualization of intact 18S and 28S rRNA.

Probe labeling

Total RNA (5μg) was used for cDNA synthesis. Superscript II (Invitrogen, Carlsbad, CA) was used to reverse-transcribe the RNA in the presence of a T7-containing oligo dT24 primer and second strand generated as recommended by Affymetrix (Santa Clara, CA). Resulting cDNA was purified using GeneChip sample clean-up modules and used as a template for in vitro transcription using the GeneChip One-Cycle Labeling Kit (Affymetrix). Generated cRNA was purified using GeneChip sample clean-up modules (Affymetrix). Quantity and purity of the cRNA was determined by absorbance at 260 nm and 260 nm/280 nm absorbance ratio, respectively. The quality of the cRNA was also evaluated on a 2100 BioAnalyzer (Agilent, Palo Akto, CA). The cRNA was fragmented as recommended by Affymetrix and quality of fragmentation verified by evaluating the sample on a 2100 BioAnalyzer.

Chip hybridization

Hybridization mix was prepared as recommended by Affymetrix. 10μg of labeled, fragmented cRNA was hybridized to individual MGU74v2.0 Murine GeneChips. The arrays were washed, stained, and scanned under low photomultiplier tube setting.

Differential Gene Array Analysis

Gene expression analysis was performed utilizing GeneSpring® software to determine differentially expressed genes (fold change ≥1.5, p<0.05). Normalization of gene expression data was processed using MAS 5.0. The primary gene expression comparisons for identifying genes of interest were WT_CFB_APAP – WT-VEH_APAP and WT_CFB_APAP – KO_CFB_APAP. Further comparisons were made to identify genes in control treated groups, which consisted of WT_VEH_VEH – KO_VEH_VEH, WT_CFB_VEH – WT_VEH_VEH, and WT_VEH_APAP – WT_VEH_VEH. Complete listings of these differentially expressed genes are present in Supplemental Table 2 through Supplemental Table 6.

Reverse Transcriptase-PCR Analysis of Hepatic Vanin-1 Expression

Total hepatic RNA (1 μg) was reverse transcribed and PCR amplified using the Promega Access RT-PCR kit. Gene specific mouse primers for vanin-1 were used as previously described (Berruyer et al., 2004). Relative amounts of cDNA synthesized were estimated by amplification of β-actin, using gene-specific primers (Xu et al., 2005). Vanin-1 and β-actin cDNA products were analyzed on agarose gels and quantified by densitometry using a KODAK Image Station 440CF (Eastman Kodak, New Haven, CT).

Reduced and Oxidized Liver Sample Preparation for ESI-LC/MS/MS Analysis

Liver homogenates were generated from 1g of liver in 4.5 ml of a reducing buffer consisting of 20mM dithiothreitol in 50mM ammonium carbonate buffer (pH8.3). Homogenates were incubated for 30 min at 60°C. Homogenates were further diluted (1:1 v/v) in an alkylating buffer to stabilize the free thiol groups. The alkylating buffer contained 200mM iodoacetamide in ammonium carbonate buffer, pH8.3. Oxidized liver homogenates were generated by diluting 1g of liver in 9ml of an alkylating buffer containing 200mM iodoacetamide in ammonium carbonate buffer, pH8.3. Ice cold acetonitrile was added to both oxidized and reduced homogenate samples (1:1 v/v). The tubes were placed at 5°C for 30 min. The samples were centrifuged at 13,000xg rpm for 10 min. Each sample was decanted and mixed in a LC/MS sample vial with 10mM ammonium formate buffer (1:1 v/v) for analysis of cysteamine and pantetheine. Another 10μL of each sample was decanted and mixed with 90μL of 0.1% formic acid for the analysis of pantothenic acid and cystamine.

HPLC-ESI-MS/MS Analysis

Chromatography was conducted with a capillary HPLC system from LC Packings/Dionex (Amsterdam, Netherlands) including an UltiMate quaternary pump and Famos autosampler. A Thermo Electron Corp. (Bellefonte, PA) Aquasil C 18 reversed-phase column (3μm particle size, 1mm x 150mm) was used for chromatography with a precolumn (1 mm x 10 mm) containing C18 reversed-phase resin. For alkylated cysteamine and pantetheine, HPLC mobile phase A consisted of 10mM ammonium formate and B was 10mM ammonium formate in 90% CH 3CN. The separation of alkylated cysteamine was done with a linear gradient starting at 90% A and 10% B to 100% B over 10 min at a flow rate of 50 μL/min. Separation of pantetheine was done by holding 2% B for 1 min then using a linear gradient to 100% B over 9 min. For pantothenic acid analysis, mobile phase A consisted of 0.1% formic acid and B consisted of 10% water, 0.1% formic acid, and 89.9% acetonitrile. Separation was done by holding 2% B for 1 min then using a linear gradient to 100% B over 9 min. A 2 μL sample injection volume was used. Analyte detection and quantification was done by ESI-MS/MS with a Waters Micromass Quattro Ultima triple quadrupole mass spectrometer (Manchester, U.K.). Quantitative analysis was done in positive ionization mode using the SRM transitions [M+H]+ →[M+H - 17]+ and [M+H]+ → [M+H - 34]+ with collision energies of 10 and 18V respectively for alkylated cysteamine. Pantetheine analysis utilized SRM transitions of [M+H] + → [M+H - 18]+ and [M+H]+ → [M+H - 130]+ with collision energies of 13V for both transitions. The same transitions were used for pantothenic acid with collision energies of 14V. The dwell time for alkylated cysteamine was set at 1 sec and 0.5 sec for pantothenic acid and pantetheine. The capillary voltage was set at 2.92 kV, the cone voltage was set at 36 V, hexapoles 1 and 2 were set at 18.1 and 0 V, respectively. The source and desolvation temperatures were 120 and 350°C. The cone gas flow rate was 99 L/h and the desolvation gas was set at 287 L/h. Argon was used as the collision gas and set at a pressure of 2.55 mTorr. Quantification was made by comparison to a calibration curve prepared by injecting standards at several concentrations.

Statistical Analysis

Results are expressed as means ± standard error (SE) for 3 to 4 mice per treatment group. Relative gene expression was compared using a student’s t-test or ANOVA and Tukey's post hoc test where appropriate. Differences were considered significant at p<0.05. A 1.5-fold change threshold was set for identifying significant changes in gene expression, as previously described (Anderson et al., 2004).

Results

Clustering strategy for differentially expressed genes in CFB-mediated hepatoprotection

To investigate PPARα-dependent pathways linked to the hepatoprotective effect of CFB, we identified differentially expressed genes between wild type (WT) and PPARα-null mice (KO) treated with vehicle (VEH) or CFB (500mg/kg) for 10 days, followed by a toxic challenge ofAPAP. Although the administered dose of APAP (400mg/kg) is considered hepatotoxic (Chen et al., 2000), 4h is not sufficient enough time to detect significant increases in plasma ALT activity (Supplemental Figure 1) or noticeable histopathology under the dosing conditions used in this study (Supplemental Table 1). The 4h time point was selected to evaluate genes modulated by CFB and APAP treatment, rather than those associated with overt pathology at later times after toxicant treatment. RNA was isolated from these livers, labeled, and hybridized to individual Affymetrix MGU74v2.0 murine GeneChips for transcriptional analysis. We selected a 1.5-fold threshold (p<0.05) for assessing differentially expressed genes as previously done by Anderson et al. (2004) in another mouse gene array study for peroxisome proliferators. The first differential analysis of genes was between WT_CFB_APAP and WT_VEH_APAP mice. This comparison was performed to identify genes modulated by the combination of CFB and APAP treatments. There were 409 differentially expressed genes in this comparison (Supplemental Table 2).

We previously showed that PPARα-null mice pretreated with CFB are not protected from APAP hepatotoxicity (Chen et al., 2000). Therefore, we compared gene expression profiles between WT_CFB_APAP and KO_CFB_APAP mice. This comparison was made to exclude changes in gene expression resulting from the combination of CFB and APAP treatment independent of PPARα activation. Similar gene sorting restrictions (1.5-fold change threshold and p<0.05) resulted in 1026 differentially expressed genes (Supplemental Table 3). Theoretically, gene(s) associated with the hepatoprotective effect of CFB are shared by these two comparisons. We focused our investigation on the 53 genes that were differentially expressed at the intersection of these comparisons (Figure 1). These 53 genes of interest were assessed for consistency across the biological replicates by heat map analysis (Figure 2).

Figure 1.

Figure 1

Open in a new tab

Clustering analysis of gene(s) associated with PPAR α-mediated hepatoprotection. GeneSpring® analysis identified 409 genes differentially expressed between vehicle and CFB pretreated wild type mice, challenged with APAP (WT_CFB_APAP – WT_VEH_APAP). 1026 genes were differentially expressed between wild type and PPAR α-null mice pretreated with CFB and challenged with APAP (WT_CFB_APAP – KO_CFB_APAP). Common genes differentially expressed in both comparisons were identified as genes of interest. These 53 genes were considered for their possible role in hepatoprotection. Only genes differentially expressed with p<0.05 and a fold change ≥1.5-fold were considered significant.

Figure 2.

Figure 2

Open in a new tab

Heat map representation of normalized, log-transformed relative intensities from the 53 genes identified for their role in PPARα-mediated hepatoprotection. Individual samples from WT_CFB_APAP – WT_VEH_APAP comparisons were normalized to the mean of WT_VEH_APAP (left box), using GeneSpring ® software. Similarly, individual samples from WT_CFB_APAP – KO_CFB_APAP comparisons were normalized to the mean of KO_CFB_APAP (right box).

Further comparisons of differentially expressed genes were made to assess changes among control groups. These comparisons included inherent differences between the WT and KO mice receiving vehicle only (WT_VEH_VEH – KO_VEH_VEH) (Supplemental Table 4), gene changes associated with CFB treatment alone in WT mice (WT_CFB_VEH – WT_VEH_VEH) (Supplemental Table 5), and the effect of APAP on gene expression (WT_VEH_APAP – WT_VEH_VEH) (Supplemental Table 6).

Differentially Regulated Genes

Table 1 shows that the vast majority of down-regulated genes are associated with immune function, including mannose-binding lectin protein c (MBL2), zeta-chain TCR associated protein kinase (ZAP70), and lymphocyte antigen 4 homolog (LY64). Decreased expression of these genes suppresses pathogen recognition, T cell signaling, and B cell proliferation, respectively (Miura et al., 1998; Dean et al., 2005; Yokosuka et al., 2005). Jun B proto-oncogene (JunB) was down-regulated approximately 11-fold. Although JunB has several cellular functions, down-regulation of this gene effectively increases c-Jun regulated cellular proliferation (Shaulian and Karin, 2001). The most significantly down-regulated gene was early growth response 1 (EGR1), which is involved in signal transduction, regulation of cell proliferation, and initiation of apoptosis (reviewed by Adamson and Mercola, 2002). Other genes regulating transcription, such as inhibitor of DNA binding 1 (ID1) and zinc finger protein 68 (ZAP68) were also down-regulated. Another gene of interest is aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme involved in ethanol metabolism and detoxification of aldehydes (Siew et al., 1976). ALDH2 is also a major target for APAP covalent binding (Landin et al., 1996).

Table 1.

Identified genes of interest investigated for their role in PPAR α-mediated protection.

Down-Regulated Genes of Interest in CFB-mediated Hepatoprotection
Gene List Abbreviation GenBank Designation Affymetrix Probeset Identification WT_CFB_APAP -WT_VEH_APAPFold Change WT_CFB_APAP -KO_CFB_APAPFold Change Gene Description Gene Function
EGR1 M28845 98579_at −13.18 −19.89 Early growth response 1 Transcriptional Regulation/ Cellular Proliferation
JUNB U20735 102362_i_at −9.48 −11.58 Jun B proto-oncogene Cell Growth and Differentiation
MCRS1 AF015309 98563_f_at −4.56 −4.80 Microspherule protein 1 Transcriptional Repression
PLK2 M96163 92310_at −4.46 −4.87 Polo-like kinase 2 (Drosophila) Cell Cycle Regulation
HNRPR AW123032 97491_at −4.40 −3.20 Heterogeneous nuclear ribonucleoprotein R Developmental
IFIT4 U43086 93956_at −3.39 −4.19 Interferon-induced protein with tetratricopeptide repeats 4 Immune Function
ID1 M31885 100050_at −3.05 −2.49 Inhibitor of DNA binding 1, dominant negative helix-loop-helix Transcriptional Regulation
HERPUD1 AI846938 95057_at −2.93 −2.70 Homocysteine-inducible, endoplasmic reticulum stress- inducible, ubiquitin-like domain member 1 Acute phase response/ protein degradation
SERPINA3K D00725 92583_at −2.79 −2.46 Serine (or cysteine) proteinase inhibitor, clade A, member 3K Acute phase protein
ZAP70 U04379 93661_at −2.89 −3.39 Zeta-chain (TCR) associated protein kinase 70kDa Immune Function
ZFP68 AB024005 95521_s_at −2.64 −1.98 Zinc finger protein 68 Transcription Repression
BAG3 AI643420 96167_at −2.60 −2.58 BCL2-associated athanogene 3 Apoptosis
GPLD1 AF050666 95566_at −2.56 −1.90 Glycosylphosphatidylinositol specific phospholipase D1 Lipid Metabolism
ALDH2 AI647493 96057_at −2.54 −2.33 Aldehyde dehydrogenase 2 family (mitochondrial) Lipid Metabolism / Aldehyde Detoxification
MAL Y07812 99089_at −2.49 −3.19 Mal, T-cell differentiation protein Membrane transport/ Apoptosis
LHB U25145 101738_at −2.47 −2.58 Luteinizing hormone beta polypeptide Reproduction
IFITM3 AW125390 160253_at −2.43 −3.10 Interferon induced transmembrane protein 3 (1-8U) Developmental
LY64 J04634 98000_at −2.43 −2.59 Lymphocyte antigen 64 homolog, radioprotective 105kDa Immune System
MAFK D42124 102919_at −2.28 −2.45 v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K Negatively Regulates Antioxidants
APCS M23552 104072_at −2.14 −3.01 Amyloid P component, serum Acute Phase Protein
MBL2 U09016 97427_at −1.97 −2.59 Mannose-binding lectin (protein C) 2, soluble (opsonic defect) Immune System
F13B D10071 97435_at −1.97 −1.72 Coagulation factor XIII, B polypeptide Clotting
TTR D00073 95350_at −1.95 −2.19 Transthyretin (prealbumin, amyloidosis type I) Albumin Production
DKK1 AF030433 99881_at −1.93 −2.43 Dickkopf homolog 1 (Xenopus laevis) Development
MAIL AA614971 98988_at −1.79 −1.95 Molecule possessing ankyrin repeats induced by lipopolysaccharide (MAIL), homolog of mouse Immune System
SERPIND1 U07425 100966_at −1.79 −1.64 Serine (or cysteine) proteinase inhibitor, clade D (heparin Acute phase protein
SPIB U87619 93388_s_at −1.73 −1.60 Spi-B transcription factor (Spi-1/PU.1 related) Immune System
ENPEP M29961 102373_at −1.71 −1.99 Glutamyl aminopeptidase (aminopeptidase A) Immune System
ARHB X99963 101030_at −1.63 −1.55 Ras homolog gene family, member B Actin cytoskeleton
CHRNB2 X82655 97010_at −1.53 −1.87 Cholinergic receptor, nicotinic, beta polypeptide 2 (neuronal) Neurologic Signaling
CD5L AF011428 93445_at −1.51 −2.07 CD5 antigen-like (scavenger receptor cysteine rich family) Immune System/ Transcriptional Regulation
Up-Regulated Genes of Interest in CFB-mediated Hepatoprotection
Gene Abbreviation GenBank Designation Affymetrix Probeset Identification WT_CFB_APAP -WT_VEH_APAPFold Change WT_CFB_APAP -KO_CFB_APAPFold Change Gene Description Gene Function
LAMB3 U43298 92759_at 37.80 34.62 Laminin, beta 3 Cellular Adhestion
EHHADH AJ011864 97316_at 18.83 46.89 Enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A Peroxisome Proliferation and Lipid Metabolism
VNN1 AJ132098 104165_at 6.79 54.09 Vanin-1 Immune System/ Lipid Metabolism/ Oxidative Stress
CD36 L23108 93332_at 5.87 7.79 CD36 antigen (collagen type I receptor, thrombospondin Cell Adhesion/ Lipid Metabolism
PLTP U28960 100927_at 4.65 2.93 Phospholipid transfer protein Peroxisome Proliferation/ Lipid Metabolism
GPD1 M25558 161753_f_at 4.50 1.63 Glycerol-3-phosphate dehydrogenase 1 (soluble) Peroxisome Proliferation/ Lipid Metabolism
ACOX1 AF006688 101515_at 3.37 7.73 Acyl-Coenzyme A oxidase 1, palmitoyl Peroxisome Proliferation/ Lipid Metabolism
DBI X61431 97248_at 3.03 2.70 Diazepam binding inhibitor (GABA receptor modulator, acyl- Coenzyme A binding protein) Peroxisome Proliferation/ Lipid Metabolism
CSNK1D AI846289 97264_r_at 2.95 5.81 Casein kinase 1, delta Microtubule Formation
CYCS X01756 98132_at 2.04 2.31 Cytochrome c, somatic Oxidative Phosphorylation/ Apoptosis
ABCD3 L28836 93045_at 2.25 4.42 ATP-binding cassette, sub-family D (ALD), member 3 Peroxisome Proliferation/ Lipid Metabolism
ME1 J02652 101082_at 1.95 4.63 Malic enzyme 1, NADP(+)-dependent, cytosolic Oxidative Phosphorylation/ Peroxisome Proliferation
TGOLN2 D50032 93882_f_at 1.90 3.80 Trans-golgi network protein 2 Membrane Trafficking
ACADL U21489 95425_at 1.81 2.02 Acyl-Coenzyme A dehydrogenase, long chain Peroxisome Proliferation/ Lipid Metabolism
PTTG1IP AW047951 94473_at 1.78 1.89 Pituitary tumor-transforming 1 interacting protein Cell Cycle Regulation
CCNI AF005886 94819_f_at 1.76 2.00 Cyclin I Cell Cycle Regulation
FABP4 M20497 100567_at 1.75 4.07 Fatty acid binding protein 4, adipocyte Peroxisome Proliferation/ Lipid Metabolism
PSMB4 U65636 98557_f_at 1.69 1.83 Proteasome (prosome, macropain) subunit, beta type, 4 Ubiquitination/ Proteosomal Degradation
MPP1 U38196 97803_at 1.68 2.96 Membrane protein, palmitoylated 1, 55kDa Tumor Suppression
YWHAB AF058797 98053_at 1.63 1.50 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide Signal Transduction
ACADM U07159 92581_at 1.61 2.33 Acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain Peroxisome Proliferation/ Lipid Metabolism
PSMA3 AF055983 92544_f_at 1.60 1.53 Proteasome (prosome, macropain) subunit, alpha type, 3 Ubiquitination/ Proteosomal Degradation
Open in a new tab

The majority of up-regulated genes were associated with peroxisome proliferation and lipid metabolism. These include enoyl-Coenzyme A hydratase (EHHADH), malic enzyme 1 (ME1), and acyl-Coenzyme A oxidase 1 (ACOX1). Two genes linked to proteosomal degradation pathways were also up-regulated approximately 2-fold, proteosomal subunitβ-type 4 (PSMB4) and proteosome subunitα-type 3 (PSMA3). Induction of these genes by peroxisome proliferators has been suggested to contribute to resistance from oxidative stress in the liver by eliminating damaged proteins (Anderson, 2004). Of interest, vanin-1 was one of the most dramatically up-regulated genes in both comparisons of differentially expressed genes. Vanin-1 is a glycosylphosphatidylinositol-anchored enzyme with pantetheinase activity, capable of hydrolyzing pantetheine into pantothenic acid (vitamin B 5) and cysteamine (2-mercapturate) (Figure 3). While vanin-1 is most commonly associated with recycling pantetheine from coenzyme A and acyl carrier proteins in the liver, de novo generation of cysteamine by vanin-1 may have a role in hepatoprotection. Cysteamine can exist in a reduced monothiol form or as the dithiol cystamine. Cysteamine and cystamine are potent antioxidants capable of ameliorating APAP toxicity when administered exogenously to rodents or humans (Prescott et al., 1974; Olinescu et al., 1982b; MacDonald et al., 1983; Miller and Jollow, 1986). The higher levels of vanin-1 gene transcript expression were further investigated in wild type and PPAR α-null mice following CFB administration (WT_CFB_APAP, KO_CFB_APAP).

Figure 3.

Figure 3

Open in a new tab

Schematic of vanin-1 pantetheinase activity. Hydrolysis of pantethenine is catalyzed by vanin-1 forming pantothenic acid and cysteamine. The reduced, monothiol cysteamine is also capable of existing in an oxidized form, the dithiol cystamine.

Gene expression of vanin-1

Gene array results show that CFB administration increased vanin-1 gene expression (7-fold) between WT_CFB_APAP and WT_VEH_APAP mice (Figure 4). The differential increase in vanin-1 was even more dramatic (53-fold) between the WT_CFB_APAP and KO_CFB_APAP groups. Greater differential expression of vanin-1 gene between wild type and PPAR α-null mice pretreated with CFB is due to lower basal levels in null mice. These findings were confirmed by RT-PCR analysis (Figure 5). Hepatic RNA was amplified and analyzed for vanin-1 using gene-specific mouse primers (Pitari et al., 2000). RT-PCR analysis showed a 2.5-fold increase in vanin-1 gene expression between WT_CFB_APAP and WT_VEH_APAP. Similarly, vanin-1 gene expression was increased 22-fold between WT_CFB_APAP and KO_CFB_APAP. No significant changes in β-actin gene expression were detected, confirming equivalent RNA loading.

Figure 4.

Figure 4

Open in a new tab

Vanin-1 gene array expression analysis in vehicle and CFB pretreated wild type mice, challenged with APAP (WT_VEH_APAP, WT_CFB_APAP). Similar analysis was conducted in PPARα-null mice (KO_VEH_APAP, KO_CFB_APAP). RNA isolated from both groups of mice was labeled and hybridized to Affymetrix GeneChips as described under the methods section. Results of vanin-1 gene expression were analyzed by GeneSpring ® software and expressed as mean optical density ± SE for 3 to 4 mice per treatment group. Asterisks (*) denote a statistical difference from control and daggers (†) indicate a statistical difference from wild type (p<0.05).

Figure 5.

Figure 5

Open in a new tab

Confirmation of vanin-1 gene array expression by RT-PCR analysis in vehicle and CFB pretreated wild type mice, challenged with APAP (WT_VEH_APAP, WT_CFB_APAP). Similar analysis was conducted in PPAR α-null mice (KO_VEH_APAP, KO_CFB_APAP). RNA from individual samples was amplified and analyzed for vanin-1 gene expression using gene specific mouse primers. The optical density (O.D.) of bands was quantified with a Kodak® Digital Science Image Station and expressed as mean O.D. x mm 2 ± SE for 3 to 4 mice per treatment group. Asterisks (*) denote a statistical difference from vehicle and daggers (†) indicate a statistical difference from wild type (p<0.05).

Intracellular hepatic concentrations of vanin-1 substrate and products

ESI-LC/MS/MS analysis of liver extracts was conducted to determine the intracellular concentrations of pantetheine, pantothenic acid, cysteamine, and cystamine in control and CFB treated WT and null mice without APAP treatment (WT_VEH, WT_CFB, KO_VEH, KO_CFB) (Figure 6). This allowed us to assess changes in hepatic levels of the enzymatic products of vanin-1 by CFB treatment prior to APAP exposure. Hepatic pantetheine levels were elevated in WT_CFB mice compared to vehicles; however, this was not statistically significant. By contrast, pantothenic acid was significantly increased in WT_CFB mice in comparison to WT_VEH (2.4-fold) and KO_CFB mice (3.6-fold). Whereas no significant changes were seen in cysteamine concentrations with any of the treatment groups, the dithiol cystamine was significantly increased (5.3-fold) in WT_CFB mice. Cystamine concentrations did not increase in KO_CFB mice. Since the mechanism by which cysteamine and cystamine elicit protection from APAP is not known, it is unclear how the ratio of cysteamine/cystamine impacts hepatoprotection.

Figure 6.

Figure 6

Open in a new tab

ESI-LC/MS/MS analysis of mouse liver for pantetheine, pantothenic acid, cystamine, and cysteamine following CFB administration. Liver extracts were generated and analyzed as described in the methods section from wild type (WT) andPPARα-null (KO) mice treated with vehicle (VEH) or CFB. These mice did not receive an APAP challenge. Results are expressed as nmoles/g of liver ± SE for 3 to 4 mice per treatment group. Statistical differences (p<0.05) from vehicle and wild type are marked by asterisks (*) and daggers (†), respectively.

Discussion

The hypolipidemic drug CFB has been used extensively to investigate the mechanisms of hepatoprotection provided by peroxisome proliferators against APAP and other hepatotoxicants. Currently, the precise mechanism has not been fully elucidated. An important clue comes from previous studies demonstrating that mice lacking the PPAR α nuclear receptor are refractory to the protective effects of CFB (Chen et al., 2000), implicating PPARα activation in hepatoprotection. In the present study, we used gene array analysis to identify differentially expressed genes that might contribute to the hepatoprotective effects of CFB. Two comparisons were made: the first one compared differentially expressed genes between CFB and vehicle pretreated wild type mice challenged with APAP (WT_CFB_APAP – WT_VEH_APAP). The second one compared changes in gene expression between wild type and PPAR α-null mice pretreated with CFB and challenged with APAP (WT_CFB_APAP – KO_CFB_APAP). We focused our investigation on the 53 common genes with similar regulation in these comparisons.

The majority of down-regulated genes within this group of common genes were associated with immune function. Given the increasing link between immune function and susceptibility to APAP toxicity (Liu et al., 2004; James et al., 2005), it is tempting to speculate that modulation of multiple genes linked to immune function may contribute to CFB-mediated hepatoprotection. However, primary hepatocytes isolated from mice dosedin vivo with CFB are also resistant to APAP toxicity (Nicholls-Grzemski, 2000). Hepatocyte isolation for culturing purposes removes a significant portion of non-parenchymal cells, thus reducing the likelihood that a change in immune function (e.g. cytokines originating from Kupffer cells) is a primary mechanism responsible for CFB-mediated hepatoprotection.

Enhanced cell proliferation is another explanation for hepatoprotection by accelerating tissue repair following chemical injury (reviewed by Mehendale, 2005). This mechanism has been suggested to contribute to CFB-mediated hepatoprotection (reviewed by Mehendale, 2000). The most dramatically down-regulated gene within the set is Egr-1, an important transcription factor modulating cellular proliferation by controlling mitotic cell progression. However, it is unlikely that down regulation of Egr-1 increases cellular proliferation because mice deficient in Egr-1 have impaired hepatic regeneration (Liao et al., 2004). Down regulation of other genes negatively regulating transcription (MCRS1, ZFP68) and inhibitors of cell growth (JunB) may impact hepatoprotection via hypertrophy and hyperplasia associated with peroxisome proliferation.

Although the events involved in the progression of APAP toxicity are not entirely known, the degree of APAP covalent binding to target hepatic proteins is directly pr oportional to the severity of toxicity (Jollow et al., 1973). Therefore, changes in the expression of APAP binding proteins may modulate toxicity. One such APAP binding protein, ALDH2, was among the down regulated genes identified in our analysis. Although the biological significance of APAP covalent binding to ALDH2 is unknown, adduct formation inhibits the activity of the enzyme (Landin et al., 1996). Presently, it is unclear how altered expression of ALDH2 may impact peroxisome proliferator-mediated hepatoprotection.

A similar study of genes differentially expressed between wild type and PPARα-null mice treated with the peroxisome proliferator Wy14,643 showed significant induction of proteasome maintenance genes (Anderson et al., 2004). The proteasome carries out ubiquitin-dependent and independent proteolysis of damaged and misfolded proteins (Goldberg, 2003). Enhanced proteasomal degradation has been suggested to contribute to peroxisome proliferator-mediated hepatoprotection (Anderson et al., 2004). Two of these proteasomal genes were also significantly up-regulated in the present analysis (PSMA3 and PSMB4). Although enhanced proteosomal activity can be beneficial in removing denatured proteins, one must consider the well-established mechanistic features of the hepatoprotection afforded by CFB. Treatment for 10 days with CFB prevents APAP covalent binding and GSH depletion. This suggests that NAPQI is being neutralized prior to causing GSH depletion and binding to or oxidizing thiols in hepatic proteins. Therefore, it seems that an enhanced removal of damaged or adducted proteins through ubiquination/proteasomal pathways is not critical to the hepatoprotective response resulting from multiple dosing with CFB. It is worth noting that a single dose of CFB provides partial protection against APAP hepatotoxicity. In contrast to the 10 day dosing regimen, a single dose does not alter GSH depletion or protein binding byAPAP. Under this scenario, enhanced proteosomal degradation of modified proteins should play a more prominent role in reducing the severity of APAP toxicity.

Vanin-1 was one of the most significantly up-regulated genes in our analysis. We hypothesized that increased vanin-1 expression translates into elevated hepatic cysteamine and cystamine levels. Interestingly, only dithiol cystamine concentrations were significantly increased in wild type mice pretreated with CFB. Although exogenous administration of cysteamine and cystamine are both hepatoprotective, it is unclear how the redox state of these compounds may affect hepatoprotection. Even though little is known about the equilibrium between cysteamine and cystamine, the present study suggests a dynamic relationship. Given the low mRNA expression of hepatic vanin-1 in KO_CFB mice, correspondingly low levels of hepatic cysteamine would be anticipated. However, PPARα-null mice had hepatic cysteamine levels comparable to wild type, suggesting a greater complexity in hepatic cysteamine regulation.

Both cysteamine and cystamine are potent antioxidants capable of ameliorating APAP toxicity when administered exogenously to rodents or humans (Prescott et al., 1974; Olinescu et al., 1982a; MacDonald et al., 1983; Miller and Jollow, 1986). There are several striking similarities between the protection afforded by cysteamine/cystamine and peroxisome proliferator-mediated hepatoprotection. First, several peroxisome proliferators have been reported to induce vanin-1 gene expression including di(2-ethylhexyl) phthalate (DEHP), Wy-14,643, and fenofibrate (Wong and Gill, 2002; Yamazaki et al., 2002). This observation is important because multiple peroxisome proliferators protect from APAP hepatotoxicity (Nicholls-Grzemski et al., 1992; Nguyen et al., 1999). In addition to its antioxidant properties, cysteamine decreases covalent binding of APAP to target hepatic proteins and reduces lipid peroxidation (Tredger et al., 1980; Stroo et al., 1985), as also seen with CFB-mediated hepatoprotection (Manautou et al., 1994; Nicholls-Grzemski et al., 2000a). Furthermore, primary hepatocytes isolated from mice treated in vivo with CFB are resistant to APAP, even with depletion of GSH by diethyl maleate (Nicholls-Grzemski et al., 2000b). Cysteamine similarly protects mice from APAP toxicity, even when GSH is depleted by butathione sulfoximine pretreatment (Miners et al., 1984). The protection afforded by cysteamine/cystamine is not limited to APAP; it also protects in vivo against toxic exposure to carbon tetrachloride and γ-irradiation (Brucer and Mewissen, 1957; Castro et al., 1973). Interestingly, CFB pretreatment also prevents carbon tetrachloride hepatotoxicity in vivo (Manautou et al., 1998). Therefore, it seems plausible that peroxisome proliferator-mediated hepatoprotection results from increased vanin-1 expression and elevated hepatic cystamine levels.

The implications of elevated vanin-1 mRNA and cystamine to hepatoprotection by CFB treatment are difficult to assess at the present time. There is only one known antibody that recognizes vanin-1. However, this antibody is not suitable for western blot analysis. Immunohistochemical localization of hepatic vanin-1 is also difficult due to low and diffuse staining (personal communication with P Naquet, Centre d'Immunologie de Marseille-Luminy CNRS-INSERM-Université de la Méditerranée, Marseille, France). There are no known specific depleting agents for cysteamine or cystamine, and inhibitors of vanin-1 are limited to general protein-thiol oxidizing agents that inactivate the enzyme (Ricci et al., 1986). A vanin-1-null mouse lacking pantetheinase activity and liver cysteamine production is available (Pitari et al., 2000). However, the use of these mice to assess the role of vanin-1 in CFB hepatoprotection would be confounded by compensatory responses in these mutants. Vanin-1-null mice have 50% more liver GSH than wild type mice, which makes them resistant to oxidative stress (Berruyer et al., 2004). The higher liver GSH content of vanin-1 mutants is due to the lack of cystamine, which is known to negatively regulates gamma-glutamylcysteine synthetase, the rate limiting enzyme in GSH synthesis (Martin et al., 2004). In the absence of liver cystamine, GSH production increases significantly.

In conclusion, differential gene analysis was used to identify genes involved in CFB-mediated hepatoprotection. Our comparisons yielded 53 genes of interest, including vanin-1. We established that CFB-mediated induction of vanin-1 gene expression is dependent upon PPAR α activation. Induction of vanin-1 results in increased levels of hepatic cystamine, a proven and potent antioxidant capable of protecting from APAP toxicity. Further mechanistic study of cysteamine and cystamine in CFB-mediated hepatoprotection is warranted as additional biochemical tools become available to modulate these potent antioxidants.

Supplementary Material

01

Supplemental Figure 1. Plasma alanine aminotransferase (ALT) activity determined 4h after APAP challenge. Male wild type and PPARα-null mice were pretreated with vehicle or CFB (500mg/kg), i.p. daily for 10d. All mice were treated with APAP (400mg/kg), po, following an overnight fast. Mice were sacrificed 4h after APAP challenge and blood was collected for measurement of plasma ALT activity. Results are expressed as mean ± SEM with n= 3–4 per treatment group. No statistical differences existed between the treatment groups.

02
03
04
05
06

Acknowledgments

We would like to thank Shibing Deng (Pfizer Inc.) for his technical assistance analyzing the gene array data. The views presented in this article do not necessarily reflect those of the U. S. Food and Drug Administration.

Funding Sources

This research was supported by the National Institutes of Health grant DK069557. Further support for the gene array study was provided by Pfizer, Inc. and ESI-LC/MS/MS analysis was funded by the National Center for Toxicological Research.

Footnotes

1

Presented at the Society of Toxicology Meeting, San Diego, CA, March 2006

Conflict of Interest Statement for Funding and Authors

There are no conflicts of interest.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Adamson ED, Mercola D. Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival. Tumour Biol. 2002;23:93–102. doi: 10.1159/000059711. [DOI] [PubMed] [Google Scholar]
  2. Anderson SP, Howroyd P, Liu J, Qian X, Bahnemann R, Swanson C, Kwak MK, Kensler TW, Corton JC. The transcriptional response to a peroxisome proliferator-activated receptor alpha agonist includes increased expression of proteome maintenance genes. J Biol Chem. 2004;279:52390–52398. doi: 10.1074/jbc.M409347200. [DOI] [PubMed] [Google Scholar]
  3. Bartolone JB, Beierschmitt WP, Birge RB, Hart SG, Wyand S, Cohen SD, Khairallah EA. Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: an in vivo and in vitro analysis. Toxicol Appl Pharmacol. 1989;99:240–249. doi: 10.1016/0041-008x(89)90006-9. [DOI] [PubMed] [Google Scholar]
  4. Berruyer C, Martin FM, Castellano R, Macone A, Malergue F, Garrido-Urbani S, Millet V, Imbert J, Dupre S, Pitari G, Naquet P, Galland F. Vanin-1−/ − mice exhibit a glutathione-mediated tissue resistance to oxidative stress. Mol Cell Biol. 2004;24:7214–7224. doi: 10.1128/MCB.24.16.7214-7224.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brucer M, Mewissen DJ. Late effects of gamma radiation on mice protected with cysteamine or cystamine. Nature. 1957;179:201–202. doi: 10.1038/179201b0. [DOI] [PubMed] [Google Scholar]
  6. Castro JA, De Ferreyra EC, De Castro CR, Diaz Gomez MI, D'Acosta N, De Fenos OM. Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride. Toxicol Appl Pharmacol. 1973;24:1–19. doi: 10.1016/0041-008x(73)90176-2. [DOI] [PubMed] [Google Scholar]
  7. Chen C, Hennig GE, Whiteley HE, Corton JC, Manautou JE. Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure. Toxicol Sci. 2000;57:338–344. doi: 10.1093/toxsci/57.2.338. [DOI] [PubMed] [Google Scholar]
  8. Chen C, Hennig GE, Whiteley HE, Manautou JE. Protection against acetaminophen hepatotoxicity by clofibrate pretreatment: role of catalase induction. J Biochem Mol Toxicol. 2002;16:227–234. doi: 10.1002/jbt.10043. [DOI] [PubMed] [Google Scholar]
  9. Dean MM, Minchinton RM, Heatley S, Eisen DP. Mannose binding lectin acute phase activity in patients with severe infection. J Clin Immunol. 2005;25:346–352. doi: 10.1007/s10875-005-4702-1. [DOI] [PubMed] [Google Scholar]
  10. Faig M, Bianchet M, Talalay P, Chen C, Winski S, Ross D, Amzel L. Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes wiht substrate binding and release. Proceedings of the National Academy of Science. 2000;97:3177–3182. doi: 10.1073/pnas.050585797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerson RJ, Casini A, Gilfor D, Serroni A, Farber JL. Oxygen-mediated cell injury in the killing of cultured hepatocytes by acetaminophen. Biochem Biophys Res Commun. 1985;126:1129–1137. doi: 10.1016/0006-291x(85)90303-1. [DOI] [PubMed] [Google Scholar]
  12. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature. 2003;426:895–899. doi: 10.1038/nature02263. [DOI] [PubMed] [Google Scholar]
  13. James LP, Simpson PM, Farrar HC, Kearns GL, Wasserman GS, Blumer JL, Reed MD, Sullivan JE, Hinson JA. Cytokines and toxicity in acetaminophen overdose. J Clin Pharmacol. 2005;45:1165–1171. doi: 10.1177/0091270005280296. [DOI] [PubMed] [Google Scholar]
  14. Jollow DJ, Mitchell JR, Potter WZ, Davis DC, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. Journal of Pharmacology and Experimental Therapeutics. 1973;187:195–202. [PubMed] [Google Scholar]
  15. Landin JS, Cohen SD, Khairallah EA. Identification of a 54-kDa mitochondrial acetaminophen-binding protein as aldehyde dehydrogenase. Toxicol Appl Pharmacol. 1996;141:299–307. doi: 10.1006/taap.1996.0287. [DOI] [PubMed] [Google Scholar]
  16. Liao Y, Shikapwashya ON, Shteyer E, Dieckgraefe BK, Hruz PW, Rudnick DA. Delayed hepatocellular mitotic progression and impaired liver regeneration in early growth response-1-deficient mice. J Biol Chem. 2004;279:43107–43116. doi: 10.1074/jbc.M407969200. [DOI] [PubMed] [Google Scholar]
  17. Liu ZX, Govindarajan S, Kaplowitz N. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology. 2004;127:1760–1774. doi: 10.1053/j.gastro.2004.08.053. [DOI] [PubMed] [Google Scholar]
  18. MacDonald JR, Gandolfi AJ, Sipes IG. Cystamine treatment of chemically induced liver injury. Dev Toxicol Environ Sci. 1983;11:463–466. [PubMed] [Google Scholar]
  19. Manautou JE, Hoivik DJ, Tveit A, Hart SG, Khairallah EA, Cohen SD. Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity. Toxicol Appl Pharmacol. 1994;129:252–263. doi: 10.1006/taap.1994.1250. [DOI] [PubMed] [Google Scholar]
  20. Manautou JE, Silva VM, Hennig GE, Whiteley HE. Repeated dosing with the peroxisome proliferator clofibrate decreases the toxicity of model hepatotoxic agents in male mice. Toxicology. 1998;127:1–10. doi: 10.1016/s0300-483x(98)00013-4. [DOI] [PubMed] [Google Scholar]
  21. Manautou JE, Tveit A, Hoivik DJ, Khairallah EA, Cohen SD. Protection by clofibrate against acetaminophen hepatotoxicity in male CD-1 mice is associated with an early increase in biliary concentration of acetaminophen-glutathione adducts. Toxicol Appl Pharmacol. 1996;140:30–38. doi: 10.1006/taap.1996.0194. [DOI] [PubMed] [Google Scholar]
  22. Martin F, Penet MF, Malergue F, Lepidi H, Dessein A, Galland F, de Reggi M, Naquet P, Gharib B. Vanin-1(−/ −) mice show decreased NSAID- and Schistosoma-induced intestinal inflammation associated with higher glutathione stores. J Clin Invest. 2004;113:591–597. doi: 10.1172/JCI19557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mehendale HM. PPAR-alpha: a key to the mechanism of hepatoprotection by clofibrate. Toxicol Sci. 2000;57:187–190. doi: 10.1093/toxsci/57.2.187. [DOI] [PubMed] [Google Scholar]
  24. Mehendale HM. Tissue repair: an important determinant of final outcome of toxicant-induced injury. Toxicol Pathol. 2005;33:41–51. doi: 10.1080/01926230590881808. [DOI] [PubMed] [Google Scholar]
  25. Miller MG, Jollow DJ. Acetaminophen hepatotoxicity: studies on the mechanism of cysteamine protection. Toxicol Appl Pharmacol. 1986;83:115–125. doi: 10.1016/0041-008x(86)90329-7. [DOI] [PubMed] [Google Scholar]
  26. Miners JO, Drew R, Birkett DJ. Mechanism of action of paracetamol protective agents in mice in vivo. Biochem Pharmacol. 1984;33:2995–3000. doi: 10.1016/0006-2952(84)90599-9. [DOI] [PubMed] [Google Scholar]
  27. Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. Journal of Pharmacology and Experimental Therapeutics. 1973a;187:185–194. [PubMed] [Google Scholar]
  28. Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. Journal of Pharmacology and Experimental Therapeutics. 1973;187:211–217. [PubMed] [Google Scholar]
  29. Miura Y, Shimazu R, Miyake K, Akashi S, Ogata H, Yamashita Y, Narisawa Y, Kimoto M. RP105 is associated with MD-1 and transmits an activation signal in human B cells. Blood. 1998;92:2815–2822. [PubMed] [Google Scholar]
  30. Nguyen KA, Carbone JM, Silva VM, Chen C, Hennig GE, Whiteley HE, Manautou JE. The PPAR activator docosahexaenoic acid prevents acetaminophen hepatotoxicity in male CD-1 mice. J Toxicol Environ Health A. 1999;58:171–186. doi: 10.1080/009841099157377. [DOI] [PubMed] [Google Scholar]
  31. Nicholls-Grzemski FA, Belling GB, Priestly BG, Calder IC, Burcham PC. Clofibrate pretreatment in mice confers resistance against hepatic lipid peroxidation. J Biochem Mol Toxicol. 2000a;14:335–345. doi: 10.1002/1099-0461(2000)14:6<335::AID-JBT6>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  32. Nicholls-Grzemski FA, Calder IC, Priestly BG. Peroxisome proliferators protect against paracetamol hepatotoxicity in mice. Biochem Pharmacol. 1992;43:1395–1396. doi: 10.1016/0006-2952(92)90193-m. [DOI] [PubMed] [Google Scholar]
  33. Nicholls-Grzemski FA, Calder IC, Priestly BG, Burcham PC. Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis. Toxicol Sci. 2000b;56:220–228. doi: 10.1093/toxsci/56.1.220. [DOI] [PubMed] [Google Scholar]
  34. Olinescu R, Nita S, Pascu N. Biochemical mechanisms involved in the hepatotoxicity of some drugs. Med Interne. 1982a;20:59–65. [PubMed] [Google Scholar]
  35. Olinescu R, Nita S, Pascu N. Biomechanical mechanisms involved in the hepatotoxicity of some drugs. Med Interne. 1982b;20:59–65. [PubMed] [Google Scholar]
  36. Pitari G, Malergue F, Martin F, Philippe JM, Massucci MT, Chabret C, Maras B, Dupre S, Naquet P, Galland F. Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice. FEBS Lett. 2000;483:149–154. doi: 10.1016/s0014-5793(00)02110-4. [DOI] [PubMed] [Google Scholar]
  37. Prescott LF, Newton RW, Swainson CP, Wright N, Forrest AR, Matthew H. Successful treatment of severe paracetamol overdosage with cysteamine. Lancet. 1974;1:588–592. doi: 10.1016/s0140-6736(74)92649-x. [DOI] [PubMed] [Google Scholar]
  38. Ricci G, Nardini M, Chiaraluce R, Dupre S, Cavallini D. Interaction of pantetheinase with sulfhydryl reagents and disulfides. Biochim Biophys Acta. 1986;870:82–91. doi: 10.1016/0167-4838(86)90011-7. [DOI] [PubMed] [Google Scholar]
  39. Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene. 2001;20:2390–2400. doi: 10.1038/sj.onc.1204383. [DOI] [PubMed] [Google Scholar]
  40. Siew C, Deitrich RA, Erwin VG. Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases. Arch Biochem Biophys. 1976;176:638–649. doi: 10.1016/0003-9861(76)90208-3. [DOI] [PubMed] [Google Scholar]
  41. Stroo WE, Olson RD, Boerth RC. Efficacy of sulfhydryl compounds as inhibitors of iron-dependent doxorubicin-enhanced lipid peroxidation. Res Commun Chem Pathol Pharmacol. 1985;48:291–303. [PubMed] [Google Scholar]
  42. Tredger JM, Smith HM, Davis M, Williams R. Effects of sulphur-containing compounds on paracetamol activation and covalent binding in a mouse hepatic microsomal system. Toxicol Lett. 1980;5:339–344. doi: 10.1016/0378-4274(80)90035-1. [DOI] [PubMed] [Google Scholar]
  43. Wendel A, Feuerstein S, Konz KH. Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo. Biochem Pharmacol. 1979;28:2051–2055. doi: 10.1016/0006-2952(79)90223-5. [DOI] [PubMed] [Google Scholar]
  44. Wong JS, Gill SS. Gene expression changes induced in mouse liver by di(2-ethylhexyl) phthalate. Toxicol Appl Pharmacol. 2002;185:180–196. doi: 10.1006/taap.2002.9540. [DOI] [PubMed] [Google Scholar]
  45. Xu Y, Cook TJ, Knipp GT. Effects of di-(2-ethylhexyl)-phthalate (DEHP) and its metabolites on fatty acid homeostasis regulating proteins in rat placental HRP-1 trophoblast cells. Toxicol Sci. 2005;84:287–300. doi: 10.1093/toxsci/kfi083. [DOI] [PubMed] [Google Scholar]
  46. Yamazaki K, Kuromitsu J, Tanaka I. Microarray analysis of gene expression changes in mouse liver induced by peroxisome proliferator- activated receptor alpha agonists. Biochem Biophys Res Commun. 2002;290:1114–1122. doi: 10.1006/bbrc.2001.6319. [DOI] [PubMed] [Google Scholar]
  47. Yokosuka T, Sakata-Sogawa K, Kobayashi W, Hiroshima M, Hashimoto-Tane A, Tokunaga M, Dustin ML, Saito T. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol. 2005 doi: 10.1038/ni1272. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Figure 1. Plasma alanine aminotransferase (ALT) activity determined 4h after APAP challenge. Male wild type and PPARα-null mice were pretreated with vehicle or CFB (500mg/kg), i.p. daily for 10d. All mice were treated with APAP (400mg/kg), po, following an overnight fast. Mice were sacrificed 4h after APAP challenge and blood was collected for measurement of plasma ALT activity. Results are expressed as mean ± SEM with n= 3–4 per treatment group. No statistical differences existed between the treatment groups.

NIHMS28093-supplement-01.pdf (53.5KB, pdf)
02
NIHMS28093-supplement-02.pdf (107.6KB, pdf)
03
NIHMS28093-supplement-03.pdf (91.3KB, pdf)
04
NIHMS28093-supplement-04.pdf (68.5KB, pdf)
05
NIHMS28093-supplement-05.pdf (59.3KB, pdf)
06
NIHMS28093-supplement-06.pdf (16.1KB, pdf)

RESOURCES