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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Interaction of Cysteine Conjugates with Human and Rabbit Organic Anion Transporter 1

Department of Physiology, University of Arizona, Tucson, Arizona (C.E.G., L.M., S.H.W.); and Zentrum für Physiologie und Pathophysiologie, Georg-August-Universitat, Göttingen, Germany (A.B., G.B.)

Received August 23, 2002; accepted October 8, 2002.

	Abstract

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Organic anion (OA) transport mediates accumulation of the zwitterionic nephrotoxic cysteine S-conjugates S-dichlorovinylcysteine (DCVC) and S-chlorotrifluoroethylcysteine (CTFC) in the rabbit renal proximal tubule (RPT). Although these cysteine conjugates are nephrotoxic to the human RPT, neither the role of OA transport nor the specific OA transport pathway(s) involved in cysteine conjugate accumulation are known. Since the OAT1 transporter has the characteristics of para-aminokippurate (PAH) transport that closely correlate to the native RPT, we examined the interaction of DCVC, CTFC, and the nontoxic benzothiazolylcysteine (BTC) with PAH transport mediated by human OAT1 and rabbit Oat1 expressed in Chinese hamster ovary and COS7 heterologous expression systems, respectively. Although the K_m values for PAH uptake by hOAT1 and rbOat1 (8.9 ± 3.6 and 20.7 ± 8 µM, respectively) were 5- to 10-fold less than the K_m for peritubular PAH transport into rabbit RPT, the IC₅₀ values for DCVC, CTFC, and BTC inhibition of PAH uptake mediated by either hOAT1 or rbOat1 were similar between these two transporters and to the IC₅₀ values for these conjugates measured in rabbit RPT. The IC₅₀ for inhibition of hOAT1- and rbOat1-mediated PAH uptake by the hydrophobic conjugate BTC was more than 5-fold lower than the IC₅₀ values seen with DCVC and CTFC, suggesting that hydrophobicity increases the affinity of OAT1 for cysteine conjugates. Finally, preloading cells transfected with hOAT1 with BTC significantly trans-stimulated the uptake of PAH, consistent with the conclusion that BTC and, hence, other cysteine S-conjugates are substrates for hOAT1.

Conjugation of xenobiotics with glutathione (GSH) increases their hydrophilicity, thereby enhancing their excretion, making conjugation an important detoxification mechanism. Conjugation of trichloroethylene and chlorotrifluoroethylene with GSH and the accumulation of GSH-conjugates or their metabolites are required to exert nephrotoxicity (Odum and Green, 1984). Glutathione conjugates of trichloroethylene and chlorotrifluoroethylene, ultimately, are cleaved to form the zwitterionic cysteine conjugates S-dichlorovinylcysteine (DCVC) and S-chlorotrifluoroethylcysteine (CTFC). These cysteine conjugates are accumulated by the RPT and biotransformed by cysteine conjugate -lyase to a reactive thiol resulting in RPT lesions (Odum and Green, 1984; Lock and Ishmael, 1985). Many cysteine conjugates, however, are detoxified by the addition of an N-acetyl, forming a negatively charged mercapturic acid (Stevens and Jones, 1989) that is accumulated by the RPT. The N-acetyl group can then be removed intracellularly by the enzyme deacetylase to reform the cysteine conjugate. Thus, accumulation of cysteine conjugates, such as DCVC and CTFC, and their N-acetyl cysteine conjugate derivatives by the RPT is paramount to the production of toxicity (Weinberg, 1993).

Although many cysteine S-conjugates appear to be nephrotoxic and even nephrocarcinogenic in humans (Chen et al., 1990; Birner et al., 1993, 1997; Bruning and Bolt, 2000; Lash et al., 2000), the role of the human organic transporter 1 (hOAT1) in the transport of these substrates has been not shown. Although the negatively charged N-acetyl derivatives of DCVC (NAC-DCVC) and of CTFC (NAC-CTFC) have been shown to be substrates for rat Oat1 (Pombrio et al., 2001), the interaction of the parent zwitterionic cysteine conjugate DCVC or CTFC with OAT1 is not known. Indeed, studies with rat renal slices and isolated renal membranes (Schaeffer and Stevens, 1987; Wolfgang et al., 1989) suggested that zwitterionic cysteine conjugates interact poorly with rat renal organic anion transport. Different results, however, have been observed with the rabbit kidney. Both DCVC and CTFC cis-inhibit and trans-stimulate basolateral membrane PAH and fluorescein transport in the rabbit RPT (Dantzler et al., 1995, 1998; Groves and Morales, 1999), suggesting that these toxicants are substrates for the basolateral OA transport pathway (including and possibly dominated by OAT1) in the native RPT. In addition, high PAH concentrations prevent DCVC-induced losses of intracellular potassium from single S2 RPT segments from the rabbit kidney (Dantzler et al., 1998), indicating that transport of this substrate via OAT1 may be involved in cysteine S-conjugate-mediated toxicity. Consequently, to further characterize and understand the molecular mechanisms of organic anion secretion in humans, the present article compared the interaction of nephrotoxic cysteine S-conjugates with hOAT1-mediated PAH transport to that observed with rbOat1. The results demonstrated that cysteine S-conjugate interaction is similar for both human and rabbit Oat1. The IC₅₀ values for inhibition of PAH uptake by DCVC, CTFC, and S-benzothiazolylcysteine (BTC) were similar to the affinities for inhibition of PAH transport previously measured for these substrates in isolated rabbit RPT. As previously reported, PAH is a high affinity substrate for hOAT1, and the affinity of rbOat1 for PAH is markedly higher than implied from studies using isolated rabbit RPT. The data from this study suggest that hOAT1 and rbOat1 share similar characteristics for PAH transport and substrate interaction.

	Materials and Methods

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Materials. [³H]PAH (specific activity, 4 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). DCVC and BTC were a gift from Dr. A. Jay Gandolfi (Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ). All other chemicals were purchased from standard sources and were of the highest purity available.

Stably and Transiently Transfected Cell Lines. CHO^hOAT1 cells were generated by stable transfection of hOAT1 cDNA into Chinese hamster ovary cells, as described by Ho et al. (2000), and were a generous gift from Dr. Thomas Cihlar (Gilead Sciences, Foster City, CA). The cells were maintained in a Ham's F-12 medium with a Kaighn's modification supplemented with 10% fetal bovine serum, 50 U/ml pen-strep, and 1 mg/ml G418. For studies with rbOat1, COS7 cells were grown in Ham's F-12 with a Kaighn's modification supplemented with 10% fetal bovine serum and 50 U/ml pen-strep.

COS7 cells were transiently transfected with rbOat1 by electroporation using a modification of the protocol of Baum et al. (1994). Briefly, COS7 cells were lifted from the culture flask by treatment with 0.25% trypsin/1 mM EDTA and resuspended at a concentration of 5 x 10⁶ cells/ml. An aliquot of 0.4 ml of cell suspension was transferred to a cuvette containing 10 µg of rbOat1 cDNA and 10 µg of salmon sperm DNA. Cells were electroporated using a BTX Electro-cell manipulator (BTX, San Diego, CA). For electroporation, COS7 cells were pulsed for approximately 25 ms at 260 mV with unlimited resistance and a capacitance of 1,050 microfarads. Electroporated cells were resuspended in Ham's F-12 medium, counted, and plated at a cell density of 84,000 cells/cm². Transport was measured 24 h after electroporation.

Measurement of [³H]PAH Transport in CHO^hOAT1 and COS7^rbOat1 Cells. CHO^hOAT1 cells were seeded into 12-well plates at a density of 350,000 cells/well. After 24 h and before measuring transport on monolayers, confluence was confirmed visibly under phase-contrast microscopy. The cells were washed once with room temperature Waymouth's buffer. To measure [³H]PAH accumulation, cells were incubated with room temperature Waymouth's buffer (135 mM NaCl, 13 mM Hepes, 2.5 mM CaCl₂ · 2H₂O, 1.2 mM MgCl₂, 0.8 mM MgSO₄ · 7H₂O, 5 mM KCl, 28 mM glucose) containing 1 µCi/ml [³H]PAH in the presence and absence of 1 mM unlabeled PAH. To examine the kinetics of [³H]PAH uptake, CHO cells were incubated for 30 s and COS7 cells for 2 min in Waymouth's buffer containing 1 µCi/ml [³H]PAH and increasing concentrations of unlabeled test substrate. To stop transport at timed intervals from 0.25 to 10 min, medium was aspirated, and cells were rinsed three times in ice-cold 0.3 mM probenecid. Cells were dissolved in 1 N NaOH, and aliquots were taken for liquid scintillation counting. Uptakes were expressed per squared centimeter of nominal cell surface area of a confluent cell layer.

Measurement of trans-Stimulation of [³H]PAH Uptake. To determine the effect of efflux of unlabeled substrate on the uptake of [³H]PAH (trans-stimulation), CHO cells and COS7 cells were incubated for 1 h in Waymouth's buffer in the absence of substrate or in the presence of 0.5 mM PAH or BTC. After 1 h, the cells were rinsed three times with room temperature Waymouth's, followed by the addition of 1 µCi/ml [³H]PAH to measure uptake. To stop transport at various time intervals, medium was aspirated and the cells rinsed and dissolved as described above.

	Results

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Kinetics of [³H]PAH Uptake in CHO Cells Stably Transfected with hOAT1 and in COS7 Cells Transiently Transfected with rbOat1. The uptake of [³H]PAH in hOAT1-expressing CHO cells and rbOat1-expressing COS7 cells increased with time (Fig. 1) and was reduced approximately 90% at all time points by the addition of 1 mM unlabeled PAH (data not shown). Uptake into the CHO cells was 2-fold more rapid than uptake into COS7 cells, probably reflecting different levels of protein expression. Estimates of the initial rate of OAT1-mediated transport in subsequent experiments were based on 30-s uptakes in the CHO cells (Fig. 1A) and 2-min uptakes in the COS7 cells (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Time course of [3H[PAH uptake by hOAT1-transfected CHO cells and rbOat1-transfected COS7 cells. Each point represents the mean ± S.E. of duplicate (hOAT1) or triplicate (rbOat1) measurements from three separate experiments.

To characterize the interaction of PAH with hOAT1, the kinetics of PAH uptake were examined in hOAT1 transfected CHO cells. As shown in Fig. 2A, increasing concentrations of unlabeled PAH decreased the accumulation of [³H]PAH. The highest concentration of unlabeled PAH failed to completely block the uptake of [³H]PAH, consistent with the presence of passive diffusion and/or nonspecific binding of radiolabel to the cells. The inhibition of the uptake of [³H]PAH by increasing concentrations of unlabeled PAH was adequately described by the kinetics of competitive inhibition using a modification of the isotope dilution procedure, as described by Malo and Berteloot (1991).

(1)

where J is the rate of cellular PAH uptake from an extracellular concentration of [³H]PAH of [T*] in the presence of an unlabeled PAH concentration of [S]. The kinetics parameters J_max, K_m, and C are defined as the maximal capacity of the carrier for PAH, the concentration of PAH at 1/2J_max, and a coefficient describing the nonsaturable accumulation of PAH (passive diffusion and/or nonspecific binding). The J_max and K_m values generated from the analysis of the kinetics of hOAT1-mediated [³H]PAH transport in three separate passages of stably transfected CHO cells were 3.8 ± 0.9 pmol · cm^-2 · min^-1 and 8.9 ± 3.6 µM, respectively. As with hOAT1-mediated PAH uptake, increasing concentrations of unlabeled PAH progressively reduced the uptake of [³H]PAH by rbOat1-transfected COS7 cells (Fig. 2B). From three separate electroporated cell passages, the J_max and K_m values measured for PAH uptake by rbOat1 were 74.9 ± 29 pmol · cm^-2 · min^-1 and 20.7 ± 8 µM, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Inhibition of [3H]PAH uptake by increasing PAH concentrations in hOAT1-transfected CHO cells and rbOat1-transfected COS7 cells. Uptakes were measured at 30 s (hOAT1) and 2 min (rbOat1). Each points represents the mean ± S.E. of duplicate (hOAT1) or triplicate (rbOat1) measurements from a representative experiment. The line fit to the data were calculated and kinetic parameters derived with a nonlinear regression algorithm (Sigma Plot; SPSS Science, Chicago, IL).

Kinetics of cis-Inhibition of PAH Uptake by Cysteine S-Conjugates in CHO Cells Stably Transfected with hOAT1 and in COS7 Cells Transiently Transfected with rbOat1. The nephrotoxic cysteine S-conjugates DCVC and CTFC and the nontoxic cysteine S-conjugate BTC have been shown to be substrates for an organic anion transport pathway in single rabbit RPT S2 segments and suspensions of rabbit RPT (Dantzler et al., 1995, 1998; Groves and Morales, 1999). Since multiple organic anion pathways have been cloned from the RPT, the interaction of these substrates directly with the cloned hOAT1 and rbOat1 transporters was examined for comparison to data we previously generated with these substrates using rabbit RPT. Increasing concentrations of DCVC, CTFC, and BTC progressively reduced the uptake of [³H]PAH in CHO cells stably transfected with hOAT1 (Fig. 3) and in COS7 cells transiently transfected with rbOat1 (Fig. 4). BTC produced the most effective inhibition of uptake, with IC₅₀ values (the concentration of inhibitor that reduced the uptake of radiolabeled substrate by 50%) for inhibition of PAH uptake of 9.9 ± 2.5 µM for hOAT1 and 14.9 ± 3 µM for rbOat1 (Table 1). The IC₅₀ values measured for the inhibition of [³H]PAH by DCVC and CTFC were substantially higher than those produced by BTC, 208 ± 62.3 and 177 ± 22.5 µM, respectively, for hOAT1 and 171 ± 53 and 184 ± 45 µM, respectively, for rbOat1 (Table 1). The apparent affinities of hOAT1 and rbOat1 for DCVC and CTFC did not differ substantially from the apparent K_i values measured for inhibition of PAH transport by these substrates in the intact rabbit RPT, 86.4 ± 7.6 and 105 ± 3 µM for DCVC and CTFC, respectively (Table 1) (Dantzler et al., 1995; Groves and Morales, 1999). Interestingly, the IC₅₀ values for DCVC inhibition of PAH uptake by hOAT1 and rbOat1 (Table 1) were similar to the K_m value of 283 ± 23.3 µM for DCVC transport in rabbit RPT S2 segments (Dantzler et al., 1998), consistent with a role for OAT1 in cysteine S-conjugate uptake by the native RPT.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Inhibition of [3H]PAH uptake by increasing concentrations of BTC (A), DCVC (B), and CTFC (C) in hOAT1-transfected CHO cells. Uptakes were measured at 30 s. Each points represents the mean ± S.E. of duplicate measurements from a representative experiment. The line fit to the data were calculated and kinetic parameters derived with a nonlinear regression algorithm (Sigma Plot).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Inhibition of [3H]PAH uptake by increasing concentrations of BTC (A), DCVC (B), and CTFC (C) in rbOat1-transfected COS7 cells. Uptakes were measured at 2 min. Each point represents the mean ± S.E. of triplicate measurements from a representative experiment. The line fit to the data were calculated and kinetic parameters derived with a nonlinear regression algorithm (Sigma Plot).

To determine whether the cysteine S-conjugates DCVC and CTFC reduced transport via a direct interaction with OAT1 or through causing cytotoxicity, CHO cells or COS7 cells were incubated for 2 to 3 min (the longest time points used to examine kinetics) with the highest concentration of these substrates (in the absence of [³H]PAH) used in the kinetic assays. The cells were then rinsed to remove the cysteine conjugate, [³H]PAH added, and transport measured. Compared with control, when cells were preincubated with the cysteine S-conjugates for 2 min and then rinsed to remove the conjugates, [³H]PAH uptake was not significantly different from control uptakes in the absence of the conjugates (data not shown) compared with the >70% inhibition in experiments involving coexposure of inhibitor and [³H]PAH. These observations indicate that a direct interaction of the inhibitors with the transporter was the probable basis of the inhibition of OAT1 activity produced by the cysteine S-conjugates.

The inhibition of [³H]PAH uptake by BTC, DCVC, and CTFC indicated an interaction between OAT1 and these cysteine S-conjugates but did not reveal whether the interaction is competitive. To determine the type of interaction between cysteine S-conjugates and transport of PAH, 30-s uptakes of [³H]PAH into CHO^hOAT1 cells were measured in the presence of increasing concentrations of unlabeled PAH; however, these measurements also were made in the presence of a single concentration (400 µM) of DCVC that should increase the K_m for PAH uptake at least 3-fold if the interaction is competitive. In four separate experiments, the presence of DCVC increased the K_m for hOAT1-mediated PAH transport approximately 4-fold (from 3.3 ± 1.7 to 13.6 ± 5.8 µM), with only a modest change in J_max (from 1.2 ± 0.3 to 2.0 ± 0.6 mol · cm^-2 · min^-1), which is consistent with a competitive interaction of this toxicant with hOAT1.

trans-Stimulation of [³H]PAH Uptake by Cysteine S-Conjugates in hOAT1 and rbOat1 Transfected Cells. Both DCVC and CTFC have been shown to trans-stimulate the efflux of PAH and/or the OA fluorescein, consistent with the conclusion that these toxicants are substrates for a common OA transporter in the native RPT (Dantzler et al., 1995; Groves and Morales, 1999). Due to the rapid uptake and efflux of [³H]PAH typically displayed in the native RPT, the ability of these toxicants to trans-stimulate PAH efflux can be measured at very short time points, effectively minimizing the threat of parallel cytotoxic effects. In OAT1-transfected CHO or COS7 cells, however, fluxes of [³H]PAH are slow (compared with those in native RPT). To measure the trans-stimulation of PAH uptake into cells requires preloading cells for at least 1 h with high concentrations (0.5 mM) of either DCVC or CTFC. Under these conditions (1-h preloading with 0.5 mM DCVC or CTFC), however, toxicity is a major concern. Consequently, our examination of trans-effects was limited to the influence of preloading cells for 1 h with the nontoxic cysteine S-conjugate BTC, which was then compared with that produced by preloading cells with unlabeled PAH. As shown in Fig. 5, hOAT1-mediated uptake of [³H]PAH increased approximately 33 and 47%, respectively, when CHO-hOAT1 cells were preloaded with 0.5 mM BTC or 0.5 mM PAH. These data support the conclusion that cysteine S-conjugates are transportable substrates of hOAT1.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Effect of preloading hOAT1-transfected CHO cells with PAH or BTC on the uptake of [3H]PAH. Cells were preincubated for1hin0.5mM PAH or BTC before measuring one min [3H]PAH uptake. Data are expressed as the mean ± S.E. of duplicate measurements from two separate experiments. #, p < 0.05 versus control.

	Discussion

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Glutathione conjugation of substrates is a mechanism employed by the body to make hydrophobic substances more water soluble, thereby facilitating their excretion. Glutathione conjugates typically undergo enterohepatic circulation and can be ultimately cleaved to their cysteine S-conjugates, which can enter renal cells to produce toxicity (Anders et al., 1988; Koob and Dekant, 1991). Since the cysteine S-conjugates DCVC and CTFC are toxic to primary cultures of human RPT cells (Chen et al., 1990), the pathways responsible for their entry in the human RPT could play a critical role in the development of cell injury. The transport pathways involved in the uptake of cysteine conjugates by human kidney are not established, however. Indeed, marked species differences appear to exist in the handling of zwitterionic cysteine conjugates. Although in native rabbit RPT, DCVC, and CTFC are substrates for OA transport (Dantzler et al., 1995, 1998; Groves and Morales, 1999) in native rat RPT uptake of ³⁵S-DCVC is not blocked by probenecid, an inhibitor of OA transport (Zhang and Stevens, 1989). In addition, although the anionic N-acetyl derivative of DCVC (NAC-DCVC) is a substrate for rat Oat1 (Pombrio et al., 2001), the zwitterionic DCVC does not interact with OA transport pathways in the native rat RPT (Zhang and Stevens, 1989). The present study is the first to document the direct interaction of nephrotoxic cysteine S-conjugates with the human renal OA transporter hOAT1. In addition, the data presented here confirm the specific interaction of cysteine S-conjugates with the rabbit ortholog of this process, rbOat1.

The cysteine S-conjugates DCVC and CTFC inhibited PAH uptake mediated by both hOAT1 and rbOat1. The apparent affinity of the OAT1s for these toxicants appeared to be lower than the affinity for the cloned transporters displayed for PAH; hOAT1 had a 20-fold lower apparent affinity and rabbit Oat1 an 8-fold lower apparent affinity for DCVC and CTFC than for PAH (Table 1). In contrast, the OA transport pathway in native rabbit RPT had a similar apparent affinity for DCVC, CTFC, and PAH (Table 1). Interestingly, the affinities for DCVC and CTFC interaction with hOAT1 and rbOat1 are similar to the affinities measured in the native rabbit RPT (Table 1). The IC₅₀ of DCVC for rbOat1-mediated PAH uptake also was not very different from the measured K_m of 283 µM for DCVC uptake in the native rabbit RPT suspensions (Dantzler et al., 1998). In the native tubule, however, the possibility still exists for cysteine S-conjugate interactions with multiple pathways, such as OAT2 to -4, which may contribute to the observed differences between these systems.

The affinities of both human and rabbit Oat1 for the nontoxic cysteine S-conjugate BTC were similar and approximately 10-fold higher than for DCVC and CTFC. As a more hydrophobic substrate, the increased interaction of BTC with OAT1 could be associated with a relationship between increasing hydrophobicity and increasing affinity as reported for OA transport in the native RPT (Somogyi, 1996; Ullrich et al., 1997). The affinity of the more hydrophilic substrate PAH, however, is similar to BTC and, hence, suggests that a more complicated interaction exists between the substrate and the OAT1 transporter than can be explained simply by hydrophobicity alone. The higher affinity of the relatively hydrophilic PAH may be due to the ability to form hydrogen bonds with the transporter (Ullrich, 1997). Although the measured affinities show variation in terms of their absolute value, the pattern of inhibition remains unaltered when comparing cysteine S-conjugate interactions with human and rabbit Oat1 with the native rabbit RPT. For hOAT1, rbOat1, and native rabbit RPT, the more hydrophobic BTC had a significantly greater affinity for inhibition of PAH transport than the more hydrophilic (compared with BTC) DCVC and CTFC, which displayed similar affinities to one another within a respective system. Although the double bond present in DCVC causes this haloalkene structure to be more hydrophobic than the haloalkane CTFC, this structural difference, in contrast to BTC, fails to have a profound effect on the inhibitory constants for inhibition of PAH uptake.

The prototypical OAT1 substrate PAH exhibited a high affinity for both hOAT1 and rbOat1. The affinity of hOAT1 for PAH was approximately 2-fold greater than for rbOat1, but the affinity of rbOat1 for PAH was also approximately 5-fold or more greater than the affinity for PAH transport measured in the native rabbit RPT (Table 1; Dantzler et al., 1995; Groves et al., 1998). Several possible bases exist for the discrepancy in apparent K_m values for PAH uptake by the native RPT and cells expressing rbOat1. First, transport of PAH in the native tubule may be influenced by parallel activity of several transporters, in addition to OAT1, that also accept PAH as a substrate (e.g., OAT2–4). PAH has been shown to be a substrate for hOAT3, with a K_m of 87 µM (Cha et al., 2001), a value that more closely resembles the K_m measured in native tubules. Second, the quantitative characteristics of OAT1 in the native tubule environment may differ markedly from those arising from expression in a cultured cell system. Finally, physical factors, including the potential influence of unstirred layers on the measurement of kinetic parameters, may complicate comparison of kinetic parameters in different experimental systems. In spite of the substantial difference in the affinity for PAH uptake noted between the rbOat1 and the native rabbit RPT, the inhibitory constants measured for BTC, DCVC, and CTFC demonstrated less variability between these two systems and suggests that OAT1 may play a significant role in cysteine S-conjugate interactions with OA transport in the native rabbit RPT.

Among the OAT transporters 1 to 4, to date only OAT1 has been shown to support a trans-stimulation of substrate transport (Sekine et al., 1998; Kusuhara et al., 1999; Cha et al., 2000). Previous work using RPT isolated from the rabbit kidney has shown that DCVC and CTFC are capable of trans-stimulating the efflux of PAH and/or fluorescein, thereby demonstrating that these toxicants are substrates for an OA carrier (Dantzler et al., 1995, 1998; Groves and Morales, 1999). Preloading hOAT1-transfected cells with the nontoxic cysteine S-conjugate BTC significantly stimulated the uptake of PAH, suggesting that cysteine S-conjugate transport is mediated by OAT1. DCVC also acts as a competitive inhibitor of hOAT1 PAH uptake, which further substantiates a role for OAT1 in the uptake of these nephrotoxicants. Transport by OAT1, however, does not preclude a role for OAT pathways 2 to 4 in cysteine S-conjugate transport. For example, the OA substrate ochratoxin A is transported by both hOAT1 and renal hOAT3 (Jung et al., 2001). In the native rabbit RPT, 5 mM PAH, a concentration 50-fold greater than the K_m for PAH transport, only reduced the uptake and the subsequent nephrotoxicity of DCVC by approximately 70%, which also indicates that DCVC uptake in the RPT could involve both OAT1 and a different OAT pathway. Since glutathione conjugation of various halogenated hydrocarbons and transport of metabolites appears to contribute significantly to their subsequent renal pathophysiology, investigation of the transport properties of all OAT transporters will be necessary to completely understand the mechanisms involved in the renal handling of cysteine S-conjugates.

In conclusion, zwitterionic cysteine S-conjugates directly inhibited the uptake of PAH mediated by the cloned OA transporters hOAT1 and rbOat1. The affinities for inhibition and the pattern of inhibition of PAH transport produced by cysteine S-conjugate interaction with rbOat1 was similar to that reported for the native rabbit RPT. Cysteine S-conjugates also competitively inhibited and are transported by hOAT1. Thus, entry into the renal cell via this pathway may contribute to the onset of nephrotoxicity and carcinogenicity associated with these toxicants.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants ES08860, DK56224, ES06694, and ES04940.

DOI: 10.1124/jpet.102.043455.

ABBREVIATIONS: OA, organic anion; RPT, renal proximal tubule; GSH, glutathione; DCVC, S-dichlorovinylcysteine; CTFC, S-chlorotrifluoroethylcysteine; hOAT1, human organic transporter 1; PAH, para-aminohippurate; BTC, S-benzothiazolylcysteine; CHO, Chinese hamster ovary; G418, geneticin.

Address correspondence to: Dr. Carlotta E. Groves, Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724. E-mail: cegroves{at}u.arizona.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Anders MW, Lash L, Dekant W, Elfarra AA, and Dohn DR (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. Crit Rev Toxicol 18: 311–341.[Medline]

Bahn A, Knabe M, Hagos Y, Rödiger M, Godehardt S, Graber-Neufeld DS, Evans KK, Burckhardt G, and Wright SH (2002) Rabbit multispecific organic anion transporter 1 (rbOat1) is involved in the detoxification of heavy metals from the kidneys. Mol Pharmacol 62: 1128–1136.[Abstract/Free Full Text]

Baum C, Forster P, Hegewisch-Becker S, and Harbers K (1994) An optimized electroporation protocol applicable to a wide range of cell lines. Biotechniques 17: 1058–1062.[Medline]

Birner G, Bernauer U, Werner M, and Dekant W (1997) Biotransformation, excretion and nephrotoxicity of haloalkene-derived cysteine conjugates. Arch Toxicol 72: 1–8.[CrossRef][Medline]

Birner G, Vamvakas S, Dekant W, and Henschler D (1993) Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure. Environ Health Perspect 99: 281–284.[Medline]

Bruning T and Bolt HM (2000) Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms and controversies. Crit Rev Toxicol 30: 253–285.[CrossRef][Medline]

Cha SH, Sekine T, Fukushima JI, Kanai Y, Kobayashi Y, Goya T, and Endou H (2001) Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol Pharmacol 59: 1277–1286.[Abstract/Free Full Text]

Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y, Kanai Y, and Endou H (2000) Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275: 4507–4512.[Abstract/Free Full Text]

Chen JC, Stevens JL, Trifillis AL, and Jones TW (1990) Renal cysteine conjugate beta-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells. Toxicol Appl Pharmacol 103: 463–473.[CrossRef][Medline]

Dantzler WH, Evans KK, Groves CE, Welborn JR, North J, Stevens JL, and Wright SH (1998) Relation of cysteine conjugate nephrotoxicity to transport by the basolateral organic anion transport system in isolated S2 segments of rabbit proximal renal tubules. J Pharmacol Exp Ther 286: 52–60.[Abstract/Free Full Text]

Dantzler WH, Evans KK, and Wright SH (1995) Kinetics of interactions of paraaminohippurate, probenecid, cysteine conjugates and N-acetyl cysteine conjugates with basolateral organic anion transporter in isolated rabbit proximal renal tubules. J Pharmacol Exp Ther 272: 663–672.[Abstract/Free Full Text]

Groves CE and Morales M (1999) Chlorotrifluoroethylcysteine interaction with rabbit proximal tubule cell basolateral membrane organic transport and apical membrane amino acid transport. J Pharmacol Exp Ther 291: 555–561.[Abstract/Free Full Text]

Groves CE, Morales M, and Wright SH (1998) Peritubular transport of ochratoxin A in rabbit renal proximal tubules. J Pharmacol Exp Ther 284: 943–948.[Abstract/Free Full Text]

Ho ES, Lin DC, Mendel DB, and Cihlar T (2000) Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1. J Am Soc Nephrol 11: 383–393.[Abstract/Free Full Text]

Hosoyamada M, Sekine T, Kanai Y, and Endou H (1999) Molecular cloning and functional characterization of a novel multispecific organic anion transporter from human kidney. Am J Physiol 276: F122–F128.

Jung KY, Takeda M, Kim DK, Tojo A, Narikawa S, Yoo BS, Hosoyamada M, Cha SH, Sekine T, and Endou H (2001) Characterization of ochratoxin A transport by human organic anion transporters. Life Sci 69: 2123–2135.[CrossRef][Medline]

Kogo K, Graves P, Leahy A, Wilson P, Stuhlmann H, and You G (1999) Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells. J Biol Chem 274: 1519–1524.[Abstract/Free Full Text]

Koob M and Dekant W (1991) Bioactivation of xenobiotics by formation of toxic glutathione conjugates. Chem-Biol Interactions 77: 107–136.[CrossRef][Medline]

Kusuhara H, Sekine T, Utsunnomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, and Endou H (1999) Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675–13680.[Abstract/Free Full Text]

Lash LH, Parker JC, and Scott CS (2000) Modes of Action of trichloroethylene for kidney tumorigenesis. Environ Health Perspect 108: 225–240.

Lock EA and Ishmael J (1985) Effect of organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-1,3-butadiene and its conjugates. Toxicol Appl Pharmacol 81: 32–42.[CrossRef][Medline]

Lu R, Chan BS, and Schuster VL (1999) Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol 276: F295–F303.

Malo C and Berteloot A (1991) Analysis or kinetic data in transport studies: new insights from kinetic studies of Na⁺-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling filtration apparatus. J Membr Biol 122: 127–141.[CrossRef][Medline]

Odum J and Green T (1984) The metabolism and nephrotoxicity of tetrafluoroethylene in the rat. Toxicol Appl Pharmacol 76: 306–318.[CrossRef][Medline]

Pombrio JM, Giangreco A, Li L, Wempe MF, Anders MW, Sweet DH, Pritchard JB, and Ballatori N (2001) Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter 1. Mol Pharmacol 60: 1091–1099.[Abstract/Free Full Text]

Pritchard JB and Miller DS (1996) Renal Secretion of organic anions and cations. Kidney Int 49: 1649–1654.[Medline]

Reid G, Wolff NA, Dautzenberg FM, and Burckhardt G (1998) Cloning of a human renal p-aminohippurate transporter, hROAT. Kidney Blood Press 21: 233–237.[CrossRef][Medline]

Schaeffer VH and Stevens JL (1987) Mechanism of transport for toxic cysteine conjugates in rat kidney cortex membrane vesicles. Mol Pharmacol 32: 293–298.[Abstract]

Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H (1998) Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179–182.[CrossRef][Medline]

Sekine T, Watanbe N, Hosoyamada M, Kanai Y, and Endou H (1997) Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272: 18526–18529.[Abstract/Free Full Text]

Somogyi A (1996) Renal transport of drugs: specificity and molecular mechanisms. Clin Exp Pharmacol Physiol 23: 986–989.[Medline]

Stevens JL and Jones DP (1989) The mercapturic acid pathways: biosynthesis, intermediary metabolism and physiological disposition, in Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin D, Poulson R, and Avramoric O eds) part B, pp 45–84, Wiley, New York.

Sweet DH, Wolff NA, and Pritchard JB (1997) Expression cloning and characterization of rOAT1. The basolateral organic anion transporter in rat kidney. J Biol Chem 272: 30088–30095.[Abstract/Free Full Text]

Ullrich KJ (1997) Renal transporters for organic anions and cations. Structural requirements for substrates. J Membrane Biol 158: 95–107.[CrossRef][Medline]

Ullrich KJ, Rumrich G, Burke TR, Shirazi-Beechey SP, and Lang H (1997) Interaction of alkyl/arylphosphonates, phosphonocarboxylates, and diphosphonates with different anion transport systems in the proximal renal tubule. J Pharmacol Exp Ther 283: 1223–1229.[Abstract/Free Full Text]

Weinberg JM (1993) The cellular basis of nephrotoxicity, in Diseases of the Kidney (Schrier RW and Gottschalk CW eds) pp 1031–1097, Little, Brown and Co., Boston.

Wolff Na, Werner A, Burkhardt S and Burkhardt G (1997) Expression cloning and characterization of a renal organic anion transporter from winter flounder. FEBS Lett 417: 287–291.[CrossRef][Medline]

Wolfgang GH, Gandolfi AJ, Stevens JL, and Brendel K (1989) N-Acetyl-S-(1,2-dichlorovinyl)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-L-cysteine in rabbit renal slices: differential transport and metabolism. Toxicol Appl Pharmacol 101: 205–219.[CrossRef][Medline]

Zhang G and Stevens JL (1989) Transport and activation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1, 2-dichlorovinyl)-L-cysteine in rat kidney proximal tubules. Toxicol Appl Pharmacol 100: 51–61.[CrossRef][Medline]

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