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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (M.J.Z.-G., K.N., X.T., A.S.B., K.L.R.B.); and Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania (K.L., M.G.B., G.D.K.)
Received June 27, 2006; accepted September 18, 2006.
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Abstract |
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20 and 20%, 4-methylumbelliferyl sulfate was reduced 50 and 65%, and harmol sulfate was decreased 30 and 45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by 96, 85, and 40%, respectively, in the livers of Abcc3–/– mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.
; Suzuki et al., 2003), and it is now known that MRP2 (ABCC2) and breast cancer resistance protein (ABCG2) mediate the biliary excretion of many phase II conjugates. In contrast to canalicular excretion, hepatic basolateral efflux mechanisms are poorly understood.
Recently, two members of the MRP family, MRP3 and MRP4 (Belinsky et al., 1998; Lee et al., 1998), have been implicated in hepatic basolateral excretion. MRP3 is expressed at low levels on the hepatic basolateral membrane in humans and rats, but it is susceptible to striking induction in cholestasis (Hirohashi et al., 1998; Konig et al., 1999). The substrate selectivity of MRP3 includes phase II metabolites, such as glucuronide and glutathione conjugates, as well as bile acids (Hirohashi et al., 1999, 2000; Zeng et al., 1999). Mrp2-deficient rats, and to a lesser extent Mrp2 knockout mice, have increased hepatic expression of Mrp3 (Hirohashi et al., 1998; Nezasa et al., 2006) and increased basolateral efflux of bilirubin glucuronides, acetaminophen glucuronide, the glucuronide conjugate of the steroid E3040, and 4-methylumbelliferyl glucuronide (Jansen et al., 1985; Takenaka et al., 1995; Xiong et al., 2000; Chu et al., 2006; Zamek-Gliszczynski et al., 2006a,c). These data implicate Mrp3 as a component of the basolateral excretory systems for glucuronide conjugates. MRP4 also has been localized to the hepatic basolateral membrane (Denk et al., 2004; Rius et al., 2003), and investigations of the pump's substrate selectivity indicate that it is able to transport glucuronide conjugates such as bilirubin glucuronide and E217
G and, to a lesser extent, glutathione conjugates (Chen et al., 2001; van Aubel et al., 2002). MRP4 transports bile acids with high affinity, but in contrast to MRP3, this activity seems to be glutathione-dependent (Rius et al., 2003, 2006). In addition, MRP4 mediates the transport of sulfated steroids, such as dehydroepiandrosterone sulfate, a property that is not shared by MRP3 (Zelcer et al., 2003).
The role of Mrp3 as a basolateral excretory system has been explored in vivo using Abcc3–/– mice. Induction of cholestasis in Abcc3–/– mice by bile duct ligation resulted in decreased concentrations of serum bilirubin glucuronide and increased hepatic concentrations of bile acid conjugates (Belinsky et al., 2005). Mice lacking Mrp3 exhibited normal unconjugated bile salt transport but decreased serum concentrations of glucuronide conjugates of bile acids (Zelcer et al., 2006). In addition, Abcc3–/– mice exhibited decreased serum concentrations of acetaminophen glucuronide and morphine glucuronide (Manautou et al., 2005; Zelcer et al., 2005). These studies demonstrating that Abcc3–/– mice have defects in sinusoidal excretion of glucuronide conjugates formed in the liver confirmed that Mrp3 is involved in the basolateral elimination of bile acids and certain glucuronide conjugates in vivo. However, because only a limited number of compounds have been investigated in Abcc3–/– mice, the full range of the pump's basolateral activities are unknown. This is particularly the case with respect to sulfate conjugates. MRP3 has the ability to transport certain sulfated bile acids in vitro (Hirohashi et al., 1999, 2000; Akita et al., 2002; Zelcer et al., 2003), which suggests that it may play a role in hepatic basolateral excretion of sulfate conjugates in vivo. However, Abcc3–/– mice exhibited only a very modest decrease in the basolateral efflux of acetaminophen sulfate (Manautou et al., 2005), raising the question whether Mrp3 is involved in basolateral transport of this important class of phase II conjugates. The in vivo activity of Mrp4 as a hepatic basolateral excretory system for xenobiotics has not been determined to any extent.
In the present studies, acetaminophen, 4-methylumbelliferone, and harmol (Fig. 1) were used as probes to study the hepatic disposition of sulfate and glucuronide conjugates in perfused livers of Abcc3–/– and Abcc4–/– mice. Results demonstrate that Mrp3 is not only involved in the basolateral excretion of glucuronide conjugates but that its activity also extends to sulfate conjugates. In addition, data demonstrate that Mrp4 is able to transport sulfate conjugates across the basolateral membrane into blood, but Mrp4 has little activity with respect to the glucuronide conjugates examined. This study thus extends our understanding of the in vivo involvement of Mrp3 in the basolateral efflux of xenobiotics and also provides the first direct evaluation of the role of Mrp4 in this process.
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Materials and Methods |
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Mice. Male C57BL/6 wild-type (age-matched heterozygotes) Abcc3–/– and Abcc4–/– mice (23–31 g) were used in this study. Generation of Abcc3–/– and Abcc4–/– mice were described previously (Belinsky et al., 2005). Mixed 129/C57BL/6 Abcc3–/– and Abcc4–/– mice were backcrossed to C57BL/6 for eight generations. Mice were maintained on a 12-h light/dark cycle with free access to water and rodent chow. All experimental procedures were performed under full anesthesia induced with ketamine/xylazine (140/8 mg/kg i.p.). The Institutional Animal Care and Use Committee at Fox Chase Cancer Center approved all animal procedures.
In Situ Liver Perfusion Experiments. The abdominal cavity of anesthetized mice was opened to expose the intestines, liver, and the gallbladder. The common bile duct was ligated above the duodenum to prevent bile from entering the intestine, and the gallbladder was cannulated with PE-10 tubing (Becton Dickinson, Parsippany, NJ). A loose suture was placed around the inferior vena cava below the liver. The portal vein was cannulated with a 20-gauge catheter (B. Braun Medical, Inc., Bethlehem, PA), and the liver was perfused (5 ml/min, drug-free continually oxygenated Krebs-Henseleit buffer containing 5 µM taurocholate). The abdominal vena cava below the liver was immediately severed by incision below the loose suture, and the inferior vena cava above the liver was cannulated with a 20-gauge catheter. Subsequently, the loose suture around the inferior vena cava was tied off to direct all perfusate outflow through the cannula inside the inferior vena cava above the liver. Following an 15-min preperfusion period for equilibration of liver temperature and bile flow, the liver was perfused with buffer containing 500 nM acetaminophen, 500 nM 4-methylumbelliferone, and 500 nM harmol for 60 min. A cassette dosing approach, which was validated previously for the study of the sulfate and glucuronide metabolites of these three compounds in mouse liver perfusions at 500 nM concentrations (Zamek-Gliszczynski et al., 2006c), was employed. Bile was collected in 10-min intervals; outflow perfusate was collected in 10- (0–30 min) and 5-min (30–60 min) intervals. At the end of the perfusion, livers were isolated and snap-frozen.
Analytical Methods. Bile, perfusate, and liver homogenate samples were analyzed by liquid chromatography with detection by tandem mass spectrometry (Applied Biosystems API 4000 triple quadrupole with TurboIonSpray interface; MDS Sciex, Concord, ON, Canada). AS, AG, 4-methylumbelliferone, 4MUS, 4MUG, HS, harmol glucuronide (HG), and the internal standard, cimetidine, were eluted from an Aquasil C18 column (2.1 x 50 mm, dp = 5 µm; Thermo Electron Corporation, Waltham, MA) using a mobile phase gradient (A, 0.05% formic acid; B, 0.05% formic acid in methanol; 0–0.75 min hold at 0% B; 0.75–2 min linear gradient to 70% B; 2–3.5 min hold at 70% B, 3.5–3.6 min linear gradient to 0% B, 3.6–4 min hold at 0% B; flow rate, 0.75 ml/min; 0.8–4 min directed to mass spectrometer) and were detected in negative ion mode using multiple reaction monitoring: AS, 230 
150 m/z; AG, 326 150 m/z; 4-methylumbelliferone, 175 133 m/z; 4MUG, 351 175 m/z; 4MUS, 255 175 m/z; HS, 277 197 m/z; HG, 374 198 m/z; and cimetidine, 251 157 m/z. Concentrations of acetaminophen, harmol, and the internal standard, cimetidine, were quantified using the chromatography conditions detailed above but were detected in positive ion mode using multiple reaction monitoring: acetaminophen, 152 110 m/z; harmol, 199 131 m/z; and cimetidine, 253 117 m/z. All analytes were quantified with standard curves prepared in the appropriate matrix, except HG, for which a pure standard was not available. Therefore, HG concentrations are expressed as the ratio of the analyte and internal standard peak areas. The lower limit of detection was 0.1 ng/ml for all analytes, standard curves ranged from 1 to 1000 ng/ml, and inter- and intraday CVs were <15%.
Immunoblot Analysis. Homogenates were prepared from perfused livers as described previously (Johnson et al., 2005). Total protein (50 µg per lane) was resolved on 4 to 12% Bis-Tris polyacrylamide gels under denaturing conditions (150 V, 2 h) and transferred to polyvinylidene fluoride membranes (38 V, 3 h). Membranes were blocked with 5% skim milk (0.5 h) and incubated (2 h) with primary antibodies (1:2000 dilution), followed by an incubation (2 h) with anti-rabbit or anti-rat horseradish peroxidase-conjugated secondary antibody (1:4000 dilution). Bands were visualized with SuperSignal West Dura chemiluminescence reagent (Pierce, Rockford, IL). Densitometric analysis of bands was conducted using Quantity One version 4.1 (Bio-Rad Laboratories, Hercules, CA).
Data Analysis. Metabolite formation clearance values were calculated as the ratio of the total metabolite recovery (sum of recovery in bile, outflow perfusate, and the liver at the end of the perfusion) and the area under the concentration-time curve of the parent compound in perfusate. The experiments were designed based on wild-type mouse pilot data, where steady-state conditions were attained for all metabolites within 20 min. Biliary and basolateral excretory unbound intrinsic clearance (Clbasolateral and biliary unbound intrinsic clearance, respectively) values for the metabolites were calculated as the ratio of the basolateral or biliary excretion rate and the hepatic unbound metabolite concentration at the end of the 60-min liver perfusion. All clearance values were normalized for liver mass.
All data are reported as mean ± S.D. (n = 3–4 per condition). Statistical significance was assessed by analysis of variance with Dunnett's post hoc test, except where the groups being compared had unequal variances or a data set failed the normality test, in which case analysis of variance on ranks with Dunn's post hoc test was used. In all cases, p < 0.05 was considered to be statistically significant.
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Results |
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Analysis of hepatic uptake and metabolism of acetaminophen, 4-methylumbelliferone, and harmol indicated that these parameters, which could affect basolateral elimination of metabolites, were comparable between mouse strains. Hepatic extraction ratios of acetaminophen [0.30 ± 0.04, 0.29 ± 0.02, and 0.32 ± 0.04 in wild-type, Abcc3–/–, and Abcc4–/– mouse livers, respectively], 4-methylumbelliferone (>0.95 in all mouse livers), and harmol (>0.94 in all mouse livers) were comparable between groups. AG accounted for the majority (92 ± 11%) of acetaminophen metabolized by the liver, whereas AS formation was less extensive (10 ± 2%). 4-Methylumbelliferone was recovered as 4MUG (77 ± 6%) and 4MUS (28.0 ± 0.2%). HS accounted for 36 ± 5% of the harmol dose. Formation clearances of metabolites (AS, 0.12 ± 0.01; AG, 1.05 ± 0.04; 4MUS, 1.1 ± 0.1; 4MUG, 3.1 ± 0.3; HS, 1.44 ± 0.03 ml/min/g liver), reflecting the volume of perfusate cleared of parent compound per unit time associated with the formation of a specific metabolite, were decreased slightly in the knockout mice, but the differences were not significant relative to wild-type mice except for the formation clearance of HS, which was significantly lower in Abcc4–/– mice (1.03 ± 0.06 ml/min/g liver).
Hepatic basolateral excretion of AS, 4MUS, and HS is summarized in Fig. 2 and Table 1. Although cumulative AS recovery in perfusate (Fig. 2A) and Clbasolateral values (Table 1) were not statistically different in livers from knockout mice, the excretion of this metabolite in perfusate of Abcc3–/– and Abcc4–/– mice was consistently reduced. Relative to wild-type mouse livers, outflow perfusate concentrations of AS were 30% lower, and the cumulative 60-min recovery of AS in perfusate was decreased 35% in both Abcc3–/– and Abcc4–/– mouse livers. These changes could be attributed to a decrease in Clbasolateral of AS both in Abcc3–/– and Abcc4–/– mice (Table 1). The Clbasolateral of 4MUS was significantly impaired in Abcc3–/–, and Abcc4–/– livers compared with wild type, resulting in lower perfusate concentrations and cumulative basolateral excretion of 4MUS in the knockout mice (Fig. 2, C and D). Like-wise, perfusate concentrations and cumulative basolateral excretion of HS were lower in the absence of hepatic Mrp3 and Mrp4 (Fig. 2, E and F) due to a decrease in Clbasolateral of HS (30 and 45% in livers from Abcc3–/– and Abcc4–/– mice, respectively; Table 1). In all cases, neither Cliver unbound (Table 1) of any sulfate metabolite nor cumulative biliary excretion was significantly different between wild-type and knockout mice.
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Hepatic basolateral excretion of glucuronide conjugates was decreased markedly in Abcc3–/– mice but was largely unaffected in Abcc4–/– mice (Fig. 3; Table 2). Perfusate concentrations and cumulative basolateral excretion of AG were reduced 80 to 85% in livers of Abcc3–/– mice (Fig. 3, A and B). The basolateral excretory clearance of AG was decreased 25-fold in Abcc3–/– mice, and there was a corresponding 6-fold increase in hepatic unbound concentrations of AG (Table 2). Perfusate concentrations and cumulative recovery of 4MUG also were markedly decreased (60–70%) in livers of Abcc3–/– mice (Fig. 2, C and D). Compared with wild-type mice, basolateral excretory clearance of 4MUG was decreased 6-fold, and the unbound hepatic concentrations were 2.5-fold higher in Abcc3–/– mice (Table 2). In addition, significant increases were observed in the cumulative biliary excretion for both AG and 4MUG in Abcc3–/– mice (7and 5-fold, respectively). HG perfusate concentrations and recovery (data not shown because concentrations were quantified as analyte to internal standard peak area ratios) and Clbasolateral (Table 2) also were decreased in Abcc3–/– mice, but not to the extent observed for the other two glucuronide conjugates. Unbound hepatic concentrations of HG were not altered significantly by the absence of Mrp3 or Mrp4 (data not shown). In striking contrast to Abcc3–/– mice, significant differences were not observed for basolateral excretion of glucuronide conjugates in Abcc4–/– mice (Fig. 3; Table 2; data not shown), although Clbasolateral of HG seemed slightly decreased (20%) in livers from Abcc4–/– mice (Table 2).
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Discussion |
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; Zelcer et al., 2005; Belinsky et al., 2006). Our results demonstrating impaired hepatic basolateral excretion of AS, 4MUS, and HS in Abcc3–/– mice established that Mrp3 not only transports glucuronide conjugates and bile acids into sinusoidal blood but that it also mediates the transport of sulfate metabolites—the products of an important metabolic pathway for both endo-and xenobiotics. In addition, our analysis of glucuronide excretion in Abcc3–/– mice also provides details on how Mrp3 functions with respect to this class of compounds. We found that basolateral excretion of all three of the glucuronide conjugates examined was impaired in Abcc3–/– mice, but not to the same extent. Basolateral excretory clearance of AG and 4MUG was profoundly affected, in accord with previous reports on AG and morphine glucuronide disposition in Abcc3–/– mice (Manautou et al., 2005; Zelcer et al., 2005); the impact of Mrp3 deficiency on HG Clbasolateral was less marked. This finding suggests that, although Mrp3 is a versatile basolateral transporter of glucuronide conjugates, its impact is dependent upon the properties of the anionic species. This notion is in accord with the physiochemical properties of AG, 4MUG, and HG. AG [logDpH7.4 =–5.9, mol. wt. = 327.3, monocyclic (ACD, v. 4.67; Advanced Chemistry Development, Inc., Toronto, ON, Canada)] and 4MUG [logDpH7.4 = –4.6, mol. wt. = 352.3, bicyclic (ACD, v. 4.67)] are both anionic and very hydrophilic at physiological pH, whereas HG (logDpH7.4 = –3.1, mol. wt. = 374.3, tricyclic) is predominantly zwitterionic, relatively less hydrophilic, and is also the largest molecule. Our results showing that loss of Mrp3 function has a greater affect on basolateral excretion of glucuronide than that of sulfate conjugates provide an explanation for why hepatic basolateral excretion of glucuronide conjugates is sensitive to cholestatic or pharmacological modulation of Mrp3 expression but has markedly less influence on the basolateral excretion of sulfate metabolites (Takenaka et al., 1995; Xiong et al., 2000; 2002; Zamek-Gliszczynski et al., 2005, 2006a).
This study is the first to directly assess the hepatic function of Mrp4 as a basolateral excretory system for xenobiotics. Our results showing that Abcc4–/– mice exhibit impaired basolateral excretion of AS, 4MUS, and HS established that Mrp4 functions to mediate the transport of sulfate conjugates into sinusoidal blood. These findings concerning Mrp4 and sulfate conjugate transport are in accord with reports that Mrp4 transports sulfated bile acids and steroids in vitro (Rius et al., 2003; Zelcer et al., 2003). Although our results demonstrate that the impact of Mrp4 on sulfate excretion is similar to that of Mrp3, there is a significant difference in the in vivo function of the two pumps with respect to glucuronide conjugates. In contrast to the marked effect of Mrp3 on glucuronide excretion, the Abcc4–/– mouse livers did not exhibit significant impairment in glucuronide excretion. The absence of impairment in glucuronide transport also was evident in a recent report (Mennone et al., 2006) showing that the Abcc4–/– mice made cholestatic by bile duct ligation have reduced basolateral excretion of bile acids but unaltered basolateral excretion of bilirubin glucuronide. Although Mrp4 did not seem to play a major role in the transport of the glucuronide conjugates of the probes we employed in our study, the in vitro activity of MRP4 toward the model glucuronide E217G seems to be comparable with that of MRP3 (Hirohashi et al., 1999; Zeng et al., 1999; Chen et al., 2001). Thus, we anticipate that Mrp4 may be involved in the basolateral excretion of certain glucuronide conjugates.
In mice, sulfate metabolites examined in the present studies are excreted into bile predominantly by the breast cancer resistance protein, whereas the glucuronide conjugates are transported by both the breast cancer resistance protein and Mrp2 (Zamek-Gliszczynski et al., 2006c). The current studies provide functional data consistent with the maintenance of breast cancer resistance protein and Mrp2 in livers of Abcc3–/– and Abcc4–/– mice (Belinsky et al., 2005; Manautou et al., 2005). Genetic deficiency of Mrp2 results in profound up-regulation of Mrp3 expression and function in rats and, to a lesser extent, in mice (Hirohashi et al., 1998; Xiong et al., 2002; Zamek-Gliszczynski et al., 2003; Nezasa et al., 2006), whereas genetic deficiency of Mrp3 does not have an appreciable effect on the expression and function of canalicular Mrp2. Furthermore, phase II metabolism may be altered in transport knockout mice (Zamek-Gliszczynski et al., 2006b). In the present studies, sulfation was 30% lower in Abcc4–/– mice, although this difference was only significant for the formation of HS. These findings are consistent with a report that Mrp4 expression in liver appears to be subject to coordinate regulation with the bile acid sulfotransferase (Schuetz et al., 2001; Zelcer et al., 2003; Assem et al., 2004). Thus, adaptive regulatory compensation that alters the expression of other transporters and/or enzymes must be considered when interpreting data from knockout models.
The present liver perfusion experiments were designed based on wild-type mouse pilot data, where steady-state conditions were attained for all metabolites within 20 min. Steady state is attained when the input rate of the metabolite into the liver is equivalent to the output rate of the metabolite from the liver, i.e., the formation rate is equivalent to the sum of biliary and basolateral excretion rates. Steady-state conditions observed in wild-type mouse livers also were attained for all sulfate conjugates and HG in both Abcc3–/– and Abcc4–/– mouse livers (output rate deviated from the input rate by less than 20%). However, Mrp3 is the predominant route of AG and 4MUG hepatic excretion; thus, the time to achieve steady state is considerably longer in Abcc3–/– mice, and steady-state conditions were not attained during the 60-min liver perfusion. The increase in biliary recovery of AG and 4MUG conjugates in Abcc3–/– mice (Fig. 3) is proportional to the increase in hepatic concentrations (Table 2); thus, there is no evidence of nonlinear kinetics.
Increased unbound hepatic concentrations and biliary recovery of AG and 4MUG were noted in Abcc3–/– mice, consistent with the fact that Mrp3 accounts for the majority of basolateral and total hepatic excretion of these conjugates. The basolateral excretion of the other metabolites was mediated by multiple transport mechanisms and/or did not account for the majority of total hepatic excretion. These data clearly demonstrate that, in knockout mice, alterations in hepatic exposure and the directionality of excretion of a metabolite depend on the importance of the specific protein governing the route of excretion that is genetically ablated.
In summary, the current study utilized Abcc3–/– and Abcc4–/– mice to investigate the role of Mrp3 and Mrp4 in the hepatic basolateral excretion of sulfate and glucuronide conjugates formed in the liver. Mrp3 was primarily responsible for the basolateral excretion of glucuronide metabolites, whereas multiple basolateral transport mechanisms, including but not limited to Mrp3 and Mrp4, were involved in the transport of sulfate conjugates.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Mrp, Abcc, multidrug resistance-associated protein; AG, acetaminophen glucuronide; 4MUS, 4-methylumbelliferyl sulfate; 4MUG, 4-methylumbelliferyl glucuronide; AS, acetaminophen sulfate; HS, harmol sulfate; HG, harmol glucuronide; Clbasolateral, basolateral excretory unbound intrinsic clearance.
1 Current affiliation: Eli Lilly and Co., Drug Disposition, Indianapolis, Indiana. 
2 Current affiliation: Shionogi & Co., Ltd., Development Research Laboratories, Toyonaka, Osaka, Japan.
Address correspondence to: Dr. Kim L.R. Brouwer, University of North Carolina School of Pharmacy, Kerr Hall, CB 7360, Chapel Hill, NC 27599-7360. E-mail: kbrouwer{at}unc.edu
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), Abcc3–/– (
), and Abcc4–/– (
) mice.