TY - JOUR
AU - Cherrington, Nathan, J.
AB - Abstract The multidrug resistance–associated proteins (Mrps) are a family of adenosine triphosphate–dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid–induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse. multidrug resistance–associated protein, cholestasis, liver, bile acid Chronic liver disease, listed as one of the top 15 causes of death in the United States (Centers for Disease Control, 2004), has become an increasingly important issue with a magnitude of over 1% of all deaths. This is underscored by the fact that chronic cholestatic liver diseases account for a large proportion of liver transplants (Sokol et al., 2006). When the liver is diseased, the normal hepatobilliary balance between bile acid uptake and efflux can become disrupted, resulting in cholestasis. This is known to be a characteristic of certain disease states of the liver, such as primary biliary cirrhosis, viral hepatitis, and alcoholic liver disease. Certain drugs, as well as genetic mutations in transport proteins (e.g., Mrp2 [multidrug resistance–associated protein 2], BSEP [bile salt export pump], MDR-3 [multidrug resistance 3]) can also cause cholestasis. Regardless of etiology, the consequent retention of hydrophobic bile acids results in accumulation of bile constituents and hepatocellular damage ensues. In contrast, a healthy liver has sufficient capacity to excrete bile constituents such as bile acids into the bile duct, primarily through the actions of the canalicular transporters Bsep and Mrp2. Bile acids are amphipathic molecules synthesized via catabolism of cholesterol. Administration of the hydrophobic bile acid LCA (lithocholic acid) has been applied as a model of intrahepatic cholestasis because it provides an accurate histologic representation of the damage that occurs in human forms of cholestatic disease (Fickert et al., 2006; Yousef et al., 1997; Zhang et al., 2004). Under normal conditions, LCA is mainly transformed into tauro- or glyco-conjugates in the liver and transported into the bile duct. However, when present in excess, LCA can cause a loss of gap-junction proteins resulting in leaky junctions and a collapse of the bile osmotic gradient resulting in decreased movement across membranes and subsequent accumulation and cellular swelling (Trauner et al., 1998). Bile acids exist as anions at physiologic pH and consequently require a carrier for transport across membranes. The Mrps are members of the ABC super family of adenosine triphosphate–dependent transporters that have broad substrate specificity, including bile acids, and are expressed in a variety of tissues including intestine, liver, and kidney (Cherrington et al., 2002). Following treatment with known inducers, several hepatic transporters were evaluated for changes in expression as well as to determine if their induction is associated with hepatoprotection from LCA-induced toxicity. Mrps 1 and 3–6 are located on the sinusoidal membrane of hepatocytes, and all are considered efflux transporters, but so far only Mrps 3 and 4 are known to transport bile acids. Bile salts known to be transported by Mrp3 include cholate, taurocholate, glycocholate, taurochenodeoxycholate-3-sulfate, and taurolithocholate-3-sulfate, hyocholate, hyodeoxycholate, which is unlikely to be an exhaustive list as both Mrp3 and Mrp4 additionally transport glucuronide- and some glutathione-conjugated bile acids (Hirohashi et al., 2000; Zelcer et al., 2006). It has also been shown that expression of these two transporters is increased during cholestasis of varying etiologies, implying a role for the extrusion of toxic bile constituents (Donner and Keppler, 2001; Wagner et al., 2003). Several Mrps, such as Mrps 2–4, have also been shown to be inducible in response to certain chemicals such as phenobarbital (PB), oltipraz (OPZ), and TCPOBOP (TC) (Cherrington et al., 2003; Slitt et al., 2003). We were most interested in bile acid efflux transporters, however, in order to better understand any protective or adaptive mechanisms, we also evaluated the expression of several organic anion uptake transporters which bring bile acids into hepatocytes as well as canalicular transporters that move bile acids from the hepatocyte into bile ducts. Importantly, under cholestatic conditions, the expression of hepatic transporters is modified as an adaptive response by the liver to protect itself. To determine the potential therapeutic effects of drug-induced transporter expression during cholestasis, known transporter inducers were administered to mice prior to LCA administration. The aim of this study was to determine whether induction of Mrp transporters can provide hepatoprotection from bile acid–induced intrahepatic cholestasis. MATERIALS AND METHODS Chemicals. PB sodium was purchased from Mallinckrodt, Inc. (Paris, KT). OPZ was purchased from LKT Laboratories, Inc. (St Paul, MN). TC was purchased from Sigma-Aldrich (St Louis, MO). Antibodies for Mrp1–6 were a generous gift from Dr. George Scheffer, VU Medical Center (Amsterdam, The Netherlands). Antibodies for Oatp1b2 (Oatp4) and Oatp1a4 (Oatp2) were a kind gift from the laboratory of Dr Curtis Klaassen at the University of Kansas Medical Center (Kansas City, KS). Mouse monoclonal β-actin antibody (SC-47778) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Animals. Ten-week-old adult male C57BL/6NHsd (Harlan, Indianapolis, IN) mice were weight matched into treatment groups (N = 4–6 mice/group). Animals were pretreated with chemical inducer for 3 days (PB 80 mg/kg, OPZ 150 mg/kg) or corn oil (CO) via ip injection in a volume of 3 ml/kg. On the fourth day, LCA administration was started (125 mg/kg bid) and inducer treatment continued for 4 days. TC is a potent and long-term inducer; therefore, mice in the TC group received one pretreatment dose (3 mg/kg) on day 3, and then LCA BID for the next four days. Animals were sacrificed 12 hours following the last treatment of LCA on day 8 and liver tissue was removed and stored at – 80°C. A minimum of four animals per treatment group were housed in metabolism cages for the duration of the study. Urine was collected during the 24-h period prior to sacrifice, and then stored at –80°C until used for bile acid analysis. Animals were maintained in a 12-h light/dark cycle at approximately 25°C, with access to food and water ad libitum. The experimental protocol was approved by the University of Arizona Institutional Animal Care and Use Committee (IACUC), and humane care of the animals was in accordance with the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (National Research Council, 1996). Liver enzymes. Blood was allowed to clot at room temperature and then centrifuged at 857 X g for 10 min using a Heraeus Biofuge pico centrifuge (Kendro Laboratory Products, Newtown, CT). Serum was removed, aliquoted into polypropylene tubes and stored at − 80°C until analyzed. Liver enzyme tests for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were performed using an Endocheck Plus Chemistry Analyzer (Hemagen Diagnostics, Inc., Columbia, MD), according to manufacturers protocol. Assessment of liver histology. Midsections of the left liver lobe were collected from each animal and fixed in 10% neutral buffered formalin. Tissue from two mice per treatment group was embedded in paraffin and 5-μm sections were stained with hematoxylin and eosin according to a standard staining protocol. Tissue was evaluated for liver injury and necrosis by an American College of Veterinary Pathology board-certified pathologist. Messenger RNA isolation. Total RNA was isolated from liver using RNA Bee reagent (Tel-Test Inc., Friendswood, TX) according to the manufacturer's instructions. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. RNA samples were analyzed by agarose gel electrophoresis with ethidium bromide staining, and integrity was confirmed by visualization of intact 18S and 28S ribosomal RNA under ultraviolet light. Branched DNA assay to measure messenger RNA expression. Specific oligonucleotide probe sets for bDNA analysis were developed as previously described: Mrp1, 2, and 3 (Cherrington et al., 2002); Mrp4 (Aleksunes et al., 2005); Mrp5, Mrp6, Ntcp, and Bsep (Leazer and Klaassen, 2003); Oatp1, Oatp2, and Oatp4 (Li et al., 2002); Bcrp (Tanaka et al., 2005). Briefly, probes were diluted in lysis buffer and then total RNA (1 μg/μl: 10 μl) was added to each well containing capture buffer and 50 μl of each diluted probe set. All reagents (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, and substrate solution) used in the analysis were supplied in the Quantigene Reagent System (Panomics, Inc., Freemont, CA). Total RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out according to manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex Data Management Software Version 5.02 (Bayer Diagnostics, East Walpole, MA). Protein analysis (Western blotting). Briefly, livers were homogenized in 5 volumes of Buffer A (pH 7.5) containing 250mM sucrose, 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH, pH 7.5, 10mM KCl, 1.5mM MgCl2, 1mM sodium ethylenediaminetetracacetic acid, 1mM sodium ethylene glycol tetraacetic acid, 1mM dithiotreitol, and 0.1mM phenylmethanesulphonylfluoride. Homogenates were centrifuged at 10,000 × g for 10 min at 4°C. Supernatants (SN) were centrifuged 100,000 × g for 60 min at 4°C. Microsomal pellets were then resuspended in Buffer A and stored at − 80°C until used. Protein concentration in microsomal preparations was determined using the BCA Protein Kit assay (Pierce Biotechnology, Rockford, IL). Liver microsome preparations containing 50 μg of total protein were separated on polyacrylamide gels (7.5% resolving, 4% stacking) using the BioRad Criterion precast gel system. Proteins were transferred onto polyvinylidene fluoride microporous membranes (Millipore, Bedford, MA). Blots were blocked with 2% nonfat dry milk (NFDM) in PBS (phosphate-buffered saline) with 0.05% Tween (PBS-T), incubated for 1 h then incubated for 2 h with primary antibody diluted in PBS-T with 2% NFDM at room temperature. After washing in PBS-T, the membranes were incubated for 1 h with a species-appropriate peroxidase-labeled secondary antibody (Sigma-Aldrich) diluted in diluted in PBS-T with 2% NFDM at room temperature. After incubation with the secondary antibody, membranes were washed in PBS-T, incubated with SuperSignal West Pico and Femto chemiluminescent substrate (Pierce Biotechnology) for 5 min. The chemiluminescent signal was captured by KODAK Image Station 2000 (Carestream Molecular Imaging, New Haven, CT). Bile acid extraction. Liver samples (approx. 50 mg) were homogenized in a 1 ml solution of t-butanol/water (1:1) and extracted overnight at room temperature. Samples were centrifuged at 9520 X g for 20 min using a Heraeus Biofuge pico centrifuge (Kendro Laboratory Products) and the SN removed and placed in a SpeedVac until dry. Samples were reconstituted with 0.9% normal saline in a volume equal to the liver weight, and stored at 8°C until used. Serum samples were extracted with acetone based the method of Daykin et al. (2002). Briefly, samples mixed with chilled acetone (1:4), shaken vigorously, and left at room temperature for 5 min before centrifugation for 3 min at 9520 X g. The SN was removed and stored at − 80°C until used. Total bile acid analysis. Total bile acids in extracted liver, extracted serum, and urine were measured enzymatically using a kit from Trinity Biotech (Wicklow, Ireland). In this assay, bile acids are first oxidized to 3-oxo bile acids in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase with equimolar amounts of nicotinamide adenine dinucleotide (NAD) being reduced to NADH. The NADH is subsequently oxidized back to NAD with a concomitant reduction of nitro blue tetrazolium salt to formazan via the enzymatic action of diaphorase. The intensity of the color produced at 530 nm by formazan is directly proportional to the concentration of bile acids in the sample (Mashige et al., 1981). Quantification of total bile acids was done according to manufacturer's instructions. Statistical analysis. For all quantitative data, the average and standard error of the mean (SEM) were calculated. Statistical differences were determined using one-way analysis of variance followed by Duncan's multiple range post hoc test using Statistica software, Version 4.5. (StatSoft, Tulsa, OK). RESULTS Effects of Treatment on Mouse Liver Histology Figure 1 illustrates that 4 days of LCA treatment results in moderate acute random multifocal hepatic necrosis with areas of diffuse vacuolization. A similar pathology was observed in the OPZ/LCA cotreated mice with areas of multifocal hepatic necrosis, diffuse vacuolization, and infiltrating neutrophils. Pretreatment with PB or the synthetic CAR agonist TC abrogated the LCA-induced hepatocellular necrosis thereby revealing livers similar in appearance to the CO control group. Thus, the livers of the PB/LCA and TC/LCA cotreated mice were completely protected from the hepatotoxicity and damage normally inflicted by LCA treatment. FIG. 1. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on liver histology in male C57BL/6 mice. A midsection of the left liver lobe was removed and fixed from each animal. Tissues were stained with hematoxylin and eosin and histopathology was determined by a board-certified veterinary pathologist (50× magnification). Two animals per treatment group were evaluated and pictures are representative of treatment group pathology. Multifocal hepatic necrosis (arrows) is easily distinguished from surrounding parenchyma and is more extensive in the LCA and OPZ/LCA cotreated mice. FIG. 1. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on liver histology in male C57BL/6 mice. A midsection of the left liver lobe was removed and fixed from each animal. Tissues were stained with hematoxylin and eosin and histopathology was determined by a board-certified veterinary pathologist (50× magnification). Two animals per treatment group were evaluated and pictures are representative of treatment group pathology. Multifocal hepatic necrosis (arrows) is easily distinguished from surrounding parenchyma and is more extensive in the LCA and OPZ/LCA cotreated mice. Effects of Treatment on Liver Enzymes Figure 2 illustrates the effect of LCA-only and the various inducer–LCA cotreatments on the concentration of liver enzymes in serum. Consistent with adverse histopathology, serum ALT levels were highest in the OPZ/LCA and LCA-only treatment groups, with 24.9- and 24.1-fold increases, respectively, relative to the CO control group. Also consistent with the histologic findings that demonstrate hepatoprotection, ALT levels in the PB/LCA and TC/LCA cotreated mice were significantly lower than those in LCA-only treated animals, by 94% and 87%, respectively. Figure 2 also demonstrates a similar trend of decreased ALP in the protected PB/LCA and TC/LCA cotreatment groups compared with LCA only, however, the changes were not statistically significant. FIG. 2. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on serum liver enzymes (ALT and ALP) in male C57BL/6 mice. Animals (N = 4/group) were dosed with inducers and LCA (125 mg/kg BID) as described in “Materials and Methods.” Results are presented as mean concentration ± SEM. † Indicates p ≤ 0.05 compared with LCA only. FIG. 2. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on serum liver enzymes (ALT and ALP) in male C57BL/6 mice. Animals (N = 4/group) were dosed with inducers and LCA (125 mg/kg BID) as described in “Materials and Methods.” Results are presented as mean concentration ± SEM. † Indicates p ≤ 0.05 compared with LCA only. Effects of Treatment on Transporter Messenger RNA Expression Figure 3 demonstrates the effect of LCA and the various inducer–LCA cotreatments on messenger RNA (mRNA) levels of uptake transporters in mouse liver. Ntcp expression was increased in the TC/LCA cotreated group compared with both CO control and LCA-only mice (64% and 83%, respectively). Oatp1 expression was markedly decreased in all chemical pretreatment groups as compared with that in the CO control group, whereas, Oatp4 mRNA expression was significantly upregulated in the PB/LCA (38%) and TC/LCA (48%) cotreated mice compared with the LCA-only group. LCA-only treatment reduced the mRNA levels of Oatp1 and Oatp4 as compared with CO controls (74% and 39%, respectively). No significant changes were observed in Oatp2 expression. FIG. 3. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on uptake transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg BID) as described in “Materials and Methods.” Hepatic Ntcp, Oatp1, Oatp2, and Oatp4 mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” Data are expressed as relative light units (RLU) ± SEM. * Indicates p ≤ 0.05 compared with CO treatment; † indicates p ≤ 0.05 compared with CO + LCA treatment. FIG. 3. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on uptake transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg BID) as described in “Materials and Methods.” Hepatic Ntcp, Oatp1, Oatp2, and Oatp4 mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” Data are expressed as relative light units (RLU) ± SEM. * Indicates p ≤ 0.05 compared with CO treatment; † indicates p ≤ 0.05 compared with CO + LCA treatment. Figure 4 illustrates a lack of effect of LCA-only and the various inducer–LCA cotreatments on mRNA levels of canalicular efflux transporters in mouse liver. Specifically, LCA-only treatment had no significant effect on mRNA levels of Bsep, Mrp2, or Bcrp. Inducer pretreatment likewise produced no significant alterations in mRNA levels of these three transporters. FIG. 4. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on canalicular transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg bid) as described in “Materials and Methods.” Hepatic Bsep, Mrp2, and Bcrp mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” There were no significant changes in canalicular efflux transporter mRNA levels in any treatment group. Data are expressed as relative light units (RLU) ± SEM. FIG. 4. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on canalicular transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg bid) as described in “Materials and Methods.” Hepatic Bsep, Mrp2, and Bcrp mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” There were no significant changes in canalicular efflux transporter mRNA levels in any treatment group. Data are expressed as relative light units (RLU) ± SEM. Figure 5 illustrates the effect of LCA-only and the various inducer–LCA cotreatments on mRNA levels of sinusoidal efflux transporters in mouse liver. mRNA levels of Mrp4 were significantly increased by all treatment groups compared with CO controls, and it is the only efflux transporter significantly affected by LCA only. Interestingly, Mrp4 mRNA levels were induced to a similar extent in liver from mice that underwent cotreatment with LCA and a chemical inducer, with the exception of TC/LCA cotreatment. Specifically, TC/LCA cotreatment resulted in 13- and 4-fold induction of Mrp4 mRNA above that of CO- and LCA-only treatment, respectively. TC/LCA cotreatment also induced Mrp3 mRNA levels by 4.8- and 2.7-fold above that of CO- and LCA-only treatment, respectively. There were no significant alterations in mRNA levels of Mrp1, Mrp5, and Mrp6 following any of the treatments. FIG. 5. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on sinusoidal efflux transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg bid) as described in “Materials and Methods.” Hepatic Mrp 1, Mrp3, Mrp4, Mrp5, and Mrp6 mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” Data are expressed as relative light units (RLU) ± SEM. * Indicates p ≤ 0.05 compared with CO treatment; † indicates p ≤ 0.05 compared with CO + LCA treatment. FIG. 5. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on sinusoidal efflux transporters in male C57BL/6 mice. Animals (N = 4–6/group) were dosed with inducers and LCA (125 mg/kg bid) as described in “Materials and Methods.” Hepatic Mrp 1, Mrp3, Mrp4, Mrp5, and Mrp6 mRNA levels in each treatment group were measured by the bDNA signal amplification assay, as described in “Materials and Methods.” Data are expressed as relative light units (RLU) ± SEM. * Indicates p ≤ 0.05 compared with CO treatment; † indicates p ≤ 0.05 compared with CO + LCA treatment. Effects of Treatment on Transporter Protein Expression Figure 6 illustrates the effect of LCA-only and inducer/LCA cotreatments on protein expression levels of Mrp transporters, as well as uptake transporters Oatp2 and Oatp4. Although Mrp1 and Mrp5 protein levels were significantly increased by OPZ/LCA cotreatment and LCA-only treatment, the PB/LCA and TC/LCA cotreatments failed to either achieve or exceed the induction that was associated with LCA-induced hepatotoxicity. Mrp2 protein levels were significantly increased by OPZ/LCA cotreatment compared with LCA-only and CO treatment, but this induction clearly fails to confer hepatoprotection (Fig. 1). Mrp3 protein levels were not significantly altered by any treatments of the current study. Mrp4 protein levels were massively induced by both OPZ/LCA and TC/LCA cotreatments, respectively. Mrp6 protein levels were decreased by all treatments compared with CO controls. There were no changes in protein expression of either Oatp2 or Oatp4. FIG. 6. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on hepatic protein expression in male C57BL/6 mice. Liver microsomal fractions were isolated and analyzed by Western blotting for protein expression of Mrp1–6 and Oatp4. Three animals per group are shown. FIG. 6. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on hepatic protein expression in male C57BL/6 mice. Liver microsomal fractions were isolated and analyzed by Western blotting for protein expression of Mrp1–6 and Oatp4. Three animals per group are shown. Effects of Treatment on Total Bile Acids Figure 7 illustrates the concentration of total bile acids in liver, serum, and urine. Changes between treatment groups in serum and urine were not appreciable, but all were increased compared with CO controls. However, of particular interest is the decrease in total bile acid levels in the liver of the PB/LCA and TC/LCA cotreatment groups compared with LCA only. These changes in the hepatoprotected groups are similar in concentration to CO controls. FIG. 7. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on total bile acid concentrations in liver, serum, and urine in male C57BL/6 mice. Bile acids from mice (N = 4–6/group) were extracted as described in “Materials and Methods” and analyzed for total bile acid concentrations in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. No significant changes in serum or urine were observed between the treatment groups. Results are presented as mean bile acid concentration ± SEM. † Indicates p ≤ 0.05 compared with CO + LCA treatment. FIG. 7. Open in new tabDownload slide Effect of treatment with LCA alone and in combination with inducers on total bile acid concentrations in liver, serum, and urine in male C57BL/6 mice. Bile acids from mice (N = 4–6/group) were extracted as described in “Materials and Methods” and analyzed for total bile acid concentrations in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. No significant changes in serum or urine were observed between the treatment groups. Results are presented as mean bile acid concentration ± SEM. † Indicates p ≤ 0.05 compared with CO + LCA treatment. DISCUSSION In this study, we report that both TC and PB pretreatments provide hepatoprotection against LCA-induced injury. The goal of the current study was to determine whether chemical induction of Mrp transporters results in hepatoprotection during LCA-induced intrahepatic cholestasis. Chemicals known to induce Mrp transporters (PB, OPZ, and TC) were administered prior to and during administration of the hepatotoxic secondary bile acid, LCA. Previous studies have shown that both Mrp3 and Mrp4 are induced during cholestasis and that they transport bile acids (Bohan et al., 2003; Donner and Keppler, 2001). The fact that both of these efflux transporters are induced in response to cholestatic conditions in humans indicates that they may function to provide protection against bile acid-induced liver damage. The degree of toxicity of bile acids generally correlates to their hydrophobicity, with the more hydrophobic bile acids (monohydroxy) being the most cytotoxic. Of common bile acids, LCA is the most hydrophobic followed by deoxycholic acid, chenodeoxycholic acid, taurocholic acid, ursodeoxycholic acid, and β-muricholic acid (Heuman et al., 1989). As the most hydrophobic and therefore most potent in terms of ability to cause hepatocellular damage, LCA administration was utilized to model intrahepatic cholestasis in the current study. LCA-induced liver damage was characterized by multifocal necrosis with vacuolization, which is similar to the findings by Fickert et al. (2006) in which necrotic liver damage was also observed in mice treated with LCA. Consistent with recent findings, we observed histologic hepatoprotection from LCA-induced injury in TC pretreated mice (Saini et al., 2004; Zhang et al., 2004) and with previously unreported PB. Increased canalicular secretion of bile acids was likely not involved in the hepatoprotection observed in the PB/LCA and TC/LCA cotreated groups as there were no significant changes in the expression of canalicular efflux transporters Bsep, Mrp2 or Bcrp. Canalicular efflux transporter expression observed in the current study is consistent with the findings of Kitada et al. (2003) after feeding mice with a 1% LCA diet for 9 days. It is interesting to note that others have found decreases in the expression of these transporters in the LPS model of cholestasis in mice (Lickteig et al., 2007). One critical component of the LPS model is that it is characterized by activation of the acute phase response and hepatic inflammation, likely leading to suppression of bile formation. The fact that the LCA and LPS models may have different consequences in terms of transporter regulation, that is, mRNA levels, likely reflects that their changes are signaled via different mechanisms. LCA-only administration did lead to a significant decrease in mRNA levels of the basolateral uptake transporter Oatp1, compared with the CO control, similar to the Kitada et al. (2003) study. Such an alteration in transcriptional regulation would seem an appropriate response of the liver to decrease its exposure to the LCA toxicant. However, this Oatp1 downregulation was accompanied by significant increases in mRNA levels of Ntcp in TC/LCA cotreated mice and Oatp4 in both PB/LCA and TC/LCA cotreated mice compared with LCA treatment alone. Although it may be counterintuitive, limiting the amount of bile acids that enter the hepatocyte does not seem to correlate with the hepatoprotection observed in the PB/LCA and TC/LCA cotreatment groups. There were no significant changes in Oatp2 mRNA levels. To address our hypothesis regarding Mrp transporter induction as a mechanism of hepatoprotection, Mrp transporters (Mrp1–6) were evaluated for changes in expression at the protein level. As bile acid uptake transporters, Oatp2 and Oatp4 were also evaluated. Treatment with TC prior to LCA-induced cholestasis caused a significant increase in protein levels of Mrp4, which as an efflux transporter may to some degree contribute to the hepatoprotection observed in the TC/LCA cotreated mice. No significant changes were observed in Mrp2 or Mrp3 protein expression. Posttranslational modifications of the Mrp3 protein cannot be ruled out to account for the differences in mRNA and protein expression observed in the present study. These novel findings indicate that although Mrp3 has been linked to hepatoprotection during cholestasis, upregulation of Mrp3 is not required for hepatoprotection against LCA-induced toxicity (Bohan et al., 2003; Teng and Piquette-Miller, 2007). Expression data from the current study are more similarly related to the findings of the mouse LCA study (1% LCA in the diet × 9 days) by Kitada et al. (2003), where they found no significant changes in the expression of Mrp3 or Mrp4 and attributed the hepatoprotective effects that they observed to increases in sulfotransferase detoxification of LCA. Results from the current study clearly indicate that changes in the expression of a single efflux transporter are not associated with the observed hepatoprotection. For example, in the TC/LCA cotreatment group, Mrp4 induction correlates with histologic hepatoprotection. In contrast, however, histologic analysis of livers in the OPZ/LCA cotreatment indicates that it failed to protect the liver, in spite of increased presence of Mrp4 protein. Additionally, in the PB/LCA cotreatment group there was no increase in Mrp3 or Mrp4 expression, yet we observed hepatoprotection in that group. On a similar note, in spite of relatively high expression of Mrp1 and Mrp5 protein in the OPZ/LCA and LCA-only groups, LCA-induced damage was not at all effectively abrogated. This indicates that transporter expression of Mrp1 and 5 may be regulated by hepatotoxicity, rather than by chemical induction. Additionally, protein expression of Oatp4 was not significantly different between groups, in contrast to the increased mRNA expression observed in the PB/LCA and TC/LCA cotreated groups, indicating a differential regulation of Oatp4. Taken together, these data would seem to indicate that transporter-mediated efflux of bile acids from the liver is not the only possible mechanism of hepatoprotection during LCA-induced cholestasis. Of additional consideration is the multiplicity of effects these inducer compounds have, amongst those being the induction of Mrp's. OPZ, for example, has been used as an antiparasitic to treat schistosomiasis as well as in the prevention of aflatoxin-induced liver cancer. It has the ability to activate multiple nuclear receptors to increase transcription of genes involved in the antioxidant response as well as xenobiotic and bile acid metabolism, thus demonstrating the diversity and range of effects of this compound. PB elicits various pleiotropic effects in liver, such as P-450 and transferase enzyme induction, increased cellular proliferation, and decreased apoptosis (Randerath et al., 1992). Ueda et al. (2002) found that PB either repressed or induced a total of 138 genes, about half of which are regulated by CAR. Like the substantial pharmacodynamic effects of drugs, when considering the multitude of factors (transporters, transcription factors, nuclear receptors, etc.) potentially involved during cholestasis it is easily conceivable to postulate that more than one mechanism could be involved in the hepatoprotection, or lack thereof. Total bile acid concentrations in the liver of the PB/LCA and TC/LCA cotreated groups were similar to the CO control concentration and were significantly decreased compared with LCA-only treated mice. The reduction in total liver bile acid concentrations in the protected groups correlates well with histopathology and liver enzymes, confirming that total hepatic bile acid concentrations are an important factor in preventing the development of cholestasis. This may explain the lack of Mrp3 induction, where the lack of bile acid accumulation in the liver needs no upregulation of efflux transporters such as Mrp3. Additionally, because the expression of hepatic efflux transporters was not increased in the PB/LCA cotreated group there was no excess movement of bile acids out of liver and into blood, thus reflecting the lack of changes seen in the serum. The lack of significant differences between groups in total urine bile acid concentrations suggests that urinary excretion of bile acids is not necessary to confer protection. This implies that mechanisms other than transport are involved in the protection, such as metabolism. Overall, the changes that occurred in the LCA model (LCA treatment alone) included marked multifocal hepatocellular necrosis, elevated ALT, downregulation of uptake transporters (Ntcp, Oatp1, Oatp4), upregulation of Mrp1 and Mrp5 expression, as well as increased concentrations of total bile acids in liver, serum, and urine indicating severe liver damage. Although conversely, in mice pretreated with PB and TC prior to LCA administration, there were effects reflecting hepatoprotection: liver histology similar to CO controls, reduced liver ALT concentration, and a decrease in total liver bile acid concentrations. This hepatoprotective effect is similar to other reports in which ALT levels are significantly decreased in LCA-only treated mice following CAR activation (Zhang et al., 2004). Although other studies conducted in mice have shown increases in efflux transporter expression during cholestasis that may contribute to hepatoprotection, our results do not support that conclusion. Future studies in this model of cholestasis should include the transgenic overexpression of Mrp3 and/or Mrp4 to specifically evaluate their role in hepatoprotection in the absence of all other secondary effects of the drug. Thus, the data here reflect that sinusoidal efflux transport does not play a significant role in the observed hepatoprotection from LCA-induced cholestasis. Rather than a single mechanism, there are likely multiple factors involved in the hepatoprotection, such as proliferation, metabolism, and nuclear receptor regulation of bile acids. 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TI - Minimal Role of Hepatic Transporters in the Hepatoprotection against LCA-Induced Intrahepatic Cholestasis
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfm287
DA - 2008-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/minimal-role-of-hepatic-transporters-in-the-hepatoprotection-against-LCtWSWn4LF
SP - 196
EP - 204
VL - 102
IS - 1
DP - DeepDyve
ER -