ToxSci Advance Access originally published online on October 20, 2004
Toxicological Sciences 2005 83(1):44-52; doi:10.1093/toxsci/kfi013
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Toxicological Sciences vol. 83 no. 1 © Society of Toxicology 2005; all rights reserved.
Differential Expression of Mouse Hepatic Transporter Genes in Response to Acetaminophen and Carbon Tetrachloride
* Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269; Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160; and Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721
1 To whom correspondence should be addressed at Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 372 Fairfield Rd. U-2092, Storrs, CT 06269. Fax: 860–486–4998. E-mail: manautou{at}uconnvm.uconn.edu.
Received October 6, 2004; accepted October 8, 2004
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ABSTRACT |
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Drug-metabolizing enzymes and membrane transporters are responsible for the detoxication and elimination of xenobiotics from the body. The goal of this study was to identify alterations in mRNA expression of various transport and detoxication proteins in mouse liver after administration of the hepatotoxicants, acetaminophen or carbon tetrachloride. Therefore, male C57BL/6 J mice received acetaminophen (APAP, 200, 300, or 400 mg/kg, ip) or carbon tetrachloride (CCl4, 10 or 25 µl/kg, ip). Plasma and liver samples were collected at 6, 24, and 48 h for assessment of alanine aminotransferase (ALT) activity, total RNA isolation, and histopathological analysis of injury. Heme oxygenase-1 (Ho-1), NAD(P)H quinone oxidoreductase-1 (Nqo1), organic anion-transporting polypeptides (Oatp1a1, 1a4 and 1b2), sodium/taurocholate-cotransporting polypeptide (Ntcp), and multidrug resistance-associated protein (Mrp 1–6) mRNA levels in liver were determined using the branched DNA signal amplification assay. Hepatotoxic doses of APAP and CCl4 increased Ho-1 and Nqo1 mRNA levels by 22- and 2.5-fold, respectively, and reduced Oatp1a1, 1a4, and Ntcp mRNA levels in liver. By contrast, expression of Mrps 1–4 was increased after treatment with APAP and CCl4. Notably, a marked elevation of Mrp4 mRNA expression was observed 24 h after APAP 400 mg/kg (5-fold) and CCl4 25 µl/kg (37-fold). Collectively, these expression patterns suggest a coordinated regulation of both transport and detoxification genes during liver injury. This reduction in expression of uptake transporters, as well as enhanced transcription of detoxication enzymes and export transporters may limit the accumulation of potentially toxic products in hepatocytes.
Key Words: acetaminophen; carbon tetrachloride; hepatotoxicity; transporters; Mrp4.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Drug-induced liver injury accounted for more than 50% of all cases of acute liver failure in the United States from 1997 to 2002, with 40% of these attributed to acetaminophen (APAP) ingestion (Lee, 2003
). Numerous chemicals, including APAP and carbon tetrachloride (CCl4), have been classically used in rodent models to investigate mechanisms of hepatotoxicity relevant to human exposure. Both APAP and CCl4 are bioactivated by Phase I cytochrome P450 enzymes to N-acetyl-p-benzoquinone imine (NAPQI) and trichloromethyl free radical, respectively. Toxicity resulting from these reactive metabolites is multifactorial and includes generation of oxidative stress and altered cellular redox status. In turn, expression of hepatic stress and detoxication genes, including microsomal heme oxygenase-1 (Ho-1) also known as heat shock protein 32, is induced in rat liver in response to chemical injury (Chiu et al., 2002; Nakahira et al., 2003). This up-regulation appears to be an adaptive mechanism to compensate for the dysregulated redox status.
Importantly, chemical-induced liver injury impairs hepatobiliary function and results in altered disposition of xenobiotics. In turn, clinicians are required to adjust dosages and dosing intervals of pharmaceuticals to compensate for reduced hepatic function in patients. Altered drug disposition in patients with liver damage has been attributed to reduced hepatic albumin production, altered protein plasma binding, poor hepatic blood flow, and altered expression and activity of Phase I and II drug-metabolizing enzymes (Verbeeck and Horsmans, 1998). However, little is known about changes in transport processes during hepatic injury, which may contribute to altered disposition and the necessity for adjustments in drug therapy.
Extraction of compounds from portal blood and subsequent excretion of the parent compound and its metabolites occurs via basolateral and canalicular transporters in hepatocyte plasma membranes (Arrese and Accatino, 2002). Constitutively expressed uptake carriers, such as organic anion-transporting polypeptides (Oatps) and the sodium/taurocholate-cotransporting polypeptide (Ntcp), transport xenobiotics and bile acids across the basolateral membrane into the hepatocyte. Subsequent excretion of these chemicals is mediated by numerous export transporters, including multidrug resistance proteins (Mdrs) also known as p-glycoprotein (Pgp), multidrug resistance-associated proteins (Mrps), bile salt export pump (Bsep), and breast cancer resistance protein (Bcrp). Canalicular transporters, such as Mrp2, Mdrs, Bcrp, and Bsep, are responsible for excretion of compounds and their metabolites from hepatocytes into bile, whereas basolateral transporters, such as Mrp 1, 3–6, are thought to mediate efflux of chemicals from hepatocytes into blood.
Despite the high incidence of drug-induced liver injury in the United States, little is known about the expression of hepatic membrane transporters and their influence on xenobiotic disposition in individuals with acute liver damage. Limited data demonstrate altered expression of xenobiotic transporters during chemical-induced hepatotoxicity. Administration of CCl4 results in reduced expression of rat Ntcp, Oatp1a1 [previously called Oatp1 (Slc21a1)], and Oatp1a4 [previously called Oatp2 (Slc21a5)] mRNA, with no change in levels of Mrp2, Bsep, and Oatp1b2 [previously called Oatp4 (Slc21a10)] (Geier et al., 2002). Altered canalicular clearance of substrates for Pgp, Bsep, and Mrp2 was observed in hepatic membrane preparations from rats given CCl4 (Song et al., 2003). Treatment with APAP results in the up-regulation of canalicular Mrp2 and Pgp protein in rat liver (Ghanem et al., 2004). Additionally, administration of the hepatotoxicant bromobenzene increases the hepatic expression of Mrp1-3 mRNA in rat liver (Heijne et al., 2004).
In order to investigate the regulation of transporter expression following chemical-induced liver injury, mice were injected with doses of APAP (200–400 mg/kg) or CCl4 (10–25 µl/kg) that resulted in varying degrees of hepatic damage over a 48-h time period. The analysis of transporter expression was extended to include numerous transporters not previously investigated during chemical-induced liver injury in rats. The inclusion of three time points (6, 24, and 48 h) for analysis of transporter expression enabled comprehensive characterization of temporal changes in relation to injury and recovery. The two hepatotoxicants were studied due to similarities and differences in their mechanisms of toxicity. Notably, APAP-induced hepatotoxicity has been associated with covalent adduct formation, depletion of cellular antioxidants such as glutathione, as well as generation of reactive oxygen and nitrogen species. Conversely, CCl4-mediated liver injury is generally characterized by formation of lipid peroxides and altered redox status. In this study, hepatotoxic doses of APAP and CCl4 resulted in the coordinated up-regulation of hepatic oxidative stress and efflux transport genes, as well as the concomitant reduction of uptake transporters.
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MATERIALS AND METHODS |
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Chemicals. APAP, CCl4, propylene glycol, and corn oil were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of reagent grade or better. RNAzol B was purchased from Tel-Test Inc. (Friendswood, TX).
Treatment of animals. Male C57BL/6 J mice, aged 10–12 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME). Mice acclimated 1 week upon arrival. Animals were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment. The mice were fed laboratory rodent diet (No. 5001, PMI Feeds, St. Louis, MO) ad libitum. APAP was dissolved in 50% propylene glycol:water. CCl4 was diluted in corn oil. Groups of mice (n = 3–7) were administered APAP (200, 300, or 400 mg/kg, 10 ml/kg, ip), CCl4 (10 or 25 µl/kg, 5 ml/kg, ip) or the respective vehicle control. The doses of APAP and CCl4 were selected in order to achieve mild to moderate, but not overt toxicity. Livers and plasma were collected 6, 24, or 48 h after APAP or CCl4 administration. Portions of each liver were removed for fixation in formalin. The remaining liver tissue was removed and snap-frozen in liquid nitrogen. Frozen tissues were stored at –80°C until assayed. All animal studies were conducted in accordance with National Institutes of Health standards and the Guide for the Care and Use of Laboratory Animals.
Alanine aminotransferase (ALT) activity. Plasma ALT activity was determined as a biochemical indicator of hepatocellular necrosis using Infinity ALT Liquid Stable Reagent (Thermotrace, Melbourne, Australia) according to the manufacturer's protocol.
Histopathology. Liver samples were fixed in 10% neutral-buffered formalin prior to routine processing and paraffin embedding. Liver sections (5 µm in thickness) were stained with hematoxylin and eosin. Sections were examined by light microscopy for the presence and severity of hepatocellular degeneration and necrosis. Centrilobular liver injury was scored using a grading system described previously (Manautou et al., 1994). Histopathology scoring was as follows: no injury = grade 0; minimal injury involving single to few hepatocytes = grade 1; mild injury affecting 10–25% of hepatocytes = grade 2; moderate injury affecting 25–40% of hepatocytes = grade 3; marked injury affecting 40–50% of hepatocytes = grade 4; or severe injury affecting more than 50% of hepatocytes = grade 5.
RNA extraction. Total tissue RNA was extracted using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) according to the manufacturer's protocol. RNA pellets were resuspended in diethyl pyrocarbonate-treated deionized water. RNA samples were analyzed by agarose gel electrophoresis, and integrity was confirmed by visualization of intact 18S and 28S rRNA under ultraviolet light.
Branched DNA signal amplification (bDNA) assay. Mouse Mrp1, 2, 3, 4, 5, 6, Bcrp, Oatp1a1, 1a4, 1b2, Ntcp, Nqo1, and Ho-1 mRNA were measured using the branched DNA signal amplification assay (Quantigene® High Volume bDNA Signal Amplification Kit, Genospectra, Fremont, CA) according to the method of Hartley and Klaassen (Hartley and Klaassen, 2000). Mouse gene sequences of interest were acquired from GenBank. Multiple oligonucleotide probe sets [capture extender (CE), label extender (LE), and blocker (BL) probes] were designed using Probe Designer software version 1.0 (Bayer Corp. Emeryville, CA), to be highly specific to a single mRNA transcript. Probesets for mouse Mrp1, 2, 4, 5, 6, Bcrp, Oatp1a1, 1a4, 1b2, Ntcp, Nqo1, and Ho-1 are listed in Supplementary Table 1. Probes to detect mouse Mrp3 have been previously described (Cherrington et al. 2003). All oligonucleotide probes were designed with a melting temperature of approximately 63°C. This enabled stringent hybridization conditions to be held constant (i.e., 53°C) during each hybridization step for each oligonucleotide probe set. Each probe designed in ProbeDesigner was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic local alignment search tool (BLASTn) to ensure minimal cross-reactivity with other mouse sequences. Oligonucleotides with a high degree of similarity to other mouse gene transcripts were eliminated from the design. Probes were synthesized by QIAGEN Operon (Alameda, CA). Briefly, 10 µl of sample RNA (1 µg/µl) were added to each well of a 96-well plate containing 50 µl of capture hybridization buffer and 100 µl of diluted probe set. Total RNA was allowed to hybridize to probe sets overnight at 53°C. Subsequent hybridization steps were carried out according to the manufacturer's protocol, and luminescence was measured with a Quantiplex® 320 bDNA Luminometer interfaced with Quantiplex® Data Management Software version 5.02. The luminescence for each well was reported as relative light units (RLU) per 10 µg total RNA.
Statistical analysis. Data from control animals at 6, 24, and 48 h were pooled and designated 0 h for gene expression analysis. No changes in basal transporter expression from control livers were seen over the 48 h time period. Quantitative results were expressed as means ± standard error of the mean (n > 3). Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (p < 0.05).
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RESULTS |
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Plasma ALT Activity after Treatment with APAP and CCl4
Administration of APAP and CCl4 to male C57BL/6 J mice resulted in hepatic injury as measured by plasma ALT levels (Fig. 1). The lower APAP doses (200 or 300 mg/kg) did not increase plasma ALT activity, whereas the highest APAP dose (400 mg/kg) increased mean plasma ALT activity at 6 h to 230 U/l. Plasma ALT activity remained elevated through the 48-h time period, although a statistical increase was only observed at 24 h. The low CCl4 dose (10 µl/kg) increased ALT values at 24 and 48 h to 160 and 290 U/l, respectively. The greatest increase in plasma ALT activity was observed after administration of 25 µl CCl4/kg. ALT increased at 6 h (mean ALT 145 U/l) and continued to increase through 48 h after CCl4 (mean ALT 4558 U/l). From these data, the highest dose of CCl4 induced a much higher plasma ALT activity compared to the highest dose of APAP.
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Liver Histopathology after Treatment with APAP and CCl4
Adverse histologic changes were absent in livers from APAP- and CCl4-treated mice at 6 h. Mild centrilobular hepatocellular injury (grade 2) was observed in livers from 50% of APAP (400 mg/kg)- and 100% of CCl4 (25 µl/kg)-treated mice at 24 h (Table 1). Resolution of APAP-induced liver injury occurred by 48 h. Conversely, centrilobular injury progressed in CCl4-treated mice, with all animals exhibiting grade 3 or 4 histological changes at 48 h (Table 2). In addition to the greater centrilobular injury, an increased number of mitotic figures indicated hepatocellular proliferation adjacent to areas of injury.
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Stress and Detoxication Gene Expression after Treatment with APAP and CCl4
Prototypical oxidative stress (Ho-1) and detoxification (NAD(P)H quinone oxidoreductase-1, Nqo1) genes were selected for analysis in both models. Up-regulation of microsomal Ho-1 has been previously documented in rats treated with hepatotoxic doses of APAP and CCl4 (Chiu et al., 2002
; Nakahira et al., 2003). This study demonstrates similar up-regulation of mouse Ho-1 mRNA levels during chemical-induced liver injury (Fig. 2). Maximal Ho-1 expression in liver was seen after APAP (400 mg/kg) and CCl4 (25 µl/kg) treatment, respectively. The up-regulation of Ho-1 in liver occurred at 6 h and returned to baseline by 48 h. Induction, but of a lesser magnitude, of Ho-1 expression in liver was also observed 6 h after administration of the lower doses of APAP (300 mg/kg) and CCl4 (10 µl/kg). Nqo1 is a cytosolic enzyme responsible for detoxication of reactive quinone species through a two-electron reduction. Expression of Nqo1 mRNA in liver was increased 2.5- and 2.8-fold at 24 h after APAP (400 mg/kg) and CCl4 (25 µl/kg), respectively (Fig. 2). Nqo1 mRNA levels in liver returned to baseline by 48 h after CCl4 treatment, but remained elevated 48 h after APAP administration.
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Uptake Transporter Gene Expression after Treatment with APAP and CCl4
In general, APAP and CCl4 treatment resulted in decreased expression of basolateral uptake transporters that are responsible for influx of chemicals from blood into hepatocytes. Specifically, transcripts for Oatp1a1, Oatp1b2, and Ntcp were reduced 24 and 48 h after treatment with the highest dose of APAP (400 mg/kg) or CCl4 (25 µl/kg) (Fig. 3). APAP treatment (400 mg/kg) decreased Oatp1a1 mRNA levels in liver to 10% of control levels at 48 h. The reduction in Oatp1a1 mRNA levels after APAP treatment was not observed in livers from mice treated with CCl4. However, the higher dose of CCl4 reduced Oatp1b2 expression by 60% at 48 h. Similarly, CCl4 decreased Ntcp expression 45 and 70% at 24 and 48 h after exposure, respectively. Interestingly, there was a 1.7-fold increase of Oatp1a4 mRNA at 6 h in both hepatotoxicity models.
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Efflux Transporter Gene Expression after Treatment with APAP and CCl4
Similar to the patterns observed with uptake transporter expression, efflux transporters responsible for basolateral (Mrp1, 3, 4) and canalicular (Mrp2) export were differentially expressed following APAP and CCl4-induced hepatic injury (Fig. 4). Increased liver Mrp1 mRNA levels were seen with CCl4 (25 µl/kg) at 24 (2.7-fold) and 48 h (4-fold). These changes were not observed with APAP treatment. Conversely, APAP, but not CCl4, increased Mrp3 expression 2-fold at 6 and 48 h. Whereas selective up-regulation of Mrp1 and Mrp3 was observed following either APAP or CCl4 administration, similar changes in Mrp2 and Mrp4 expression were observed with both hepatotoxicants. APAP and CCl4 treatment increased Mrp2 mRNA levels in liver 2-fold. Mrp4 mRNA levels were increased by APAP (400 mg/kg) and CCl4 (25 µl/kg) at time points for which hepatotoxicity was observed. APAP administration increased Mrp4 mRNA levels in liver 5-fold at 24 h and 3-fold at 48 h. A more marked elevation of Mrp4 mRNA expression (37-fold) was associated with CCl4-induced injury at 24 h and remained elevated (6.4-fold) at 48 h. APAP and CCl4 treatment did not alter the expression of Bcrp, Mrp5, or Mrp6 (data not shown). Notably, minimal changes in Mrp expression were seen with nonhepatotoxic doses of APAP and CCl4, suggesting a dependency on hepatic injury for altered mRNA levels.
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DISCUSSION |
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Other models of hepatic injury are accompanied by similar changes in transporter expression. Obstructive cholestasis and lipopolysaccharide-induced cholestasis in humans, mice, and rats results in the differential up-regulation of Mrp isoforms and down-regulation of Oatp/Ntcp isoforms (Cherrington et al., 2004
; Donner and Keppler, 2001; Gartung et al., 1996; Wagner et al., 2003). Reduced levels of rat and human Ntcp/NTCP, Mrp2/MRP2, and Oatp/OATP isoforms are observed following intrahepatic and obstructive cholestasis (Donner and Keppler, 2001; Gartung et al., 1996; Kullak-Ublick et al., 2004; Zollner et al., 2003). Additionally, up-regulation of MDR1, MRP1, and MRP3 is documented in patients with hepatitis or chronic cholestasis (Ros et al., 2003). Similar patterns in the expression of mouse transporters have been observed during cholestasis. Common bile duct ligation in mice results in elevated levels of Mrp3 and Mrp4 with reduced expression of Ntcp (Wagner et al., 2003). Furthermore, feeding cholic acid to mice also increases expression of Mrp2 and Bsep (Fickert et al., 2001). The induction of Bsep and Mrp isoforms in cholestatic mice appears to be an adaptive mechanism to enhance excretion of toxic bile acids and conjugates into the bile and/or portal blood.
Similarly, CCl4 treatment reduces the expression of Oatp1a1, Oatp1a4, and Ntcp in rat liver. More recent work shows the up-regulation of Mrp2 and Pgp expression and function following APAP treatment of rats (Ghanem et al., 2004). The work presented in this manuscript more comprehensively documents the down-regulation of mouse uptake carriers (Oatp and Ntcp) and up-regulation of Mrp efflux and stress (Ho-1 and Nqo1) genes. Collectively, these data support the hypothesis that the liver alters gene expression following injury to limit the accumulation of chemicals within the hepatocyte.
To date, this is the first study that documents a marked induction of hepatic Mrp4 transcript in mouse liver. Limited data exists regarding the potential role of Mrp4 in liver injury. Mrp4 mRNA is up-regulated in bile duct-ligated mice (Wagner et al., 2003), although to a much lesser extent (three-fold). Mrp4 substrates include nucleotide chemicals, including cyclic AMP and GMP, prostaglandin E1 and E2, as well as some HIV antiviral drugs (Borst et al., 2000; Reid et al., 2003; Sampath et al., 2002). Additionally, Mrp4 transports sulfated compounds, including the steroid, dehydroepiandrosterone 3-sulphate (Assem et al., 2004). The coordinated transcriptional regulation of Mrp4 and sulfotransferase 2a1 has been reported to occur through constitutive androstane receptor (CAR)-mediated signaling pathways (Assem et al., 2004). Interestingly, modulation of CAR alters susceptibility of mice to APAP-induced hepatotoxicity (Zhang et al., 2002). Therefore, activation of CAR during liver injury may represent one mechanism contributing to the up-regulation of Mrp4 in these studies.
Although the up-regulation of efflux and down-regulation of uptake transporters during hepatotoxicity appears to represent a general pattern in response to injury, some of the changes were specific to either APAP or CCl4. In some instances, different isoforms of Mrp and Oatp were altered by APAP (Mrp3, Oatp1b2) and CCl4 (Mrp1, Oatp1a1, Oatp1b2), while similar changes in Mrp2, Mrp4, Oatp1a4 were seen by both. This differential regulation may reflect the different pathogenic and transcriptional pathways elicited by APAP and CCl4. For example, down-regulation of Oatp1b2 and Ntcp during CCl4-induced hepatotoxicity may represent an attempt to limit influx of potentially harmful bile acids. On the contrary, the increase in Mrp1 and Mrp2 expression in response to CCl4 may enable efficient removal of the lipid peroxide 4-hydroxynonenal generated during injury (Reichard et al., 2003; Renes et al., 2000).
It should be noted that APAP and CCl4 are not thought to be substrates for uptake transporters and instead appear to enter the hepatocyte by diffusion (McPhail et al., 1993). The induction of Mrps may be an attempt by the hepatocyte to remove residual APAP metabolites, such as APAP-glucuronide, which is a substrate of Mrp2 and Mrp3 (Chen et al., 2003; Slitt et al., 2003; Xiong et al., 2002). It is presently unknown if Mrp4 transports APAP-glucuronide. Based upon the temporal up-regulation of Mrp genes at 24 and 48 h in this study, these changes would be futile, because these conjugates are efficiently cleared in mice during the first 24 h (Wong et al., 1981). Instead, these changes in transport may represent an attempt to better dispose of these metabolites upon a second challenge with APAP.
In addition to the transporter isoform-specific changes, the magnitude of altered expression differs between APAP and CCl4-treated mice. Presently, it is difficult to determine whether the marked increase (37-fold) in Mrp4 expression following CCl4 treatment compared to the moderate increase (5-fold) with APAP is related to differences in the mechanisms of injury, intrinsic xenobiotic properties, or the extent of hepatic injury achieved in these studies. Further analysis of additional hepatotoxicants, such as bromobenzene and chloroform, as well as higher doses of APAP may address this discrepancy in the magnitude of change in Mrp4 expression between the two hepatotoxicity models.
Inclusion of Ho-1 and Nqo1 in our analysis may offer additional insight into the regulatory mechanisms underlying the observed changes in transporter expression. Inducible expression of Ho-1 in mouse liver is in part regulated by cytokine signaling, including interleukin-6 (IL-6) (Masubuchi et al., 2003). Studies with IL-6 null mice demonstrate a role for IL-6 in the regulation of Mrp2, Mrp3, and Ntcp expression following lipopolysaccharide treatment (Siewert et al., 2004). Similarly, exogenous administration of IL-6 reduces murine expression of Mrp2, Oatp1a1, and Oatp1a4 mRNA (Hartmann et al., 2002). The critical role of IL-6 extends to modulation of hepatotoxicity, proliferation, and repair pathways in response to APAP and CCl4 (James et al., 2003; Masubuchi et al., 2003). Coordinated regulation of both detoxication and transport genes through IL-6 signaling during liver toxicity may represent a recovery mechanism by the injured hepatocyte.
Nuclear transcription factor-E2 p45-related factor 2 (Nrf2) is a transcription factor that regulates expression of multiple hepatic detoxification and stress genes including Nqo1, Ho-1, and glutathione-S-transferase during oxidative stress. Mice deficient in Nrf2 are more sensitive to the toxic effects of APAP (Chan et al., 2001; Enomoto et al., 2001). The enhanced sensitivity of Nrf2-deficient mice results, in part, from impaired compensatory up-regulation of these detoxication genes. Interestingly, Nrf2 is required for the constitutive and inducible expression of Mrp1 in mouse embryo fibroblasts (Hayashi et al., 2003). Further investigation is necessary to determine the potential role of Nrf2 in regulating hepatic membrane transporters in mice.
This study comprehensively characterizes the temporal and dose-related changes in transporter expression during chemical-induced hepatotoxicity. A better understanding of this altered expression is necessary to address the contribution of transport mechanisms to the impaired hepatic clearance of xenobiotics during liver injury. Clinical management of patients with drug-induced liver disease should consider the role of altered transporter expression when selecting doses and dosing regimens for administration of pharmaceuticals.
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SUPPLEMENTARY DATA |
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Supplementary data is available online.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Health Grant ES10093 and the University of Connecticut Research Foundation. Lauren Aleksunes is a Howard Hughes Medical Institute Predoctoral Fellow.
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REFERENCES |
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Arrese, M., and Accatino, L. (2002). From blood to bile: Recent advances in hepatobiliary transport. Ann. Hepatol. 1, 64–71.[Medline]
Assem, M., Schuetz, E. G., Leggas, M., Sun, D., Yasuda, K., Reid, G., Zelcer, N., Adachi, M., Strom, S., Evans, R. M., et al. (2004). Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. J. Biol. Chem. 279, 22250–22257.
Borst, P., Evers, R., Kool, M., and Wijnholds, J. (2000). A family of drug transporters: The multidrug resistance-associated proteins. J. Natl. Cancer Inst. 92, 1295–1302.
Chan, K., Han, X. D., and Kan, Y. W. (2001). An important function of Nrf2 in combating oxidative stress: Detoxification of acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 98, 4611–4616.
Chen, C., Hennig, G. E., and Manautou, J. E. (2003). Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats. Drug Metab. Dispos. 31, 798–804.
Cherrington, N. J., Slitt, A. L., Li, N., and Klaassen, C. D. (2004). Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug Metab. Dispos. 32, 734–741.
Cherrington, N. J., Slitt, A. L., Maher, J. M., Zhang, X. X., Zhang, J., Huang, W., Wan, Y. J., Moore, D. D., and Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 (mrp3) in vivo is independent of constitutive androstane receptor. Drug Metab. Dispos. 31, 1315–1319.
Chiu, H., Brittingham, J. A., and Laskin, D. L. (2002). Differential induction of heme oxygenase-1 in macrophages and hepatocytes during acetaminophen-induced hepatotoxicity in the rat: Effects of hemin and biliverdin. Toxicol. Appl. Pharmacol. 181, 106–115.[CrossRef][Web of Science][Medline]
Donner, M. G., and Keppler, D. (2001). Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 34, 351–359.[CrossRef][Web of Science][Medline]
Enomoto, A., Itoh, K., Nagayoshi, E., Haruta, J., Kimura, T., O'Connor, T., Harada, T., and Yamamoto, M. (2001). High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59, 169–177.
Fickert, P., Zollner, G., Fuchsbichler, A., Stumptner, C., Pojer, C., Zenz, R., Lammert, F., Stieger, B., Meier, P. J., Zatloukal, K., et al. (2001). Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 121, 170–183.[CrossRef][Web of Science][Medline]
Gartung, C., Ananthanarayanan, M., Rahman, M. A., Schuele, S., Nundy, S., Soroka, C. J., Stolz, A., Suchy, F. J., and Boyer, J. L. (1996). Down-regulation of expression and function of the rat liver Na + /bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 110, 199–209.[CrossRef][Web of Science][Medline]
Geier, A., Kim, S. K., Gerloff, T., Dietrich, C. G., Lammert, F., Karpen, S. J., Stieger, B., Meier, P. J., Matern, S., and Gartung, C. (2002). Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride. J. Hepatol. 37, 198–205.[CrossRef][Web of Science][Medline]
Ghanem, C. I., Gomez, P. C., Arana, M. C., Perassolo, M., Ruiz, M. L., Villanueva, S. S., Ochoa, E. J., Catania, V. A., Bengochea, L. A., and Mottino, A. D. (2004). Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein. Biochem. Pharmacol. 68, 791–798.[CrossRef][Web of Science][Medline]
Hartley, D. P., and Klaassen, C. D. (2000). Detection of chemical-induced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology. Drug Metab. Dispos. 28, 608–616.
Hartmann, G., Cheung, A. K., and Piquette-Miller, M. (2002). Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J. Pharmacol. Exp. Ther. 303, 273–281.
Hayashi, A., Suzuki, H., Itoh, K., Yamamoto, M., and Sugiyama, Y. (2003). Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem. Biophys. Res. Commun. 310, 824–829.[CrossRef][Web of Science][Medline]
Heijne, W. H., Slitt, A. L., van Bladeren, P. J., Groten, J. P., Klaassen, C. D., Stierum, R. H., and van Ommen, B. (2004). Bromobenzene-induced hepatotoxicity at the transcriptome level. Toxicol. Sci. 79, 411–422.
James, L. P., Lamps, L. W., McCullough, S., and Hinson, J. A. (2003). Interleukin 6 and hepatocyte regeneration in acetaminophen toxicity in the mouse. Biochem. Biophys. Res. Commun. 309, 857–863.[CrossRef][Web of Science][Medline]
Kullak-Ublick, G. A., Stieger, B., and Meier, P. J. (2004). Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126, 322–342.[CrossRef][Web of Science][Medline]
Lee, W. M. (2003). Acute liver failure in the United States. Semin. Liver Dis. 23, 217–226.[CrossRef][Web of Science][Medline]
Manautou, J. E., Hoivik, D. J., Tveit, A., Hart, S. G., Khairallah, E. A., and Cohen, S. D. (1994). Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity. Toxicol. Appl. Pharmacol. 129, 252–263.[CrossRef][Web of Science][Medline]
Masubuchi, Y., Bourdi, M., Reilly, T. P., Graf, M. L., George, J. W., and Pohl, L. R. (2003). Role of interleukin-6 in hepatic heat shock protein expression and protection against acetaminophen-induced liver disease. Biochem. Biophys. Res. Commun. 304, 207–212.[CrossRef][Web of Science][Medline]
McPhail, M. E., Knowles, R. G., Salter, M., Dawson, J., Burchell, B., and Pogson, C. I. (1993). Uptake of acetaminophen (paracetamol) by isolated rat liver cells. Biochem. Pharmacol. 45, 1599–1604.[CrossRef][Web of Science][Medline]
Nakahira, K., Takahashi, T., Shimizu, H., Maeshima, K., Uehara, K., Fujii, H., Nakatsuka, H., Yokoyama, M., Akagi, R., and Morita, K. (2003). Protective role of heme oxygenase-1 induction in carbon tetrachloride-induced hepatotoxicity. Biochem. Pharmacol. 66, 1091–1105.[CrossRef][Web of Science][Medline]
Reichard, J. F., Doorn, J. A., Simon, F., Taylor, M. S., and Petersen, D. R. (2003). Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal. Arch. Biochem. Biophys. 411, 243–250.[CrossRef][Web of Science][Medline]
Reid, G., Wielinga, P., Zelcer, N., van der Heijden, I., Kuil, A., de Haas, M., Wijnholds, J., and Borst, P. (2003). The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc. Natl. Acad. Sci. U.S.A. 100, 9244–9249.
Renes, J., de Vries, E. E., Hooiveld, G. J., Krikken, I., Jansen, P. L., and Muller, M. (2000). Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal. Biochem. J. 350(2), 555–561.[CrossRef]
Ros, J. E., Libbrecht, L., Geuken, M., Jansen, P. L., and Roskams, T. A. (2003). High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease. J. Pathol. 200, 553–560.[CrossRef][Web of Science][Medline]
Sampath, J., Adachi, M., Hatse, S., Naesens, L., Balzarini, J., Flatley, R. M., Matherly, L. H., and Schuetz, J. D. (2002). Role of MRP4 and MRP5 in biology and chemotherapy. AAPS PharmSci 4, E14.[CrossRef][Medline]
Siewert, E., Dietrich, C. G., Lammert, F., Heinrich, P. C., Matern, S., Gartung, C., and Geier, A. (2004). Interleukin-6 regulates hepatic transporters during acute-phase response. Biochem. Biophys. Res. Commun. 322, 232–238.[CrossRef][Web of Science][Medline]
Slitt, A. L., Cherrington, N. J., Maher, J. M., and Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 in rat liver is associated with altered vectorial excretion of acetaminophen metabolites. Drug Metab. Dispos. 31, 1176–1186.
Song, I. S., Lee, Y. M., Chung, S. J., and Shim, C. K. (2003). Multiple alterations of canalicular membrane transport activities in rats with CCl(4)-induced hepatic injury. Drug Metab. Dispos. 31, 482–490.
Verbeeck, R. K., and Horsmans, Y. (1998). Effect of hepatic insufficiency on pharmacokinetics and drug dosing. Pharm. World Sci. 20, 183–192.[CrossRef][Web of Science][Medline]
Wagner, M., Fickert, P., Zollner, G., Fuchsbichler, A., Silbert, D., Tsybrovskyy, O., Zatloukal, K., Guo, G. L., Schuetz, J. D., Gonzalez, F. J., et al. (2003). Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterology 125, 825–838.[CrossRef][Web of Science][Medline]
Wong, L. T., Whitehouse, L. W., Solomonraj, G., and Paul, C. J. (1981). Pathways of disposition of acetaminophen conjugates in the mouse. Toxicol. Lett. 9, 145–151.[CrossRef][Web of Science][Medline]
Xiong, H., Suzuki, H., Sugiyama, Y., Meier, P. J., Pollack, G. M., and Brouwer, K. L. (2002). Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment. Drug Metab. Dispos. 30, 962–969.
Zhang, J., Huang, W., Chua, S. S., Wei, P., and Moore, D. D. (2002). Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science 298, 422–424.
Zollner, G., Fickert, P., Silbert, D., Fuchsbichler, A., Marschall, H. U., Zatloukal, K., Denk, H., and Trauner, M. (2003). Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis. J. Hepatol. 38, 717–727.[CrossRef][Web of Science][Medline]
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