Materials and Methods

Chemicals.

APAP and taurocholate (TC) were purchased from Sigma Chemical Co. (St. Louis, MO). AG and AS were gifts from McNeil Pharmaceuticals (Ft. Washington, PA). [³H]AG (12.4 Ci/mmol) was synthesized by Amersham Life Sciences (Buckinghamshire, England) and was >99% pure as determined by HPLC. [³H]TC (2.4 Ci/mmol) was purchased from New England Nuclear Research Products (Boston, MA). All other chemicals were of analytical reagent grade.

Animals.

Male Sprague-Dawley rats (∼250 g; Charles River Laboratories, Raleigh, NC) were used to prepare liver plasma membrane vesicles. For isolated perfused liver studies, male Wistar rats (240–255 g; Charles River Laboratories) and TR⁻rats (240–265 g; breeding colony of the Academic Medical Center, Amsterdam, The Netherlands) were used as liver donors. Male retired breeders (Wistar; Charles River Laboratories) were used as blood donors. Rats were maintained on a 12-h light/dark cycle. Access to rat chow and water was allowed ad libitum. Rats were allowed to acclimate for at least 5 days before experimentation. The Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill approved all procedures.

Preparation of Rat Liver Plasma Membrane Vesicles.

Livers (∼40 g) from four rats were minced and homogenized in 720 ml of 1 mM sodium bicarbonate/0.1 mM phenylmethylsulfonyl fluoride (pH 7.4) with a loose-fitting Dounce tissue grinder. The homogenate was filtered through cheesecloth and centrifuged at 1500g_av for 15 min. The resulting pellet was mixed with 2.2 volumes of 70% sucrose. The mixture was overlaid with discontinuous sucrose gradients (44%/36.5%; 8 ml/8 ml) and centrifuged at 39,500g_av for 1.5 h. The mixed LPM at the 44%/36.5% interface was collected. Separation of cLPM and bLPM subfractions was performed as described by Meier and Boyer (1990). The resulting purified cLPM and bLPM fractions were resuspended in standard membrane suspension buffer [250 mM sucrose, 10 mM Hepes/Tris (pH 7.4), and 0.2 mM CaCl₂], homogenized, and stored at −80°C. The specific activities of Na⁺,K⁺- ATPase, Mg²⁺-ATPase, and alkaline phosphatase were determined for each preparation according to Schoner et al. (1967) andBessey et al. (1946). Glucose-6-phosphatase (microsomal marker) was assessed by monitoring production of inorganic phosphate (Fiske and Subbarow, 1925). Mitochondrial contamination (succinate dehydrogenase) was determined by the method of Shephard and Hübscher (1969). Vesicle orientation was assessed by measuring sialic acid liberation from membrane vesicles (Warren, 1959; Steck and Kant, 1974).

Measurement of Solute Transport in cLPM Vesicles.

Frozen membrane suspensions were thawed quickly in a 37°C water bath, and passed repeatedly (15 times) through a 27-gauge needle. [³H]TC was used as a positive control to verify integrity of ATP-dependent and membrane potential-dependent solute uptake by cLPM vesicles. In studies where preloading of the vesicles with potassium thiocyanate or potassium bicarbonate (KHCO₃) was required, the compound was added before revesiculation of the thawed membranes. Uptake of [³H]TC and [³H]AG into cLPM vesicles was measured by a rapid Millipore filtration system (Millipore Corp., Bedford, MA). Aliquots of membrane suspensions (20 μl; 30–60 μg of protein for TC; 60–100 μg of protein for AG) were preincubated for 5 min at 37°C, and uptake was initiated by the addition of 80 μl of prewarmed incubation medium to the membrane suspensions. The incubation buffer for AG transport consisted of 250 mM sucrose, 10 mM Hepes/Tris (pH 7.4), and 0.2 mM CaCl₂. In studies examining the effects of ATP on solute transport, the incubation buffer also contained an ATP-regenerating system (10 mM phosphocreatine, 100 μg/ml creatine phosphokinase, and 10 mM MgCl₂) with or without ATP. Nonspecific binding of [³H]TC and [³H]AG was determined in each experiment. These values were subtracted from all determinations.

Isolated Perfused Liver Experiments.

Livers were isolated and perfused by standard techniques (Brouwer and Thurman, 1996). After anesthesia (ketamine, 60 mg/kg, and xylazine, 12 mg/kg i.p.), the bile duct and portal vein were cannulated and the liver was perfused in situ with oxygenated Krebs-Ringer bicarbonate buffer maintained at 37°C. After the liver was transferred to a 37°C perfusion chamber, perfusion was continued with 80 ml of recirculating oxygenated Krebs-Ringer bicarbonate buffer containing 1% dextrose (w/v) and 20% (v/v) heparinized (1000 U/ml) male rat blood at a constant flow rate of 20 ml/min. A constant infusion of sodium taurocholate (30 μmol/h) was delivered into the perfusate reservoir at a rate of 2 ml/h. The liver and perfusate were allowed to equilibrate for ∼15 min before administration of APAP (10 mg in 1 ml). The liver was perfused for 120 min. Bile was collected continuously and 0.5 ml of perfusate samples was collected at 15-min intervals. After the 120-min perfusion period, livers were removed from the chamber, blotted, and weighed. All samples were frozen at −20°C until assayed. Liver viability was determined by bile flow rate (≥0.8 and ≥0.13 μl/min/g of liver for control and TR⁻ rats, respectively), constant inflow perfusion pressure (<15 cm of H₂O), and the release of lactate dehydrogenase from the liver (<0.1 I.U./g of liver/h; assayed with Sigma lactate dehydrogenase diagnostic kit, catalog 500).

Assay Methodology.

APAP, AG, and AS concentrations in perfusate and bile were quantitated by the HPLC method of Brouwer and Jones (1990). Standard curves for APAP, AG, and AS in perfusate (5–500 μg/ml) and bile (25–1000 μg/ml) were linear (r ≥ 0.997) and were prepared daily. The lower limit of quantitation for APAP, AG, and AS was 2 μg/ml. AG and AS concentrations were expressed as APAP equivalents by determining the conversion factors (0.374 for AG and 0.544 for AS) as described by Brouwer and Jones (1990).

Pharmacokinetic Modeling and Simulations.

A compartmental modeling approach was used to describe the hepatobiliary disposition of APAP, AG, and AS in the isolated perfused rat liver. APAP perfusate concentration versus time data were analyzed by a one-compartment model; the elimination rate constant for APAP initially was estimated from the slope of the terminal phase of the log-concentration versus time profile. APAP was assumed to be eliminated from the system via the formation of AG, AS, and a non-AG/AS pathway, including the biliary excretion of APAP. Rate constants for the formation of AG and AS, as well as the non-AG/AS pathway, were estimated based on the amount of each metabolite recovered at the end of the perfusion. AG and AS biliary excretion rates were calculated as the product of bile flow and biliary concentration during each collection period. To describe the disposition of AG and AS, the liver, perfusate, and bile were considered as three distinct compartments. Differential equations based on the mass balance of APAP, AG, and AS in each compartment were resolved simultaneously by nonlinear least-squares regression (WinNonlin 1.1; Pharsight Corporation, Mountain View, CA). A weighting scheme of 1/(Y_predicted)²was used. The apparent volume of distribution for APAP was restricted within a physiologically meaningful range (85–110 ml) and was based on the perfusate volume plus the estimated distribution volume of APAP in the liver assuming a liver/body partition coefficient of ∼1.5 (Ara and Ahmad, 1980). Various models using linear and saturable processes were fit to the data. The goodness of fit of each model was assessed by visual examination of the distribution of residuals, the condition number, and Akaike's information criterion (Akaike, 1976).

The cumulative excretion of AG in bile, and perfusate AG concentration versus time profiles, were simulated with the model that best described the hepatobiliary disposition of APAP, AG, and AS in isolated perfused livers from control rats to examine the possibility that impaired biliary excretion of AG in isolated perfused TR⁻rat livers was due solely to an increase in the basolateral egress of AG, instead of to a deficiency in Mrp2-mediated biliary excretion. The basolateral egress rate constant for AG (K_PAG) was increased until the simulated cumulative biliary excretion of AG approximated the actual mass excreted in bile, and the impact on the appearance of AG in perfusate was examined. Simulations of the perfusate AG concentration versus time profiles with the model that best described the hepatobiliary disposition of APAP, AG, and AS in isolated perfused TR⁻ rat livers were conducted to test the hypothesis that the increased basolateral egress of AG in isolated perfused TR⁻ rat livers was due to up-regulation of the rate process and not due solely to an increased driving force because AG biliary excretion was impaired. In these simulations,K_PAG was fixed at the mean value estimated for control livers, and the impact on the appearance of AG in perfusate was assessed.

Statistics.

Two-way ANOVA tables were constructed to examine the effects of time and ATP concentration on AG uptake in rat cLPM vesicles. When significant effects were noted with no interactions between independent variables, one-way ANOVA was performed to examine the effects of a single factor on AG uptake. The Student'st test was used to determine statistically significant differences between the control and TR⁻ livers with respect to the total amount of each metabolite recovered in perfusate and bile, and the model parameter estimates. In all cases, data were presented as mean ± S.D. and the criterion for statistical significance was P < .05.

Results

cLPM Vesicle Uptake Studies.

cLPM fractions were enriched ∼37- and ∼59-fold in the canalicular membrane markers alkaline phosphatase and Mg²⁺-ATPase, respectively; enrichment of the basolateral membrane marker Na⁺,K⁺-ATPase was ∼4-fold. The low enrichment of marker enzymes glucose-6-phosphatase (∼0.7) and succinate dehydrogenase (∼0.03) indicated minimal microsomal and mitochondrial contamination of the cLPM fractions, respectively. Assessment of sialic acid liberation from canalicular membranes indicated that 81.9 ± 6.4% of cLPM vesicles were oriented right-side out.

Uptake of 1 μM [³H]TC into cLPM vesicles was stimulated markedly by 1 mM ATP; uptake exceeded equilibrium values at early time points in the presence of ATP (data not shown). These data demonstrate functional integrity of ATP-dependent solute transport in cLPM vesicles. Accumulation of [³H]AG in cLPM vesicles was temperature dependent, but did not exceed equilibrium values in the absence and presence of ATP. [³H]AG uptake increased significantly with both duration of uptake (P < .0001) and ATP (P = .0012; two-way ANOVA). The effect of ATP on [³H]AG uptake was most evident at 5 and 8 min (P = .0290, P = .0189, respectively; one-way ANOVA) (Fig. 1). Uptake of 1 μM [³H]TC in cLPM vesicles was stimulated by an inside-positive diffusion potential generated by preloading the vesicles with potassium thiocyanate followed by incubation of the vesicles with potassium gluconate (data not shown). [³H]TC uptake at 20 s was significantly greater than equilibrium values (60 min) (P = .0337; one-way ANOVA). In contrast, uptake of 5 μM [³H]AG in the same cLPM preparations was not stimulated by an inside-positive diffusion potential. To determine whether [³H]AG uptake was facilitated by exchange with bicarbonate, cLPM vesicles were preloaded with KHCO₃, and either KHCO₃(control) or potassium gluconate (treated) was added to the incubation medium with [³H]AG. Uptake of 5 μM [³H]AG in cLPM vesicles was not stimulated by an outwardly directed bicarbonate gradient (Table1). In additional studies, uptake of 5 μM [³H]AG in cLPM vesicles preloaded with standard membrane suspension buffer (pH 7.4) was not significantly different when incubated under the following inside/outside pH conditions: 7.4/6.0, 7.4/7.4, and 7.4/8.0 (Table 1).

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Figure 1

Time course and temperature dependence of [³H]AG uptake in rat cLPM vesicles incubated with 5 μM [³H]AG in the absence (open symbols) or presence (closed symbols) of 1 mM ATP in incubation medium containing an ATP-regenerating system at either 4°C (■, ▪) or 37°C (○,●). [³H]AG uptake was influenced significantly by time (P < .0001) and by treatment (P = .0012). Values represent mean ± S.D. (n = 3); *P < .05.

Isolated Perfused Liver Studies.

Livers from TR⁻ rats (12.35 ± 0.93 g) were significantly larger than control livers (9.72 ± 1.10 g,P < .01) even though body weights were similar. The bile flow rate in TR⁻ rat livers (0.28 ± 0.07 μl/min/g of liver) was significantly lower than in control livers (1.13 ± 0.10 μl/min/g of liver, P < .0001).

The percentage of the APAP dose recovered as APAP, AG, and AS in perfusate and bile during the 2-h perfusion is shown in Table2. In both control and TR⁻ rat livers, the amount of APAP recovered in bile was very low, and the concentration of APAP in the perfusate at the end of the 2-h perfusion was below the limit of quantitation. Although the total recovery of AG and AS was not significantly different between the control and TR⁻ rat livers, the percentage of the APAP dose recovered as AG and AS in perfusate was significantly higher, and the percentage of the APAP dose recovered as AG and AS in bile was significantly lower in TR⁻ versus control rat livers.

Pharmacokinetic Modeling.

The model shown in Fig.2 best described the disposition of APAP, AG, and AS in isolated perfused livers from control rats. For the APAP dose used in this study, AG formation and the non-AG/AS pathway (K_OTHER) were described best as first order processes, and AS formation was described best as a saturable process. The Michaelis-Menten constant for AS formation (K_mAS) was assigned a value of 17 μg/ml based on previous work (Watari et al., 1983; Lin and Levy, 1986; Tone et al., 1990). Data from TR⁻ rats were best described by a model that excluded both the non-AG/AS elimination pathway for APAP (characterized by K_OTHER) and AG biliary excretion (K_BAG) because the biliary excretion of unchanged APAP and the formation of other metabolites, as well as the amount of AG excreted in bile, were negligible. Observed data points and model-generated best fit curves of APAP, AG, and AS perfusate concentration versus time profiles, and AG and AS biliary excretion rate versus time profiles from representative control and TR⁻ rat livers, are shown in Fig.3. APAP perfusate concentration versus time profiles in control and TR⁻ rat livers were similar. However, AG and AS perfusate concentrations were higher, and AS biliary excretion rates were markedly lower, in livers from TR⁻ rats.

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Figure 2

Model scheme depicting the disposition of APAP, AG, and AS in the isolated perfused rat liver. Processes characterized byK_OTHER and K_BAGwere excluded in isolated perfused TR⁻ rat livers. Parameters are designated as follows:K_AG, first order rate constant for AG formation; K_mAS andV_maxAS, Michaelis-Menten constant and maximum velocity for AS formation; K_OTHER, first order rate constant for all elimination pathways other than the formation of AG and AS; K_PAG andK_PAS, rate constants for the basolateral egress of AG and AS, respectively; K_BAG andK_BAS, rate constants for the canalicular egress of AG and AS, respectively; CL_AS, clearance of AS from perfusate to hepatocytes; V, apparent volume of distribution of APAP; and V_R, volume of perfusate.

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Figure 3

Representative perfusate concentration versus time profiles (A and B) and biliary excretion rate versus time profiles (C and D) for APAP (■), AG (●), and AS (▴) in isolated perfused livers from control and TR⁻ rats. Symbols denote observed perfusate concentrations or biliary excretion rates. Lines represent the best fit of the model depicted in Fig. 2 to the data. Perfusate concentrations and biliary excretion rates of AG and AS were expressed as APAP equivalents. The amount of AG in bile was negligible in TR⁻ rat livers.

The pharmacokinetic parameters describing the hepatobiliary disposition of APAP, AG, and AS in control and TR⁻ rat livers are provided in Table 3. In TR⁻ rat livers, the rate constant for the basolateral egress of AG (K_PAG) was increased ∼7-fold, and the rate constant for the biliary excretion of AS (K_BAS) was decreased ∼10-fold. TheV_max for AS formation (V_maxAS) and the clearance of AS from perfusate to hepatocytes (CL_AS) tended to increase in TR⁻ rat livers, but these trends were not statistically significant.

Based on the model depicted in Fig. 2, and using the mean values of the parameter estimates shown in Table 3, the simulated perfusate concentration versus time profiles for AG in isolated perfused livers from control and TR⁻ rats described the mean data well (Fig. 4, A and B). WhenK_PAG was increased 10-, 100- and 1000-fold, the cumulative biliary excretion of AG decreased from 13% to 2.5%, 0.26% and 0.03% of the dose, respectively, in control livers. In all cases, the AG concentrations in perfusate increased more rapidly and were markedly higher than the observed values (Fig. 4A). The simulated perfusate concentration versus time profiles for AG in TR⁻ livers were significantly underestimated when K_PAG was fixed at the mean value associated with control livers (Fig. 4B).

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Figure 4

Observed and simulated perfusate AG concentration versus time profiles in isolated perfused livers from control (A) and TR⁻ rats (B). Observed values (symbols) represent mean ± S.D. A, perfusate AG concentration versus time profiles were simulated with the model that best described the data from isolated perfused control rat livers, whereK_PAG was fixed at 0.028 min⁻¹ (solid line), 0.28 min⁻¹ (long-dashed line), 2.8 min⁻¹ (short-dashed line), and 28 min⁻¹ (dotted line), respectively. The mean parameter estimates for control rat livers, as shown in Table 3, were used for all other model parameters. B, perfusate AG concentration versus time profiles were simulated with the model that best described the data from isolated perfused TR⁻ rat livers.K_PAG was fixed at the mean value estimated for TR⁻ rat livers (0.206 min⁻¹, solid line) or control rat livers (0.028 min⁻¹, dashed line). The mean parameter estimates for TR⁻ rat livers, as shown in Table3, were used for all other model parameters.

Discussion

Initial studies were conducted in rat cLPM vesicles. The specific activity and relative enrichment of membrane marker enzymes in cLPM preparations were consistent with literature values (Meier et al., 1984b). Taurocholate uptake was stimulated markedly by ATP in cLPM vesicles, consistent with previously published data (Müller et al., 1991; Nishida et al., 1991). AG uptake in cLPM vesicles was temperature dependent, and enhanced modestly by ATP (significant differences in AG uptake were noted at 5 and 8 min); however, AG uptake did not exceed equilibrium values at any time through 60 min. Similar results were reported for S-ethylglutathione (Ballatori and Truong, 1995). S-ethylglutathione uptake in cLPM vesicles exhibited minimal stimulation by ATP (10–20% above control values), and the stimulatory effects of ATP were evident only at later time points. Typical Mrp2 substrates such as dinitrophenyl glutathione and bilirubin diglucuronide exhibit rapid initial uptake and extensive accumulation above equilibrium values in the presence of ATP at early time points; uptake declines at later times as ATP concentrations in the incubation medium decrease (Kobayashi et al., 1990; Nishida et al., 1992).

Temperature-dependent transport of AG in the absence of ATP in cLPM vesicles suggested that an ATP-independent canalicular transport mechanism also might be involved. Taurocholate was used as a positive control for ATP-independent canalicular transport mechanisms. As expected, taurocholate uptake in cLPM vesicles was stimulated by an inside-positive diffusion potential (Meier et al., 1984a). In contrast, AG uptake in cLPM vesicles was not stimulated by an inwardly directed positive diffusion potential. Alternate mechanisms for AG transport also were examined. Meier et al. (1985) reported electroneutral chloride/bicarbonate exchange in rat cLPM vesicles, and postulated that canalicular translocation of organic anions may be facilitated by a secondary exchange process with bicarbonate. However, AG uptake in cLPM vesicles was not facilitated by the presence of an outwardly directed bicarbonate gradient. Furthermore, changes in extravesicular pH over a range of 6.0 to 8.0 had no effect on AG uptake into cLPM vesicles with an intravesicular pH of 7.4.

Modest stimulation of AG uptake in cLPM vesicles by ATP was consistent with the hypothesis that AG is a low-affinity substrate for Mrp2, an ATP-dependent organic anion transporter on the canalicular membrane. Rat cLPM vesicles may not be a practical system, from an animal consumption standpoint, for investigating transport mechanisms for low-affinity substrates due to a relatively low intra- to extravesicular concentration ratio. The TR⁻Wistar rat, an Mrp2/cMOAT-deficient mutant strain that exhibits an autosomal recessive defect in the biliary excretion of many multivalent organic anions (Jansen et al., 1985), was used to test the hypothesis that AG is an Mrp2 substrate. Molecular cloning of rat Mrp2 revealed that a one base-pair deletion at amino acid 393 introduces a premature stop codon in TR⁻ rats, which results in decreased mRNA levels and complete absence of the protein from the canalicular membrane in the liver (Paulusma et al., 1996). Mrp2 is responsible for the biliary excretion of a variety of endogenous and exogenous organic anions, including many glucuronide conjugates, some glutathione conjugates and a few sulfate conjugates (Oude Elferink et al., 1995). The majority of Mrp2 substrates identified to date have at least two negative charges (Oude Elferink et al., 1995). AG is an organic anion with one negative charge at physiological pH. Although the formation of AG was not altered (Tables 2 and 3), the biliary excretion of AG in isolated perfused TR⁻ rat livers decreased over 300-fold (from 13.5% to only 0.04% of the dose). Simulation studies were consistent with the hypothesis that this decrease in the biliary excretion of AG could not result solely from increased basolateral egress of AG. For the biliary excretion of AG to be reduced to the observed level (0.04% of the dose), the rate constant for the basolateral egress of AG (K_PAG) would need to be increased by three orders of magnitude. Not only is this increase in the rate constant unrealistic but also the simulated perfusate AG concentrations markedly overestimated the observed data (Fig. 4A). The negligible biliary excretion of AG in TR⁻ rat livers, in conjunction with the simulation studies, clearly demonstrated that AG is an Mrp2 substrate, consistent with previous reports that multiple negative charges are not an absolute requirement for Mrp2 substrates (de Vries et al., 1989; Cui et al., 1999).

AS biliary excretion was significantly decreased in livers from TR⁻ rats, whereas AS formation remained unchanged (Tables 2 and 3). The amount of AS recovered in bile was decreased ∼5-fold, and the estimated rate constant for AS biliary excretion was decreased ∼10-fold, in TR⁻ rat livers, suggesting that AS also may be an Mrp2 substrate. AS has two negative charges and a molecular weight of 269, similar to other Mrp2 substrates. The fact that measurable concentrations of AS were detected in bile from TR⁻ rat livers suggests that AS may traverse the hepatic canalicular membrane by multiple mechanisms.

The majority of the APAP dose administered in the present study was accounted for as glucuronide and sulfate conjugates; a small fraction is metabolized by P450 enzymes and further conjugated with glutathione (Jollow et al., 1974, Miner and Kissinger, 1979). Glucuronidation and sulfation are both saturable processes at low doses. In vivo studies in rats have revealed that theK_m and V_max for APAP glucuronidation are ∼138 μg/ml and 417 μg/min/kg, respectively (Watari et al., 1983). TheV_max/K_m ratio (3.02 ml/min/kg) is consistent with our estimate for the formation clearance of AG in control livers (2.64 ml/min/kg) based onK_AG and V values in Table 3 and the average body weight of control rats (246 ± 4 g). TheK_m for in vivo APAP sulfation has been estimated independently by Watari et al. (1983), Lin and Levy (1986), and Tone et al. (1990), with mean values ranging from 15.5 μg/ml to 16.9 μg/ml. In the present single-dose study, theK_m for AS formation was assigned a value of 17 μg/ml, and V_maxAS was estimated based on the data.

Impaired AG and AS biliary excretion due to the absence of canalicular Mrp2 was not the only difference in the hepatobiliary disposition of APAP and metabolites between control and TR⁻ rat livers. Increased basolateral egress of AG from the liver to perfusate (∼7-fold increase in K_PAG) in TR⁻ rat hepatocytes was indicative of elevated levels of an AG transporter on the basolateral membrane. Further simulations demonstrated that impaired AG biliary excretion alone could not account for the increased basolateral egress of AG in isolated perfused TR⁻ rat livers. Although the final AG concentration in perfusate was similar, the simulated curves withK_PAG fixed at the control value significantly underestimated AG perfusate concentrations at early time points (Fig. 4B). Hirohashi et al. (1998) reported that Mrp3 was up-regulated in Mrp2-deficient Eisai hyperbilirubinemic rats and by bile duct ligation in Sprague-Dawley rats. König et al. (1999)have reported up-regulation of a basolateral MRP isoform (MRP3) in patients who are deficient in MRP2. Mrp3 shares some substrates with Mrp2, such as E3040 glucuronide, 4-methylumbelliferone glucuronide, 1-naphthol-β-d-glucuronide, and methotrexate (Hirohashi et al., 1999). In addition, Ogawa et al. (2000) reported a 10.6-fold increase in the expression of Mrp3 protein in Eisai hyperbilirubinemic rat livers, which is in close agreement with the 7-fold increase in K_PAG observed in the present study. Increased basolateral egress of AG in TR⁻ rat livers would be consistent with the hypothesis that AG is a substrate for basolateral Mrp3.

The application of pharmacokinetic modeling in this study provided important insights regarding the hepatobiliary disposition of APAP, AG, and AS that were not evident by mass balance analysis. By comparing the percentage of the APAP dose recovered as AG and AS in perfusate and bile (Table 2), it was not possible to determine whether differences in AG and AS recovery in control and TR⁻ rat livers were due to changes in transporter function in the canalicular membrane alone or due to changes in transporter function in both basolateral and canalicular membrane domains. Pharmacokinetic modeling provided estimates of rate constants for the formation and egress of AG and AS across the basolateral and canalicular membranes, thereby enabling a direct comparison of discrete transport processes.

In conclusion, results from these studies clearly demonstrate that AG, a monovalent organic anion, is a substrate for Mrp2, consistent with the finding of modest ATP-dependent transport of AG in cLPM vesicles. Data from the present study also suggest that AS is excreted into bile by multiple mechanisms, one of which may involve Mrp2. Increased basolateral egress of AG is consistent with up-regulation of an organic anion transporter on the basolateral membrane, which serves as a compensatory mechanism to avoid the accumulation of organic anions in TR⁻ rat livers. Finally, pharmacokinetic modeling and simulation studies emphasize the importance of recognizing potential alterations in substrate disposition at other sites when evaluating data generated in transport-deficient animal models.