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Research ArticleMetabolism, Transport, and Pharmacogenomics

How Organic Anions Accumulate in Hepatocytes Lacking Mrp2: Evidence in Rat Liver

Philippe Millet, Marcelle Moulin, Bruno Stieger, Youssef Daali and Catherine M. Pastor
Journal of Pharmacology and Experimental Therapeutics March 2011, 336 (3) 624-632; DOI: https://doi.org/10.1124/jpet.110.175406

Abstract

In the liver, the accumulation of hepatobiliary contrast agents is a crucial issue to understand the images of liver scintigraphy or magnetic resonance (MR) imaging. Thus, depending on the regulation of uptake and exit membrane systems in normal and injured hepatocytes, these contrast agents will accumulate differently within cells. Gadobenate dimeglumine (Gd-BOPTA) is a hepatobiliary MR contrast agent that distributes to the extracellular space and enters into rat hepatocytes through the sinusoidal transporters, organic anion-transporting polypeptides. Gd-BOPTA is not metabolized during its transport to the canalicular membrane where it is excreted into bile through multiple resistance protein-2 (Mrp2). It is not well known how Gd-BOPTA accumulates in normal livers and in livers lacking Mrp2. We perfused livers from normal rats and from rats lacking Mrp2 with 153Gd-BOPTA at increasing concentrations and assessed the hepatic accumulation of this agent using a gamma probe placed above the livers. By use of a pharmacokinetic model that best described the amounts of Gd-BOPTA in perfusate, bile, and hepatic tissue over time, we showed how increasing concentrations and the absence of Mrp2 modify the hepatic accumulation of the contrast agent. It is noteworthy that despite the absence of Gd-BOPTA bile excretion and a similar efflux back to sinusoids in livers lacking Mrp2, the maximal hepatic accumulation of contrast agent was similar to normal rats. We also showed how hepatic accumulation relies on the concomitant entry into and exit from hepatocytes. Such information improves our understanding of liver imaging associated with the perfusion of hepatobiliary contrast agents, which was recently introduced in clinical practice.

Introduction

The liver is an essential organ for detoxification and forms, after the intestine, the second barrier before the systemic circulation. Clearance of drugs from the systemic circulation is a complex process that involves a large array of transporters in both the kidney and the liver. For substances to be eliminated via the liver, the first transport step occurs at the sinusoidal membrane of hepatocytes (Hagenbuch, 2010). Drug detoxification activity of the liver is very important to predict their pharmacokinetic properties, as well as their pharmacological and adverse effects in extrahepatic organs (Kusuhara and Sugiyama, 2010; Rodrigues, 2010). After drug uptake, hepatocytes are also responsible for the hepatic metabolism and the excretion of drugs into bile through transporters located in the canalicular membrane formed between two adjacent hepatocytes. Finally, the sinusoidal membrane of hepatocytes possesses transporters used for the release of hepatic substances into the sinusoidal blood.

Besides the detoxification activity, other drugs such as statins act within hepatocytes; thus, the hepatic accumulation is important to understand. Once inside hepatocytes, the accumulation of statins will depend on bile excretion (Kusuhara and Sugiyama, 2010; Rodrigues, 2010). The accumulation of hepatobiliary contrast agents is another important issue to understand the images of liver scintigraphy or magnetic resonance (MR) imaging. Thus, depending on the regulation of uptake and exit membrane systems in normal and injured hepatocytes, these contrast agents will accumulate differently within cells and the better the understanding of their hepatic accumulation, the better the diagnosis of tumors.

Thus, liver images obtained with the hepatobiliary MR contrast agents Gd-BOPTA (gadobenate dimeglumine, MultiHance; Bracco, Milan, Italy) and Gd-EOB-DTPA (gadoxetic acid, Primovist; Bayer Schering Pharma, Berlin, Germany) have been correlated with the expression of human organic anion-transporting peptides (OATPs) and multiple resistance protein-2 (MRP2) transporters in hepatocellular carcinomas (Marin et al., 2009; Narita et al., 2009; Tanimoto et al., 2009; Tsuboyama et al., 2010).

We previously studied the transport of the hepatobiliary MR contrast agent Gd-BOPTA in isolated rat hepatocytes, hepatocytes injected into a bioreactor perfused with the contrast agent, or in isolated rat liver perfusions. In these experimental systems, Gd-BOPTA was measured by signal intensities (MR liver imaging) or radioactive counts. We also showed that, in rats, Gd-BOPTA distributes to the extracellular space and enters into hepatocytes through the sinusoidal transporters Oatps 1, 2, and 4 (Planchamp et al., 2007). Gd-BOPTA was not modified during its transport to the canalicular membrane, and its time transit through hepatocytes was lower than that observed with horseradish peroxidase, suggesting that Gd-BOPTA is not transported through vesicles as observed with the enzyme. Gd-BOPTA is excreted into bile through Mrp2 (de Haën et al., 1996; Planchamp et al., 2007).

By use of MR liver imaging, we also showed that Gd-BOPTA accumulates in mild cirrhotic livers similar to normal livers until the expression of Oatps is so low that Gd-BOPTA behaves as the extracellular contrast agent Gd-DTPA (gadopentetate dimeglumine, Magnevist; Bayer Schering Pharma) (Planchamp et al., 2005b). In the model of isolated rat liver perfusion, the aim of the present study is to investigate how Gd-BOPTA accumulates in normal livers and in livers from TR− rats that do not possess Mrp2. In contrast to most pharmacokinetic investigations that used plasma and hepatic clearance over time to estimate hepatobiliary transport, we directly measured the hepatic concentrations of Gd-BOPTA over time. By use of the pharmacokinetic model that best described the amounts of Gd-BOPTA in perfusate, bile, and hepatic tissue over time, we showed 1) how increasing Gd-BOPTA concentrations in in-flow perfusate and 2) how the absence of Mrp2 modifies the hepatic accumulation of the contrast agent.

Materials and Methods

Animals

Before liver perfusion, Sprague-Dawley rats that lack the canalicular transporter Mrp2 (TR− rats) were anesthetized with pentobarbital (50 mg/kg i.p.). The protocol was approved by the animal welfare committee of the University of Geneva and the veterinary office and followed the Guidelines for the Care and Use of Laboratory Animals.

Rat Liver Perfusion

Livers were perfused in situ as described previously (Pastor et al., 1996). In brief, the abdominal cavity was opened and the portal vein was cannulated and secured. A G16 catheter was introduced into the portal vein up to 2 to 3 mm from the liver. A ligature was placed around the inferior vena cava above the left renal vein. After the cannulation of the portal vein, the abdominal vena cava was transected and the Krebs-Henseleit-bicarbonate (KHB) solution was pumped without delay into the portal vein. The flow rate was increased slowly over 1 min up to 30 ml/min. In a second step, the chest was opened and a second cannula was inserted through the right atrium into the thoracic inferior vena cava and secured with a ligature. Finally, the ligature around the abdominal inferior vena cava was tightened. The KHB solution was perfused to the liver through the portal catheter and eliminated by the catheter placed in the thoracic inferior vena cava. In each experiment, the common bile duct was cannulated with a PE10 catheter.

The entire perfusion system consisted of a reservoir, a pump, a heating circulator, a bubble trap, a filter, and an oxygenator. The perfusate was equilibrated with a mixture of 95% O2, 5% CO2 during the protocol. The livers were perfused with a KHB buffer with or without contrast agents during the entire protocol using a nonrecirculating system (Fig. 1). The concentrations of contrast agents entering the portal vein were steady.

Fig. 1.
Fig. 1.

A, experimental protocol and data measured during liver perfusion with the contrast agents. B, pharmacokinetic modeling of Gd-BOPTA transport in rat perfused livers. Entry of Gd-BOPTA (B) and Gd-DTPA (D) into the liver is modeled as a zero-order infusion rate Kin. Compartment 1 illustrates the dilution of Gd-BOPTA at the beginning and at the end of perfusion. Compartments 2, 4, and 5 describe the liver: compartment 2 is the extracellular space, compartment 4 depicts the compartment of hepatocytes, and compartment 5 is an additional hepatocyte-associated space. Compartment 3 represents the amount of contrast agents in perfusate, and compartment 6 represents that in bile. First-order rate constants kij are defined with respect to compartment of origin i and compartment of convergence j.

Quantification of Hepatic BOPTA Accumulation

BOPTA was labeled by adding 153GdCl3 to a 0.5 M BOPTA solution (1 MBq/ml; MultiHance; Bracco), which contained a slight excess of the ligand BOPTA (Planchamp et al., 2005a,c). The contrast agent then was diluted in KHB solution to obtain various concentrations of perfusion: 40, 100, 200, 400, 800, and 1600 μM. To assess the exact intracellular concentrations of Gd-BOPTA, Gd-DTPA (Magnevist; Bayer Schering Pharma) was perfused at similar doses before Gd-BOPTA. Gd-DTPA has the same extracellular distribution as Gd-BOPTA but does not enter into hepatocytes. Thus, in the liver, the difference between both contrast agents corresponds to the accumulation of Gd-BOPTA within hepatocytes. To quantify hepatic Gd-BOPTA, a gamma scintillation probe that measures radioactivity every 20 s was placed 1 cm above the liver (Fig. 1). To transform radioactivity counts into contrast agent amounts, the radioactivity in the entire liver at the end of each experiment was measured (Activimeter Isomed 2000; MCD Nuklear Medizintechnik, Dresden, Germany) and related to the last count measured by the probe. Samples were also collected every 15 min for perfusate and every 5 min for bile to measure 153Gd-BOPTA.

Experimental Protocols

Perfusion of Increasing Concentrations of 153Gd-DTPA and 153Gd-BOPTA.

To study Gd-BOPTA transport in the entire liver, we perfused livers from 21 normal rats and 18 TR− rats with KHB solution (recovery period, 15 min), 153Gd-DTPA (15 min), KHB solution (rinse period, 30 min), 153Gd-BOPTA (30 min), and KHB solution (rinse period, 30 min) (Fig. 1). 153Gd-DTPA and 153Gd-BOPTA were perfused at various concentrations: 40, 100, 200, 400, 800, and 1600 μM.

During each perfusion, we measured bile flow (microliters per minute per gram), 153Gd-BOPTA bile accumulation (nanomoles per gram), 153Gd-BOPTA bile excretion rate (nanomoles per minute per gram), and 153Gd-BOPTA accumulation within the liver (nanomoles per gram) (Figs. 1 and 2).

Fig. 2.
Fig. 2.

Effects of 200 μM Gd-BOPTA perfusion (between 45 and 75 min, black horizontal bar) on liver and bile Gd-BOPTA accumulation (nanomoles per gram), Gd-BOPTA bile excretion rate (nanomoles per min per gram), and bile flow (microliters per minute per gram). From 0 to 15 min, livers were perfused with 200 μM Gd-DTPA (gray horizontal bar) and from 15 to 45 min and 75 to 105 min, livers were perfused with a Krebs-Henseleit bicarbonate solution (white horizontal bar). Liver perfusions from control rats (black squares) and from rats without Mrp2 expression (white squares) are illustrated.

Pharmacokinetic Modeling.

In perfused livers isolated from control rats, the compartment model that best described the amounts of contrast agents over time in perfusate, bile, and liver was published previously and illustrated in Fig. 1 (Planchamp et al., 2005c). Each compartment describes the amount of contrast agent over time. Compartment 1 (dilution) reflects the transition between the perfusion of both contrast agents and the KHB solution that induces a mixture of the two solutions before a steady state is reached. Compartment 2 shows the amount of contrast agent in the extracellular space, and compartments 4 and 5 show the amounts in two distinct hepatocytes compartments (hepatocytes A and B). The transfer between compartments 4 and 5 is unidirectional, and both compartments have access to bile (compartment 6). Compartment 3 represents the amount of contrast agent in the outflow perfusate leaving the liver. Entry of contrast agent into the system is modeled by a zero-order infusion rate Kin over a time period. In the first-order rate constant kij, i represents the compartment of origin and j represents the compartment of convergence. Differential equations based on the amount of contrast agents in each compartment were fit simultaneously to the experimental data of liver accumulation, perfusate, and bile excretion. The model was implemented in MATLAB software (2010b; MathWorks, Natick, MA). It was assumed that the rate constant k24 was associated with Oatp-mediated hepatic uptake of Gd-BOPTA, and the possibility that Gd-DTPA minimally enters into hepatocytes with rate constant k24 (Gd-DTPA) was considered. k46 and k56 described Mrp2-dependent canalicular excretion through both hepatic compartments, and k42 was associated with the efflux of Gd-BOPTA from hepatocytes back to sinusoids. The model was applied in livers isolated from control and TR− rats that were perfused with increasing concentrations of Gd-DTPA and Gd-BOPTA.

Immunofluorescence.

Liver biopsies were embedded in Tissue-Tech O.C.T. compound (Sakura Finetek Europe, Zoeterwoude, the Netherlands), frozen, and kept at 20°C. After fixation, the sections (5 μm) were covered with Triton X-100 [0.1% in phosphate-buffered saline (PBS) for 10 min], washed with PBS, and covered with 1% bovine serum albumin in PBS for 15 min. A mixture of polyclonal rabbit anti-rat Mrp2 antibody (1:500) and monoclonal rat anti-mouse ZO-1 antibody (1:500; Biozol, Eching, Germany) or PBS for control staining was applied for 1 h in a wet chamber. After rinsing and washing, slides were incubated for 45 min with a mixture of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:100) and TR-conjugated goat anti-rat IgG (1:200) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). After a final washing, sections were covered with mounting medium and a coverslip. Images were acquired from two channels at wavelengths of 488 nm to visualize Mrp2 and 568 nm to visualize ZO-1 with a confocal laser-scanning microscope.

Statistics

Parameters are means ± S.D. Mann Whitney U test compared means in normal rats and rats lacking Mrp2. One-way ANOVA compared parameters at increasing concentrations in a single group and two-way ANOVA determined whether the evolution of the parameter at increasing concentrations was different in the two groups of rats.

Results

Experimental Setup.

Livers isolated from normal or TR− rats were perfused with the extracellular Gd-DTPA (15 min), KHB solution (30 min), Gd-BOPTA (30 min), and KHB solution (30 min) (Fig. 1). Such a protocol successively measures the extracellular accumulation of Gd-BOPTA (similar to that of Gd-DTPA) and the additional accumulation within hepatocytes. Gd-BOPTA bile excretion rate, Gd-BOPTA bile accumulation, and modifications of bile flow associated with the organic anion bile excretion are also measured (Fig. 2).

Hepatic Pharmacokinetic of Gd-BOPTA (200 μM perfusion) in Normal Rats.

During the perfusion of 200 μM Gd-DTPA (Fig. 2, 0–15 min), the hepatic accumulation rapidly increased to a low steady state (<100 nmol/g). Gd-DTPA then was rapidly washed out during the subsequent KHB perfusion (Fig. 2, 15 to 45 min), and no Gd-DTPA was detected in bile. At the 45-min time point, Gd-BOPTA was added to the perfusate and taken up into the liver at a fast rate, and hepatic accumulation at T75 was much higher than that observed with Gd-DTPA (Fig. 2, 45–75 min). The washout of Gd-BOPTA after switching to the KHB solution started promptly but took a longer period of time than that for Gd-DTPA (Fig. 2, 75–105 min). To assess the time necessary for the first molecules of Gd-BOPTA to be detected into hepatocytes with the probe, we measured the time necessary to obtain a radioactive count above that for Gd-DTPA. The time was 1.06 ± 0.13 min and was similar for all doses of Gd-BOPTA in either normal or TR− rats. Because bile sample was collected each 5 min, the time necessary to reach bile could not be measured precisely. However, the time for Gd-BOPTA to reach bile was ≤5 min (Fig. 2).

Characteristics of hepatic Gd-BOPTA accumulation are shown in Table 1. During the first 10 min (T45–T55), the Gd-BOPTA accumulation rate (resulting from the concomitant entry into hepatocytes and excretion through the canalicular membrane) was 37 ± 3 nmol · min−1 · g−1. The accumulation rate then decreased to 12 ± 3 (T55–T65) and 7 ± 2 nmol · min−1 · g−1 (T65–T75). At the end of Gd-BOPTA perfusion (T75), the hepatic accumulation was maximal: 514 ± 74 nmol/g. When Gd-BOPTA perfusion was replaced by a KHB solution, hepatic Gd-BOPTA emptying rate was −29 ± 2 (T75–T85), −11 ± 2 (T85–T95), and −5 ± 1 nmol · min−1 · g−1 (T95–T105). Gd-BOPTA residual accumulation at T105 was 114 ± 49 nmol/g.

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TABLE 1

Parameters measured at increasing Gd-BOPTA concentrations in normal rats

Parameters are means ± S.D.

It is noteworthy that we were able to detect the efflux of Gd-BOPTA back into the perfusate from T95 to T105 (Table 1 and Fig. 3). During this period, livers had been perfused with a KHB solution for 20 min, and the radioactivity measured in perfusate within 10 min corresponds to the efflux back from hepatocytes to sinusoids: 89 ± 37 nmol in 10 min. During this period, bile excretion was 464 ± 101 nmol in 10 min.

Fig. 3.
Fig. 3.

Effects of increasing doses of Gd-DTPA on hepatic accumulation (top left) and hepatic distribution (top right) in control rats. During the last rinse period (from T95 to T105), we measured Gd-BOPTA efflux back into sinusoids (left bottom, in nanomoles) and into bile (right bottom, in nanomoles). Black bars, normal rats; gray bars, rats without Mrp2.

Hepatic Pharmacokinetics of Gd-BOPTA (200 μM perfusion) in TR− Rats.

The absence of Mrp2 expression in TR− rats is illustrated in Fig. 4. In rats lacking Mrp2, baseline bile flow was lower than in control rats (Fig. 2). No Gd-BOPTA was detected in bile, and bile flow did not increase during Gd-BOPTA perfusion.

Fig. 4.
Fig. 4.

Immunofluorescence of Mrp2 (green) and ZO-1 (red) in livers from control rats and rats without Mrp2 (TR−). In livers from control rats, Mrp2 immunostaining was confined to the space lined by the two rows of tight junctions stained by the anti-ZO-1 antibody (red) showing the location of Mrp2 within the canalicular membranes. No Mrp2 staining was observed in TR- rats.

In livers from TR− rats, the hepatic accumulation rate of Gd-BOPTA was steady over time from T45 to T75 (Table 2) and did not decline over time as observed in normal livers. The accumulation rate was lower in TR− than control rats from T45 to T55 (p = 0.02) but higher from T55 to T65 and from T65 to T75 (p = 0.02). Liver accumulation at T75 was similar in TR− and control rats. Gd-BOPTA then remained trapped within hepatocytes from T75 to T105, with the decline of Gd-BOPTA accumulation within livers being very slow (1–2 nmol · min−1 · g−1; Table 2). The small decline was associated with an efflux back into sinusoids during T95–105 (132 ± 40 nmol in 10 min), which was not different from that measured in control rats.

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TABLE 2

Parameters measured at increasing Gd-BOPTA concentrations in TR− rats

Values are presented as mean ± S.D.

Perfusion of Increasing Concentrations of Gd-DTPA.

The extracellular accumulation of Gd-DTPA within the liver increased when the concentrations of contrast agents perfused were increased from 40 to 1600 μM, but the ratio between the extracellular accumulation and the perfusate concentrations remained steady at all concentrations, the volume of hepatic distribution being unchanged in livers isolated from normal and TR− rats (p = 0.75) (Fig. 3).

Perfusion of Increasing Concentrations of Gd-BOPTA in Normal Livers.

Gd-BOPTA bile excretion rate increased with the concentrations perfused (Fig. 5 and Table 1). At each concentration, the bile excretion rate increased during the first 15 min of perfusion and then remained steady within the next 15 min. The increased bile flow over baseline values was correlated to Gd-BOPTA bile excretion rate (Fig. 6A).

Fig. 5.
Fig. 5.

Effects of increasing doses of Gd-BOPTA perfusion (between 45 and 75 min, black horizontal bar) on liver and bile Gd-BOPTA accumulation (nanomoles per gram), Gd-BOPTA bile excretion rate (nanomoles per minute per gram), and bile flow (microliters per minute per gram) in control rats. From 0 to 15 min, livers were perfused with increasing doses of Gd-DTPA (gray horizontal bar) and from 15 to 45 min, and from 75 to 105 min, livers were perfused with a Krebs-Henseleit bicarbonate solution (white horizontal bar). White squares, 40 μM Gd-DTPA and Gd-BOPTA; black squares, 200 μM Gd-DTPA and Gd-BOPTA; and black circles, 800 μM Gd-DTPA and Gd-BOPTA. Other concentrations are omitted to increase the clarity of the graph.

Fig. 6.
Fig. 6.

A, relationship between increased bile flow over baseline values (microliters per minute per gram) and Gd-BOPTA bile excretion rates (nanomoles per minute per gram). B and C, maximal Gd-BOPTA accumulation within hepatocytes (nanomoles per gram per 30 min) at increasing concentrations in TR− (B) and control rats (C) is shown.

The hepatic accumulation rate significantly increased with the concentrations perfused (p = 0.002; Fig. 5 and Table 1). The time to peak accumulation (T75) was similar at all concentrations. Hepatic Gd-BOPTA then decreased during the rinse period until a residual concentration that was correlated to the perfused concentrations. From T95 to T105, the efflux back into sinusoids was much lower than the bile excretion at each concentration, and the ratio between bile and perfusate excretion did not change with the concentrations (p = 0.14) (Fig. 3 and Table 1).

The overall hepatic extraction of Gd-BOPTA was also measured by the following formula: Gd-BOPTA amount (micromoles) perfused during each experiment − Gd-BOPTA amount (micromoles) measured during each experiment in the effluent perfusate (Fig. 7). The overall hepatic excretion was also expressed as the percentage of the perfused amount (Fig. 7). Hepatic extraction in micromoles increased with the concentrations perfused; however, the percentage extraction was higher at low concentrations.

Fig. 7.
Fig. 7.

Total hepatic extraction of Gd-BOPTA in each experiment in micromoles (top) and percentage (bottom). Total hepatic extraction was measured by the following formula: Gd-BOPTA amount (micromoles) perfused during each experiment − Gd-BOPTA amount (micromoles) measured during each experiment in the effluent perfusate. Normal rats (black bars) and rats without Mrp2 (gray bars) are indicated.

Perfusion of Increasing Concentrations of Gd-BOPTA in TR− Rats.

At each concentration, the hepatic accumulation rate of Gd-BOPTA was lower in TR− than in control rats from T45 to T55 but higher from T55 to T65 and T65 to T75 (Tables 1 and 2). Moreover, Gd-BOPTA remained trapped within hepatocytes from T85 to T105 (Fig. 8). From T75 to T85, the higher decline corresponded to the washout of the extracellular space by the KHB solution and, consequently, the higher the concentrations infused, the higher the decline. From T95 to T105, the efflux back into sinusoids increased, and the efflux was similar to that of control rats (Table 2 and Fig. 3).

Fig. 8.
Fig. 8.

Effects of increasing doses of Gd-BOPTA perfusion (between 45 and 75 min, black horizontal bar) on liver and bile Gd-BOPTA accumulation (nanomoles per gram), Gd-BOPTA bile excretion rate (nanomoles per gram per minute), and bile flow (micromoles per minute per gram) in rats lacking TR−. From 0 to 15 min, livers were perfused with increasing doses of Gd-DTPA (gray horizontal bar), and from 15 to 45 min and 75 to 105 min, livers were perfused with a Krebs-Henseleit bicarbonate solution (white horizontal bar). White squares (40 μM Gd-DTPA and Gd-BOPTA); black squares (200 μM); black circles (800 μM). Other concentrations are omitted to increase the clarity of the graph.

In TR− rats, the relation between the maximal hepatic accumulation within hepatocytes in 30 min and the concentrations perfused showed a Km of 654 μM and a Vmax of 2945 nmol/g per 30 min (Fig. 6). Because no exit from hepatocytes was possible in this group of rats, this relation characterizes the transport through the sinusoidal membrane. However, the overall hepatic extraction was lower in TR− than in normal rats, in accordance with the lower hepatic accumulation rate into hepatocytes. The relationship between the maximal hepatic accumulation within hepatocytes in 30 min and the concentrations perfused was different in control rats (Fig. 6), and this difference is related to the exit of Gd-BOPTA from normal hepatocytes into bile.

Pharmacokinetic Analysis.

In normal livers, the Gd-BOPTA k24 (or hepatic uptake of Gd-BOPTA through Oatps) significantly decreased when the perfused concentrations increased (Table 3). The k24 of Gd-DTPA was 100 times lower than the k24 of Gd-BOPTA, showing a very low level of hepatocyte uptake or nonspecific binding of Gd-DTPA to hepatocytes. Moreover, this Gd-DTPA k24 did not change with the concentrations. k46 slightly increased with the concentrations without reaching significance. It is noteworthy that the distribution toward the secondary hepatocyte-associated space (k45) increased with the concentrations. However, bile excretion from this compartment (k56) was not modified by concentrations. Efflux back to perfusate (k42 for Gd-BOPTA) was 10 times lower than entry into hepatocytes (k24 for Gd-BOPTA).

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TABLE 3

Hepatic pharmacokinetics of Gd-DTPA and Gd-BOPTA by compartmental analysis

Values are presented as means ± S.D.

The compartmental model described for normal livers did not fit with the experimental data in TR− rats, and k45, k46, k56, and Gd-DTPA k24 had to be set to 0 to fit the experimental data. Similar to control rats, the Gd-BOPTA k24 in TR− rats decreased when the perfused concentrations increased (Table 3). In accordance with the decreased overall hepatic extraction of Gd-BOPTA, the k24 was lower in TR− rats than in normal rats at each concentration. k12 and k23 were similar in normal and TR− rats (Table 3). The absence of Mrp2 modified the distribution of Gd-BOPTA within hepatocytes, and a single compartment of hepatocytes was depicted.

Discussion

Online Measurement of Gd-BOPTA Accumulation in Livers.

In this study, we analyzed the time-dependent hepatic accumulation of Gd-BOPTA during a steady-state perfusion and the hepatic elimination during the washout period. The hepatic accumulation of Gd-BOPTA is the main pharmacokinetic parameter to investigate because the differential accumulation of MR contrast agent in normal and injured hepatocytes will characterize focal lesions and injured tissues. Such an understanding is also important for the hepatic pharmacological effects of statins (Rodrigues, 2010). One way to measure accumulation of compounds within hepatocytes is to collect tissue samples and measure the hepatic concentrations of compounds at various time points in experimental models. To avoid tissue damage during sample collection, we measured online the hepatic accumulation of the organic anion 153Gd-BOPTA within liver using a gamma probe placed over the liver. Hepatic accumulation was studied in perfused rat livers because it is easy to control hepatic perfusate flow that was set to 30 ml/min in all experiments. Moreover, the composition of solutions to be perfused was well controlled, and interference with extrahepatic organs was avoided. It is noteworthy that we showed that despite the absence of Gd-BOPTA bile excretion and a similar efflux back to sinusoids observed in rats lacking Mrp2, the maximal hepatic accumulation of contrast agent was similar to normal rats. We also showed how hepatic accumulation relies on the concomitant entry into and exit from hepatocytes.

Pharmacological Properties of Gd-BOPTA in Rat Livers.

Gd-BOPTA distributes to the extracellular space and enters into hepatocytes through the sinusoidal transporter Oatps 1, 2, and 4 (Planchamp et al., 2007). With the probe used in our study, the time to detect Gd-BOPTA within hepatocytes was 1.06 ± 0.13 min. Such time is similar for all doses of Gd-BOPTA in either normal or TR− rats. The time for Gd-BOPTA to reach bile is ≤5 min, as observed with the bile salt taurocholate (Crawford et al., 1988). Gd-BOPTA is not modified during its transport to the canalicular membrane (de Haën et al., 1999). Its time transit through hepatocytes is lower than that observed with horseradish peroxidase, suggesting that Gd-BOPTA is not transported through vesicles as observed with this enzyme (Planchamp et al., 2007). Moreover, Gd-BOPTA is excreted into bile through Mrp2, and rats lacking Mrp2 do not excrete Gd-BOPTA. The increase of bile flow during the perfusion of the contrast agent correlates with the bile excretion rate and the higher the bile excretion rate, the higher the osmotic gradient that drives water through both sinusoidal and canalicular membranes (Tietz et al., 2005; Mottino et al., 2006; Lehmann et al., 2008).

Gd-BOPTA efflux into sinusoids is much lower than efflux into bile and the higher the concentration within hepatocytes, the higher the efflux. Such sinusoidal efflux is not modified in TR− rats. This result differs from other organic anions, such as acetaminophen glucuronide (Xiong et al., 2002) and valerenic metabolites (Maier-Salamon et al., 2009) whose efflux to perfusate is increased in TR− rats. The efflux of Gd-BOPTA may use Oatps that are bidirectional (Li et al., 2000) or Mrp transporters of the sinusoidal membrane whose expression is increased in TR− rats (Akita et al., 2001).

Accumulation of Gd-BOPTA within Hepatocytes.

We have evidence proving that hepatic Gd-BOPTA (nanomoles per gram) accumulation results from simultaneous entry into and exit from hepatocytes during the period of perfusion (T45–T75) in normal rats. This accumulation increased over the time perfusion until T75. However, the accumulation rate (in nanomoles per minute per gram) corresponding to the slope of Gd-BOPTA accumulation over time was maximal from T45 to T55 but decreased from T55 to T75. At a 1600 μM concentration, the maximal accumulation rate was 105 ± 7 nmol · min−1 · g−1. In TR− rats, the initial accumulation rate was lower from T45 to T55, suggesting a lower initial Gd-BOPTA transport through Oatps. Thereafter, the accumulation remains steady and higher than that of normal rats from T55 to T75. This steady accumulation over time reflects the absence of bile excretion. The maximal hepatic accumulation at T75 was similar in both groups, suggesting that the main parameter to be regulated in hepatocytes was the cellular accumulation. The decreased transport through Oatps in TR− rats is shown at all concentrations by: 1) the initial lower accumulation rate from T45 to T55; 2) the decreased overall hepatic extraction; and 3) the decreased k24 in the pharmacokinetic model. Such decreased uptake might compensate for the absence of bile excretion.

Pharmacokinetic analysis found two pools of Gd-BOPTA accumulation within hepatocytes, as published previously by Chandra et al. (2005). However, in this last study, the authors describe a bidirectional transfer between the two compartments and a single bile efflux from the second compartment. We found a unidirectional transfer between the two compartments that significantly increased with the accumulations of Gd-BOPTA (Planchamp et al., 2005c). The bile efflux was possible from the two compartments, but the efflux from compartment 5 remained steady, whereas efflux from compartment 4 increased with the hepatic accumulation. Compartment 5 did not exist in livers lacking Mrp2; in normal rats, this might correspond to a storage zone of Gd-BOPTA before exit through Mrp2.

During the hepatic accumulation phase, Gd-BOPTA bile excretion rate increased during the first 15 min of perfusion and then remained steady within the next 15 min, suggesting a saturation of the transport through Mrp2 that contributes to the hepatic accumulation. The saturation of Mrp2 function is also proven by the fact that the ratio of Gd-BOPTA concentrations between bile and hepatocytes at T75 decreases with the concentrations perfused (data not shown). During the rinse period, hepatic Gd-BOPTA bile excretion and hepatic emptying were correlated. At T105, residual Gd-BOPTA in hepatocytes increased with the concentrations perfused.

Relevance of Hepatic Accumulation of Gd-BOPTA and Other Organic Anions.

The transport of drugs across hepatocytes relies on the expression and function of transporters located on the sinusoidal and canalicular membranes. It is easy to determine the expression of the transporters in normal and injured hepatocytes. However, it is more difficult to measure concomitantly the function of both sinusoidal and canalicular transporters and determine the overall effect on hepatocyte accumulation. Entry of Gd-BOPTA through sinusoidal transporters can be decreased; however, the hepatic accumulation would depend on the bile excretion and efflux back to sinusoids either through bidirectional sinusoidal transporters or through sinusoidal Mrp transporters. Tsuboyama et al. (2010) recently showed that when human OATPs are present in hepatocellular carcinoma, the cellular accumulation of Gd-EOB-DTPA (a contrast agent close to Gd-BOPTA) relies on the expression and location of MRP2. Localization of MRP2 on canalicular membrane allows exit of the contrast agent into bile canaliculi, and enhancement of signal intensities at liver MR imaging is low. The enhancement is much higher when the canalicular membrane lacks MRP2, the contrast agent being trapped within hepatocytes. Moreover, the phosphorylation of Mrp2 retrieves the transporter from the canalicular membrane and traps Gd-BOPTA within hepatocytes (Planchamp et al., 2007). On the other hand, the entry of Gd-BOPTA may be impaired by drug-drug interactions or decreased expression of Oatps in the sinusoidal membrane. Gd-BOPTA behaves as an extracellular contrast agent when Gd-BOPTA is perfused with bromosulfophthalein (0.5 mM) (Pastor et al., 2003) or when the expression of Oatps is decreased by cirrhosis (Planchamp et al., 2005b). Finally, modification of solution pH probably would modify hepatic accumulation, emphasizing the complexity of Gd-BOPTA accumulation within hepatocytes (Leuthold et al., 2009).

Authorship Contributions

Participated in research design: Millet, Daali, Stieger, and Pastor.

Conducted experiments: Millet, Moulin, and Pastor.

Performed data analysis: Millet and Pastor.

Wrote or contributed to the writing of the manuscript: Millet, Moulin, Stieger, Daali, and Pastor.

Acknowledgments

We thank Joëlle Bourquin for excellent technical assistance.

Footnotes

  • The work was supported by the “Fonds National Suisse de la Recherche Scientifique” [Grant 310030-126030] (to C.M.P.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.110.175406.

  • ABBREVIATIONS:

    MR
    magnetic resonance
    Gd-BOPTA
    gadobenate dimeglumine
    Gd-EOB-DTPA
    gadoxetic acid
    OATPs
    organic anion-transporting peptides
    Mrp2 or MRP2
    multiple resistance protein-2
    Gd-DTPA
    gadopentetate dimeglumine
    KHB
    Krebs-Henseleit-bicarbonate
    ANOVA
    analysis of variance
    PBS
    phosphate-buffered saline
    TR−
    rats without Mrp2 transporter.

  • Received September 27, 2010.
  • Accepted December 1, 2010.

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Research ArticleMetabolism, Transport, and Pharmacogenomics

How Organic Anions Accumulate in Hepatocytes Lacking Mrp2: Evidence in Rat Liver

Philippe Millet, Marcelle Moulin, Bruno Stieger, Youssef Daali and Catherine M. Pastor
Journal of Pharmacology and Experimental Therapeutics March 1, 2011, 336 (3) 624-632; DOI: https://doi.org/10.1124/jpet.110.175406
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