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Modulation of Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP2 (ABCC2) Transport Activities by Endogenous and Exogenous Glutathione-Conjugated Catechol Metabolites

Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics, Queen's University, Kingston, Ontario, Canada (A.J.S., R.G.D., S.P.C.C.); and Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, Tucson, Arizona (D.D.W., T.J.M.)

(Received November 6, 2007; Accepted December 12, 2007)

	Abstract

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Members of the multidrug resistance protein (MRP/ABCC) subfamily of ATP-binding cassette proteins transport a wide array of anionic compounds, including sulfate, glucuronide, and glutathione (GSH) conjugates. The present study tested the ATP-dependent vesicular transport of leukotriene C₄ and 17β-estradiol 17-(β-D-glucuronide) (E₂17βG) mediated by the MRP1 and MRP2 transporters in the presence of six potential modulators from three different classes of GSH-conjugated catechol metabolites: the ecstasy metabolite 5-(glutathion-S-yl)-N-methyl--methyldopamine (5-GS-N-Me--MeDA), the caffeic acid metabolite 2-(glutathion-S-yl)-caffeic acid (2-GS-CA), and four GSH conjugates of 2-hydroxy (OH) and 4-OH estrogens (GS estrogens). MRP1-mediated E₂17βG transport was inhibited in a competitive manner with a relative order of potency of GS estrogens (IC₅₀ <1 µM) > 2-GS-CA (IC₅₀ 3 µM) > 5-GS-N-Me--MeDA (IC₅₀ 31 µM). MRP2-mediated transport was inhibited with a similar order of potency, except the 2-hydroxy-4-(glutathion-S-yl)-estradiol and 4-hydroxy-2-(glutathion-S-yl)-estradiol conjugates were approximately 50- and 300-fold less potent, respectively. Transport activity was unaffected by N-acetylcysteine conjugates of N-Me--MeDA and CA. The position of GSH conjugation appears important as all four GS estrogen conjugates tested were potent inhibitors of MRP1 transport, but only the 2-hydroxy-1-(glutathion-S-yl)-estradiol and 2-hydroxy-1-(glutathion-S-yl)-estrone conjugates were potent inhibitors of MRP2-mediated transport. In conclusion, we have identified three new classes of MRP1 and MRP2 modulators and demonstrated that one of these, the estrogen conjugates, shows unanticipated differences in their interactions with the two transporters.

MRP2 shares 49% sequence identity with MRP1; however, unlike MRP1, it is expressed in a limited number of tissues such as liver, kidney, and placenta and in polarized cells it localizes to apical membranes (Leslie et al., 2005). The most important physiological role of MRP2 is mediating the biliary excretion of organic anion conjugates, primarily bilirubin glucuronides (König et al., 1999; Leslie et al., 2005). Despite the differences in their membrane and tissue localization, MRP1 and MRP2 transport many of the same conjugated metabolites, including LTC₄ and 17β-estradiol 17-(β-D-glucuronide) (E₂17βG). However, the relative affinity of MRP2 for these metabolites is significantly lower than the affinity of MRP1 (Cui et al., 1999; König et al., 1999).

The ring-substituted methamphetamine derivative, methylenedioxy-methamphetamine (MDMA, ecstasy) (Fig. 1A), is a popular drug of abuse among Western youth. Although systemic administration of MDMA to rats leads to selective serotonin neurotoxicity, direct injection of MDMA or one of its primary catechol metabolites, -methyldopamine, fails to produce any long-lasting neurotoxic effects (McCann and Ricaurte, 1991). It has now been established that GSH-conjugated metabolites of MDMA contribute to the observed neurotoxicity. Thus, administration of the GSH conjugate 5-(glutathion-S-yl)-N-methyl--methyldopamine (5-GS-N-Me--MeDA) to rats reproduces both the behavioral and short-term alterations in brain catecholamine levels observed after systemic MDMA administration (Miller et al., 1996). Little is known about the disposition of MDMA metabolites, but previous research has suggested that a GSH conjugate uptake mechanism present at the blood-brain barrier is probably involved (Bai et al., 2001).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Metabolic pathways and chemical structures of the GSH-conjugated catechol metabolites examined in the present study. Shown are (A) MDMA, its primary phase I metabolite N-Me--MeDA, and its GSH conjugate 5-GS-N-Me--MeDA; (B) CA and its GSH conjugate 2-GS-CA; (C) 17β-estradiol and two phase I metabolites, 2-OH-17β-estradiol (2-OH-E2) and 4-OH-17β-estradiol (4-OH-E2), and some of their corresponding GSH-conjugated metabolites, 2-OH-1-GS-E2, 2-OH-4-GS-E2, and 4-OH-2-GS-E2. Demethylation of MDMA by CYP2D, CYP2B, and CYP3A yields N-Me--MeDA. Hydroxylation of 17β-estradiol by CYP1A yields 2-OH-E2, and hydroxylation by CYP1B yields 4-OH-E2.

Caffeic acid (3,4-dihydroxycinnaminic acid, CA) (Fig. 1B) is a nonflavonoid catecholic acid found in relatively large quantities in fruits and vegetables. Absorbed CA appears to be mainly glucuronidated, sulfated, or O-methylated to ferulic acid (Mateos et al., 2006). Incubation of CA with liver microsomes and isolated hepatocytes also leads to the formation of several GSH conjugates (Galati et al., 2002; Moridani et al., 2002). Both mono- and di-substituted GSH conjugates of caftaric acid and CA are present in relatively high amounts in grape juice and wine (Singleton et al., 1985). Otherwise, little is known about the metabolism and disposition of CA and its metabolites. However, CA can act as an anti-inflammatory agent by inhibiting 5-lipoxygenase, which in turn inhibits the synthesis of proinflammatory cytokines, such as the MRP1 substrate LTC₄ (Koshihara et al., 1984).

Catechol estrogens (Fig. 1C), although structurally dissimilar from MDMA metabolites or CA, have a similar potential to form conjugates with GSH when oxidized to their corresponding quinones (Butterworth et al., 1996). Prolonged exposure to catechol estrogens is thought to play a role in carcinogenesis, although the underlying mechanisms are unknown. However, 4-OH estrogen quinones are known to form covalent adducts with guanine, leading to depurinating DNA adducts (Roy and Liehr, 1999; Yue et al., 2003; Abel et al., 2004). Furthermore, catechol estrogens can enhance endogenous DNA adduct formation and free radical generation attributable to redox cycling between the catechol and quinone forms, which ultimately leads to DNA damage (Roy and Liehr, 1999; Yue et al., 2003).

Beyond the shared ability of the three chemically distinct groups of catechol metabolites described above to form conjugates with GSH, relatively little is known about their further disposition. However, it is reasonable to presume that some membrane transport proteins are involved. Because both MRP1 and MRP2 play important roles in the distribution and elimination of a variety of GSH conjugates, we investigated the ability of GSH-conjugated catechol metabolites of MDMA, CA, and estrogens to interact with MRP1 and MRP2 by testing whether they could modulate the transport activity of these homologous transporters in vitro, using the model substrates LTC₄ and E₂17βG.

	Materials and Methods

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Materials. [14,15,19,20-³H]LTC₄ (194.6 and 166.8 Ci/mmol) and [6,7-³H]E₂17βG (53.0 and 46.9 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Woodbridge, ON, Canada). LTC₄ was purchased from Calbiochem (San Diego, CA). Creatine kinase and creatine phosphate were obtained from Roche Diagnostics (Laval, QC, Canada). AMP, ATP, CA, and E₂17βG were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal antibody M₂I-4, specific for MRP2, was purchased from Alexis Laboratories (San Diego, CA). Monoclonal antibody QCRL-1, specific for MRP1, was derived in this laboratory (Hipfner et al., 1994). 2- and 4-OH-17β-estradiol and 2-OH-17β-estrone were purchased from Steraloids (Newport, RI). N-Me--MeDA was synthesized by standard procedures. Briefly, MDMA was demethylated with a 2-fold molar excess of boron trichloride in methylene chloride under argon. MDMA was kindly provided by the Research Technology Branch, National Institute on Drug Abuse (Rockville, MD). All other chemicals and reagents were of analytical grade.

Synthesis of Catechol GSH Conjugates. GSH conjugates of CA, N-Me--MeDA, 2-OH-17β-estradiol, and 4-OH-17β-estradiol were prepared as described previously (Butterworth et al., 1996; Jones et al., 2005). Briefly, each parent catechol compound was oxidized to its corresponding quinone using sodium periodate. Quinones were further reacted with excess GSH. The resulting conjugates were then purified by reverse-phase semipreparative high-performance liquid chromatography [LC-6A (Shimadzu, Kyoto, Japan) and Ultrasphere ODS-5 (Beckman Coulter, Fullerton, CA) columns] and characterized by their retention times and ultraviolet-visible spectroscopic parameters (Butterworth et al., 1996; Cao et al., 1998). The identity of the purified conjugates was confirmed by electrospray ionization-mass spectrometry (Southwest Environmental Health Sciences Center Proteomics Facility Core, Tucson, AZ).

Cell Culture and Transfection of MRP1 and MRP2 Expression Vectors. pcDNA3.1(-) expression vectors containing human MRP1 and MRP2 cDNAs were transfected into simian virus 40-transformed human embryonic kidney (HEK293T) cells (Létourneau et al., 2007). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 4 mM l-glutamine and 7.5% fetal bovine serum. Approximately 18 x 10⁶ cells were seeded per 150-mm plate; 24 h later cells (at 60–85% confluence) were transfected with 20 µgof DNA using 50 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 48 h at 37°C, the cells were collected, snap-frozen in liquid nitrogen, and stored at -80°C until needed.

Membrane Vesicle Preparation and Determination of MRP1 and MRP2 Protein Expression Levels. Pellets of transfected cells were thawed and disrupted by argon cavitation at 300 psi, and membrane vesicles were prepared as described previously (Loe et al., 1996b; Létourneau et al., 2007). Membrane vesicles were aliquoted and stored at -80°C. Vesicular protein concentrations were determined using the Bradford method (Bio-Rad Laboratories, Mississauga, ON, Canada) with bovine serum albumin as the standard. Levels of MRP1 and MRP2 protein expressed by the transfected cells were determined by immunoblot analysis. Briefly, proteins were resolved on a 7% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride membranes (Pall Corporation, Pensacola, FL). Membranes were subsequently blocked with 4% (w/v) skim milk powder in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h followed by overnight incubation at 4°C with the human MRP1-specific murine monoclonal antibody QCRL-1 (1:10,000) or the MRP2-specific murine monoclonal antibody M₂I-4 (1:10,000) in blocking solution (Létourneau et al., 2007). After washing with TBS-T, immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Pierce, Edmonton, AB, Canada) in blocking solution, followed by application of Western Lightning chemiluminescence blotting substrate (PerkinElmer Life and Analytical Sciences) and exposed to film (Ultident, St. Laurent, QC, Canada).

View larger version (35K): [in this window] [in a new window]   FIG. 2. Expression of MRP1 and MRP2 proteins in membrane vesicles. Immunoblots of membrane vesicles prepared from transfected (0.75, 1.0, and 1.25 µgof protein) and untransfected (1.25 µg of protein) HEK293T cells are shown. Monoclonal antibodies (MAbs) QCRL-1 and M2I-4 were used to detect MRP1 (A) and MRP2 (B), respectively. HEK refers to control membrane vesicles prepared from untransfected cells.

For MRP1-mediated LTC₄ uptake, 2 µg of vesicle protein was incubated with [³H]LTC₄ (50 nM, 10 nCi) for 60 s at 23°C. MRP1-mediated E₂17βG uptake was measured by incubating 2 µg of vesicle protein with [³H]E₂17βG (400 nM, 20 nCi) for 60 s at 37°C. For MRP2-mediated E₂17βG uptake, 6 µg of vesicle protein was incubated with [³H]E₂17βG (400 nM, 40 nCi) for 5 min at 37°C (Létourneau et al., 2007). MRP1-mediated E₂17βG uptake kinetics were typically measured by incubating 2 µg of vesicle protein with a range of [³H]E₂17βG concentrations (100 nM–30 µM) (40 nCi) for 60 s at 37°C in the presence or absence of catechol conjugates at the concentrations indicated.

Data Analysis. IC₅₀ and kinetic parameters were computed using GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA). When shown, error bars represent the S.D. from the mean.

	Results

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Modulation of MRP1-Mediated [³H]E₂17βG and [³H]LTC₄ Transport by GSH-Conjugated Catechol Metabolites. To determine whether any of the GSH-conjugated catechol metabolites shown in Fig. 1 could modulate MRP1-mediated transport of E₂17βGor LTC₄, uptake assays were performed using MRP1-enriched membrane vesicles prepared from transiently transfected cells. Levels of MRP1 expression in membrane vesicles were determined by immunoblot analysis before transport experiments (Fig. 2A). As shown in Fig. 3, the MDMA and CA metabolites inhibited both E₂17βG (Fig. 3, A and B) and LTC₄ (Fig. 3, C and D) uptake by MRP1 in a concentration-dependent fashion with IC₅₀ values ranging from 3 to 137 µM. In contrast, the corresponding N-acetylcysteine conjugates [5-(N-acetylcystein-S-yl)-N-methyl--methyldopamine and 5-(N-acetylcystein-S-yl)-caffeic acid] had no effect on organic anion uptake (up to 1 mM) (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effect of GSH-conjugated metabolites of MDMA and CA on MRP1-mediated vesicular uptake activity. Shown are representative concentration-response curves demonstrating the effect of 5-GS-N-Me--MeDA on (A) [3H]E217βG uptake () and (C) [3H]LTC4 uptake () and the effects of 2-GS-CA on (B) [3H]E217βG uptake () and (D) [3H]LTC4 uptake () by MRP1-enriched membrane vesicles. MRP1 transport activity is expressed as a percentage of the activity in the absence of metabolite. Transport conditions were as described under Materials and Methods. Data points represent the means ± S.D. of triplicate determinations in a single experiment. Similar results were obtained in two additional independent experiments.

When the effects of GSH-conjugated estrogen catechols were examined, each of the four metabolites was found to inhibit both E₂17βG (Fig. 4, A–D) and LTC₄ (Fig. 4, E–H) uptake in a concentration-dependent fashion with all IC₅₀ values less than 2 µM. The IC₅₀ values from multiple repeat experiments are summarized in Table 1.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effect of GSH-conjugated catechol estrogen metabolites on MRP1-mediated vesicular uptake activity. Shown are representative concentration response curves for (A–D) [3H]E217βG uptake () and (E–H) [3H]LTC4 uptake () in the presence of the four estrogen conjugates indicated on the x-axis of each graph. MRP1 transport activity is expressed as a percentage of the activity in the absence of metabolite. Transport conditions were as described under Materials and Methods. Data points represent the means ± S.D. of triplicate (E217βG) or duplicate (LTC4) determinations in a single experiment. Similar results were obtained in two (E217βG) or one (LTC4) additional independent experiments.

Kinetic Parameters of MRP1-Mediated [³H]E₂17βG Transport in the Presence of GSH-Conjugated Catechol Metabolites. To determine more precisely how GSH-conjugated catechol metabolites modulate MRP1-mediated E₂17βG transport, the kinetic parameters of E₂17βG uptake were determined in the presence (or absence) of two concentrations of 5-GS-N-Me--MeDA (30 and 100 µM), 2-GS-CA (3 and 10 µM), and 2-OH-1-GS-E₂ (0.3 and 1 µM) (Fig. 5). As shown in Fig. 5A, 5-GS-N-Me--MeDA competitively inhibited E₂17βG transport by MRP1 as the apparent K_m of E₂17βG increased from 1.6 to 9.2 µM in the presence of 100 µM of the metabolite, whereas the V_max remained relatively unchanged. 2-GS-CA and 2-OH-1-GS-E₂ similarly inhibited MRP1-mediated E₂17βG transport in a competitive manner (Fig. 5, B and C). The K_i values for the inhibition of MRP1-mediated E₂17βG transport by 5-GS-N-Me--MeDA, 2-GS-CA, and 2-OH-1-GS-E₂ are summarized in Table 2.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Kinetic analysis of MRP1-mediated E217βG uptake in the presence of GSH-conjugated catechol metabolites. Shown are representative Michaelis-Menten and Eadie-Hofstee plots of [3H]E217βG uptake in the absence or presence of catechol metabolites. A, [3H]E217βG uptake in the absence () (Km 1.6 µM, Vmax 827 pmol/mg/min) or presence of 30 µM() (Km 3.4 µM, Vmax 820 pmol/mg/min) or 100 µM ()(Km 9.2 µM, Vmax 775 pmol/mg/min) 5-GS-N-Me--MeDA. B, [3H]E217βG uptake in the absence ()(Km 2.2 µM, Vmax 387 pmol/mg/min) or presence of 3 µM()(Km 3.1 µM, Vmax 411 pmol/mg/min) or 10 µM()(Km 8.4 µM, Vmax 403 pmol/mg/min) 2-GS-CA. C, [3H]E217βG uptake in the absence ()(Km 3.7 µM, Vmax 626 pmol/mg/min) or presence of 0.3 µM()(Km 4.8 µM, Vmax 650 pmol/mg/min) or 1 µM()(Km 12.3 µM, Vmax 818 pmol/mg/min) 2-OH-1-GS-E2. Uptake was measured at E217βG concentrations ranging from 0.10 to 30 µM. Km and Vmax values were determined from Eadie-Hofstee analysis. Transport conditions were as described under Materials and Methods. Data points represent the means of duplicate determinations in a single experiment. All r2 values for the Michaelis-Menten analysis were >0.97, and r2 values for the Eadie-Hofstee analysis were >0.77.

Modulation of MRP2-Mediated [³H]E₂17βG Transport by GSH-Conjugated Catechol Metabolites. We also investigated whether the GSH-conjugated catechol metabolites could inhibit MRP2-mediated transport. Vesicular uptake experiments were performed using membrane vesicles prepared from transiently transfected HEK cells and the expression of MRP2 was confirmed by immunoblot analysis before transport experiments as before (Fig. 2B). As shown in Fig. 6, A and B, the MDMA and CA metabolites inhibit MRP2-mediated E₂17βG uptake in a concentration-dependent fashion, with IC₅₀ values ranging from 10 to 145 µM. Similar to MRP1, the corresponding N-acetylcysteine conjugates [5-(N-acetylcystein-S-yl)-N-methyl--methyldopamine and 5-(N-acetylcystein-S-yl)-caffeic acid] had no effect on either E₂17βG or LTC₄ uptake by MRP2 (data not shown). When the effects of the GSH-conjugated estrogen catechols were examined (Fig. 6, C–F), the 2-OH-1-GS-E₂ and 2-OH-1-GS-E₁ metabolites potently inhibited E₂17βG uptake by MRP2 with IC₅₀ values of 2.1 and 1.6 µM, respectively, whereas the 2-OH-4-GS-E₂ and 4-OH-2-GS-E₂ conjugates were approximately 50- and 300-fold less potent (IC₅₀ values of approximately 95 and 580 µM, respectively). A summary of IC₅₀ values from multiple repeat experiments is provided in Table 3.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Effect of GSH-conjugated MDMA, CA, and estrogen metabolites on MRP2-mediated vesicular uptake activity. Shown are concentration response curves for [3H]E217βG uptake by MRP2 in the presence of different concentrations of (A) 5-GS-N-Me--MeDA, (B) 2-GS-CA, (C) 2-OH-1-GS-E2, (D) 2-OH-1-GS-E1, (E) 2-OH-4-GS-E2, and (F) 4-OH-2-GS-E2. MRP2 transport activity is expressed as a percentage of activity in the absence of metabolite. Transport conditions were as described under Materials and Methods. Data points represent the means ± S.D. of triplicate determinations in a single experiment. Similar results were obtained in at least one additional independent experiment.

	Discussion

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The potential of several structurally diverse GSH-conjugated catechol metabolites to modulate the transport activity of MRP1 and MRP2 was investigated to gain insight into which of these ABC transporters might be involved in the disposition of these metabolites in vivo. The results presented here show for the first time that GSH-conjugated catechol metabolites of MDMA, CA, and estradiol are inhibitors of MRP1 and MRP2.

In general, the six GSH conjugates were more potent at inhibiting MRP1-mediated E₂17βG transport than LTC₄ transport. This finding could be expected because MRP1 exhibits a 15-fold higher affinity for LTC₄ than for E₂17βG (Loe et al., 1996a). The GSH conjugates were also more potent at inhibiting MRP1-than MRP2-mediated E₂17βG transport. This observation was again not surprising, because MRP1 exhibits a 5- and 10-fold higher affinity for E₂17βG and LTC₄ than for MRP2, respectively (Cui et al., 1999; König et al., 1999). Lastly, the N-acetylcysteine conjugates investigated had no observable effect (up to 1 mM) on MRP1- or MRP2-mediated vesicular transport, as would be expected, as previous studies have demonstrated the critical importance of the -glutamyl residue of GSH for interaction with these transporters (Loe et al., 1996b; Leslie et al., 2003).

Many experimental inhibitors of MRP1 and related proteins have been described previously, but very few of them are highly specific for MRP1 or its homologs (Boumendjel et al., 2005). One exception is the highly potent and specific tricyclic isoxazole inhibitor LY475776 that inhibits LTC₄ transport by MRP1 with an IC₅₀ of 50 nM (in vitro) in the presence of millimolar concentrations of GSH (Mao et al., 2002). The widely used inhibitor MK571 is considerably less potent than LY475776 and also inhibits MRP2 (and other MRPs), whereas LY475776 does not (Gekeler et al., 1995; Mao et al., 2002).

In addition to small molecule modulators, established substrates of MRP1 and MRP2, e.g., E₂17βG, are often competitive inhibitors of the transport of other substrates (Loe et al., 1996a; Dantzig et al., 2004). In the present study, all six GSH-conjugated catechol metabolites were shown to inhibit both E₂17βG and LTC₄ transport by MRP1, and, in the case of E₂17βG, inhibition was demonstrated to be competitive. The observed IC₅₀ and K_i values both showed a rank order of inhibitory potency of the metabolites as glutathion-S-yl (GS) estrogens > 2-GS-CA > 5-GS-N-Me--MeDA (Table 1). This rank order was similar for inhibition of MRP2-mediated transport with the notable exception that 2-OH-4-GS-E₂ and 4-OH-2-GS-E₂ were markedly less potent (50- and 300-fold, respectively) than 2-OH-1-GS-E₁ or 2-OH-1-GS-E₂ (Table 3). This difference in inhibitory potency for MRP1 and MRP2 was somewhat surprising because the estrogen conjugates are structurally quite similar to one another and their potencies were comparable with respect to inhibition of MRP1-mediated transport (Table 1). Further studies are ongoing to elucidate whether the catechol metabolites exert their inhibitory effects simply by binding to MRP1 or MRP2 without being transported or by competing for active transport.

MDMA was first identified as a potential neurotoxicant in 1986 and, much later, the cysteinyl-S-conjugates of primary MDMA metabolites were shown to be the causative agents (Stone et al., 1986; Miller et al., 1996). Neurotoxicity was observed at nanomolar concentrations, and increases in brain concentrations of the conjugates were shown to be directly proportional to the amount of selective serotonergic neurotoxicity observed (Jones et al., 2005). Furthermore, GSH was shown to modulate the entry of GSH-conjugated metabolites of MDMA into rat brains, and GSH-conjugated metabolite degradation could be prevented by inhibiting the ectoenzyme -glutamyltranspeptidase with acivicin (Bai et al., 1999). GSH-conjugated metabolites of MDMA are also detected in the bile of rats after systemic administration of this agent (Bai et al., 2001). As MRP2 is the predominant canalicular GSH conjugate efflux transporter, it seems likely that it mediates this transport, although in vivo studies are needed to confirm this theory. On the other hand, because hepatocytes do not normally express significant levels of MRP1 (Leslie et al., 2005), another transporter seems likely to be responsible for the basolateral efflux of GSH-conjugated MDMA metabolites into the blood circulation. However, the identity of this hepatic transporter remains unknown. At the blood-brain barrier, several MRP-related proteins, including MRP1, have been localized to the apical membrane of endothelial cells as well as in astrocytes (Dallas et al., 2006). Thus, it has been proposed that these MRPs may play a role in preventing the accumulation of toxic GSH conjugates in the brain. However, if MDMA metabolites are inhibitors of MRP-mediated transport in vivo, they may account for increased brain concentrations as their efflux activity is inhibited. The results of the present study show for the first time that MRP1 and MRP2 can interact with 5-GS-N-Me--MeDA, at least in vitro and may thus be involved in the disposition of this neurotoxic metabolite in vivo.

It is presumed that CA is metabolized to 2-GS-CA, although the extent to which this metabolism occurs is not known. In the present study, 2-GS-CA proved to be a moderately potent inhibitor of MRP1-mediated E₂17βG and LTC₄ transport with IC₅₀ values of 3 and 20 µM, respectively. Like 5-GS-N-Me--MeDA, 2-GS-CA was more potent at inhibiting E₂17βG than LTC₄. Similarly, 2-GS-CA more potently inhibited E₂17βG transport by MRP1 than by MRP2 (IC₅₀ of 3 versus 10 µM). Relatively little is known about the metabolism and distribution of CA or its metabolites. Although CA is metabolized to dihydrocaffeic acid and ferulic acid, these compounds may be converted back to CA by hydrogenase activity and CYP1A activity, respectively (Moridani et al., 2002). However, this cycle is disrupted when CA is conjugated to GSH, and this conjugation reaction appears to be preceded by metabolic activation of CA by CYP2E1 (Fig. 1B) (Moridani et al., 2002).

Our data are the first to demonstrate that the GSH conjugate of CA can inhibit MRP1-mediated transport of LTC₄. CA noncompetitively inhibits 5-lipoxygenase activity, which is critical for biosynthesis of LTC₄ and other leukotrienes (Koshihara et al., 1984). Thus, the anti-inflammatory actions of CA and its metabolites may be 2-fold. They may inhibit both the formation of LTC₄ through action on 5-lipoxygenase and release of LTC₄ via MRP1 from proinflammatory cells. Further study is needed to determine human plasma concentrations of this conjugated GSH catechol metabolite to establish whether its ability to inhibit MRP1-mediated LTC₄ transport in vitro is physiologically relevant for the anti-inflammatory potential of CA.

Catechol estrogens have been associated with an increased risk of breast and endometrial cancers (Rogan et al., 2003; Yue et al., 2003; Doherty et al., 2005). However, the biological properties of their corresponding GSH conjugates have not been thoroughly investigated, although several GSH-conjugated estrogens have been detected in tumor tissues (Devanesan et al., 2001; Rogan et al., 2003; Yue et al., 2003). On the other hand, administration of 2-OH-4-GS-E₂ or 2-OH-1-GS-E₂ to Syrian hamsters can produce mild nephrotoxicity. Moreover, repeated daily administration of 2-OH-4-GS-E₂ causes a sustained elevation in urinary markers of renal damage and in the concentration of renal protein carbonyls and lipid hydroperoxides (Butterworth et al., 1998). Because catechol estrogens are conjugated with GSH (Roy and Liehr, 1999; Bolton et al., 2000; Yue et al., 2003), it is likely that there is a physiological system in place that prevents these conjugates from accumulating to toxic levels. In addition to endogenous catechol estrogens, GSH conjugates are also formed from equine estrogens that are often used for hormone replacement therapy (Zhang et al., 2001). Furthermore, disruption of the cellular efflux of GSH catechol estrogen metabolites may exacerbate the carcinogenic potential of 4-OH estrogens as many GSH conjugates retain their ability to produce reactive oxygen species and form covalent adducts (Roy and Liehr, 1999; Bolton et al., 2000; Yue et al., 2003). Thus, knowledge of the transporters involved in the disposition of both exogenous and endogenous catechol estrogen GSH conjugates is of interest.

Whereas all four of the estrogen conjugates tested were potent inhibitors of MRP1-mediated E₂17βG transport (IC₅₀ 0.08–0.29 µM) (Table 1), only the two metabolites conjugated at the 1-position of the steroid nucleus strongly inhibited MRP2 transport (IC₅₀ 1.6–2.1 µM), compared with those conjugated at position 2 (IC₅₀ 582 µM) and position 4 (IC₅₀ 94 µM) (Table 3). Thus, the 4-GS and 2-GS conjugates were approximately 300- and 1500-fold less potent at inhibiting MRP2-mediated E₂17βG transport than MRP1, compared with 10- to 20-fold for the 1-GS conjugates. Whether any of these conjugates interact with MRP2 at a substrate binding site and/or an allosteric binding site remains to be elucidated. Nevertheless, it seems apparent that the 2-OH-4-GS-E₂ and 4-OH-2-GS-E₂ conjugates interact in a different way with the substrate and/or modulatory binding sites of MRP2 than metabolites conjugated at position 1 (2-OH-1-GS-E₂ and 2-OH-1-GS-E₁).

In summary, the results of this study show that several structurally and biologically distinct classes of both endogenous and exogenous GSH-conjugated catechol metabolites can inhibit both MRP1- and MRP2-mediated transport in vitro. However, the potencies of the metabolites vary substantially, most notably with respect to the 2-GS and 4-GS estrogen conjugates. Thus, all six conjugates are potential substrates for MRP1, and to a lesser extent, MRP2. Studies aimed to determine whether these metabolites are simply modulators of MRP1 and MRP2 or whether they compete for substrate transport are currently underway. Furthermore, it should be noted that our studies do not exclude the possibility that these metabolites could interact with other ABC proteins such as ABCG2 and MRP3 (ABCC3), but this remains to be tested experimentally (Suzuki et al., 2003; Haimeur et al., 2004; Leslie et al., 2005). Because the parent compounds and some of the metabolites themselves exert biological effects in humans, understanding the activities of these and other metabolites is crucial to understanding their full pharmacological and toxicological potential.

	Acknowledgments

 
We thank Drs. Gwenaëlle Conseil, Alice Rothnie, and Isabelle Létourneau for helpful advice during the execution of this work and Gladys Erives, Kathy Sparks, and Maureen Hobbs for technical assistance.

	Footnotes

 
This work was supported by Grant MOP-10519 from the Canadian Institutes of Health Research (CIHR). A.J.S. is the recipient of a CIHR Doctoral Research Award and a Queen's University Robert John Wilson Fellowship. S.P.C.C. is the recipient of a Canada Research Chair in Cancer Biology and is the Queen's University Bracken Chair in Genetics and Molecular Medicine.

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

doi:10.1124/dmd.107.019661.

ABBREVIATIONS: MRP, multidrug resistance protein; ABC, ATP-binding cassette; LTC₄, leukotriene C4; GSH, glutathione; E₂17βG, 17β-estradiol 17-(β-D-glucuronide); MDMA, methylenedioxymethamphetamine; CA, caffeic acid, 3,4-dihydroxycinnaminic acid; OH, hydroxy; N-Me--MeDA, N-methyl--methyldopamine; HEK, human embryonic kidney; 5-GS-N-Me--MeDA, 5-(glutathione-S-yl)-N-methyl--methyldopamine; 2-GS-CA, 2-(glutathion-S-yl)-caffeic acid; 2-OH-1-GS-E₂, 2-hydroxy-1-(glutathion-S-yl)-17β-estradiol; 2-OH-1-GS-E₁, 2-hydroxy-1-(glutathion-S-yl)-estrone; 2-OH-4-GS-E₂, 2-hydroxy-4-(glutathion-S-yl)-17β-estradiol; 4-OH-2-GS-E₂, 4-hydroxy-2-(glutathion-S-yl)-17β-estradiol; LY475776, (N-(4-azido-3-phenyl)-2-[3-(9-chloro-3-methyl-4-ozo-4H-isoxazolo[4,3-c]quinolin-5-yl)-cyclohexyl]-acetamide); MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; GS, (glutathion-S-yl).

Address correspondence to: Dr. Susan P. C. Cole, Division of Cancer Biology & Genetics, Queen's University Cancer Research Institute, Kingston, ON, Canada K7L 3N6. E-mail: spc.cole{at}queensu.ca

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