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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand (C.S., V.C.); and Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (C.S., A.G.A., J.B.P.)
Received November 8, 2004; accepted January 6, 2005.
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Abstract |
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). Therefore, it is a popular sugar substitute in a variety of foods and often used as a food supplement in Japan, China, South Korea, and Taiwan. Stevioside can be degraded to steviol, an aglycone, by the intestinal microflora from various animal species, including man (Hutapea et al., 1997; Koyama et al., 2003b). Stevioside is a large neutral molecule with both polar and hydrophobic regions, whereas steviol contains a hydrophobic ring and one negative charge on the carboxylic group. The chemical structures of stevioside and steviol are shown in Fig. 1. In addition to its sweetening properties, stevioside has been shown to have potential therapeutic value as a contraceptive (Melis, 1999) and an antihypertensive agent (Chan et al., 1998; Jeppesen et al., 2002, 2003). Continued consumption of stevioside extract for 3 months reduced blood pressure in hypertensive patients (Chan et al., 2000). Both stevioside and steviol alter glucose metabolism (Suanarunsawat and Chaiyabutr, 1997) and glucose absorption (Toskulkao et al., 1995). They have also been proposed to have a potential role as antihyperglycemic agents by stimulating insulin secretion from pancreatic beta cells (Jeppesen et al., 2000, 2002, 2003). Stevioside was not effectively absorbed from everted sacs of rat intestine, whereas steviol was rapidly absorbed (Koyama et al., 2003b). Similarly, stevioside showed very limited absorption by the Caco-2 monolayers and steviol was much better absorbed (Geuns et al., 2003). In addition, limited intestinal secretion of steviol was also observed in these studies.
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The renal handling of stevioside and steviol has also been evaluated. It has been reported that stevioside clearance was higher than inulin clearance, indicating that stevioside is secreted by renal tubular epithelium (Melis, 1992). After oral administration of steviol-17-[14C] into rats, its excretion was found in the feces and urine. In addition, steviol-derived radioactivity was largely found in the urine following bile duct ligation after oral or intracecal administration, indicating that steviol was absorbed from lower bowel and excreted by both renal and biliary systems (Wingard et al., 1980). However, the underlying mechanisms of stevioside and steviol excretion are still unknown. Stevioside and steviol have also been shown to induce diuresis and natriuresis without a significant change in glomerular filtration rate or renal plasma flow (Melis, 1992, 1997). Furthermore, steviol was shown to inhibit accumulation of PAH in rat renal cortical slices (Toskulkao et al., 1994). Both stevioside and steviol have shown to inhibit transepithelial transport of PAH in isolated S2 segments of rabbit renal proximal tubule by interfering with the basolateral entry step (Jutabha et al., 2000; Chatsudthipong and Jutabha, 2001). However, this experimental model did not permit the clear elucidation of the interactions of stevioside and steviol with specific basolateral organic anion transporters.
Two organic anion transporters have been cloned in man that are expressed in the basolateral membranes of the renal proximal tubule, hOAT1 and hOAT3, and both act as organic anion exchangers (Hosoyamada et al., 1999; Cha et al., 2001; Sweet et al., 2003). Thus, they mediate the energetically uphill transport of organic anions from the blood into the cells of the proximal tubule in exchange for intracellular 
-ketoglutarate and drive tubular secretion of a wide variety of endogenous and exogenous anions including nucleotide antiviral agents, nonsteroidal anti-inflammatory drugs, methotrexate, and urate (Cihlar et al., 1999; Khamdang et al., 2002; Takeda et al., 2002).
In the present study, cloned hOAT1 and hOAT3 expressed in Xenopus laevis oocytes were used to establish whether these transporters were involved in the transport of stevioside and steviol. These data indicate that steviol, but not stevioside, is an effective inhibitor of both hOAT1 and hOAT3 transport. Furthermore, as assessed by trans-stimulation and electrophysiological studies, steviol is itself a substrate for these carriers.
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Materials and Methods |
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Oocyte Expression. Adult female X. laevis were purchased from Xenopus I (Ann Arbor, MI). The animals were anesthetized with 0.3% tricaine (Sigma-Aldrich), decapitated, and pithed. All animal procedures were conducted according to protocols approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee. Stage V and VI oocytes were selected and isolated by collagenase digestion as previously described (Cihlar et al., 1999). Capped cRNA of hOAT1, hOAT3, and fOat1 was synthesized from purified plasmid cDNA using mMESSAGE mMACHINE T7 ultra kit for ultra high yields of capped RNA (Ambion, Inc., Austin, TX). Defolliculated oocytes were injected with 20 ng of cRNA for transport and electrophysiological studies.
Measurement of PAH and ES Uptake in Oocyte-Expressing hOAT1 and hOAT3. The cis-inhibitory effects (i.e., inhibitor and labeled substrate were presented to the same side of the membrane or cell) of stevioside, steviol, and various compounds on PAH and ES uptake mediated by hOAT1 and hOAT3 were assessed 3 days postinjection. Oocytes were incubated with 1 ml of oocyte ringer 2 (OR-2) buffer [82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 1 mM MgCl2, 1 mM CaCl2, 1 mM sodium-pyruvate, 5 mM HEPES/sodium hydroxide (NaOH), pH 7.6] containing either 10 µM [3H]PAH (hOAT1) or 100 nM [3H]ES (hOAT3) in the absence or presence of stevioside, steviol, and other test compounds for 60 min at room temperature. Both stevioside and steviol were dissolved in dimethyl sulfoxide (DMSO) and diluted with OR-2 buffer. The final concentration of DMSO in the incubation medium was adjusted to 1% or less, and control measurements were made in the presence of equal DMSO concentrations. Uptake was terminated by aspiration of the incubation medium and addition of 1 ml of ice-cold OR-2 buffer. Oocytes were washed four more times with ice-cold OR-2 and placed in individual scintillation vials, lysed with 1 N NaOH, and neutralized with 1 N hydrochloric acid (HCl). Four milliliters of scintillation fluid (Ecolume; MP Biomedicals, Irvine, CA) were added, and radioactivity was measured using a Tri-Carb 2900TR Liquid Scintillation Analyzer (PerkinElmer Life and Analytical Sciences). The uptake of PAH and ES was expressed as a mean percentage of the control.
Kinetic Analysis. Human OAT1- and hOAT3-expressing oocytes were incubated with OR-2 buffer containing either [3H]PAH (hOAT1) or [3H]ES (hOAT3) at various concentrations in the absence or presence of 50 µM steviol for 30 min (approximates initial rate of uptake for either substrate into oocytes) (data not shown). Data were plotted as double reciprocal plots. The inhibitory constants (IC50) were calculated from sigmoidal dose-response analysis using GraphPad Prism version 4.00 for windows (GraphPad Software Inc., San Diego, CA). The Michaelis-Menten constants (Km) were calculated from the interception of x-axis, whereas the maximum rate of organic anions uptake (Vmax) was estimated from y-axis intercept. The Michaelis-Menten inhibition constants (Ki) were calculated by fitting the data to the following equation for competitive inhibition (Cheng and Prusoff, 1973).
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Determination of PAH Efflux from hOAT1- and hOAT3-Expressing Oocytes. Three days postinjection, oocytes were loaded with OR-2 buffer containing 10 µM[3H]PAH for 120 min to generate an intracellular accumulation of [3H]PAH. Oocytes were then rinsed with room temperature OR-2 buffer. Subsequently, oocytes were incubated with OR-2 buffer in the presence or absence of 1 mM unlabeled PAH, probenecid, and various concentrations of steviol for 90 min. At the end of the efflux period, [3H]PAH in the efflux medium and intracellular accumulation were measured using liquid scintillation counter as described above. Results were expressed as a mean percentage of the total PAH that appeared in the efflux medium after 90 min of efflux.
Electrophysiology. When human or flounder Oat1 were expressed in Xenopus oocytes, inward currents were generated upon the presentation of substrate, indicating that both isoforms exhibit electrogenic exchange of organic anion for dicarboxylate (Burckhardt et al., 2000; Aslamkhan et al., 2003). However, the magnitudes of the currents seen were considerably greater for the flounder clone. Thus, we used fOat1-expressing oocytes to directly investigate whether steviol was itself transported by the basolateral organic anion transporters. Three to 4 days postinjection, fOat1-expressing oocytes were placed in the 1.0-mm well of a RC-1Z oocyte recording chamber (Warner Instruments, Hamden, CT) and continuously bathed at a rate of 4 ml/min with OR-2 buffer at room temperature. The conventional two-electrode (3 M KCl; resistance of 0.5–5 M
) voltage-clamp method (Geneclamp 500B; Axon Instruments Inc., Union City, CA) was used for measuring oocyte membrane currents. The oocyte membrane potential was held at –80 mV, which is lower than the physiological membrane potential of the proximal tubule cell in situ to increase current evoked by added organic anion substrates, e.g., PAH or steviol. OR-2 buffer containing either PAH or steviol was applied to the oocytes in a pulsed manner via solenoid-controlled valves. Recordings were sampled at 50 Hz with a 10-Hz filter frequency. After sampling, current signals were filtered with a Gaussian low band pass filter (2 Hz cut-off).
Statistics. Data were expressed as means ± S.E. Statistical differences were assessed using one-way analysis of variance with Dunnett's test. In the efflux experiment, differences in efflux were analyzed using two-way analysis of variance with randomized-block design. Differences were considered to be significant when *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.
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To compare the effectiveness of stevioside and steviol with known inhibitors of hOAT1 and hOAT3, dose-response experiments were conducted. As shown in Fig. 3A, 50 µM to 1 mM stevioside had no effect on PAH uptake in hOAT1-expressing oocytes. In contrast, 10 µM to 1 mM steviol significantly inhibited hOAT1-mediated PAH uptake in a dose-dependent manner with an IC50 of 11.1 µM. When the effect of stevioside on ES uptake was investigated in oocytes expressing hOAT3, no inhibition was seen, even at 1 mM stevioside. In contrast, steviol significantly inhibited hOAT3-mediated ES uptake in a dose-dependent manner (IC50, 62.6 µM) (Fig. 3B).
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Kinetics of Steviol Inhibition. To determine the affinity of hOAT1 and hOAT3 for steviol, kinetic analyses were performed. As shown in Fig. 4A, [3H]PAH concentrations were varied from 3 to 30 µM. The rate of PAH uptake by the hOAT1-expressing oocytes was linear for 30 min (data not shown). Thus, this time was used to estimate the initial rate for Lineweaver-Burk analysis of hOAT1-mediated PAH uptake to determine the kinetic parameters (Km, Vmax, and Ki) in the presence or absence (control) of steviol (summarized in Table 1). The calculated Km of hOAT1-mediated PAH uptake doubled in the presence of 50 µM steviol. The calculated Ki value for steviol was 2.0 ± 0.3 µM. In the hOAT3 experiments, the [3H]ES concentration was varied from 2 to 9 µM (Fig. 4B). The estimated Km for ES in the presence of 50 µM steviol was 2-fold higher than the control (Table 1), with the calculated Ki 5.4 ± 2.0 µM.
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Trans-Stimulation of hOAT1- and hOAT3-Mediated PAH and ES Uptake. Although the above results demonstrated that steviol effectively inhibited both hOAT1 and 3, it remained uncertain whether steviol could be transported by these basolateral OATs. Therefore, trans-stimulation of hOAT1 and hOAT3 transport by steviol was investigated. Trans-stimulation takes place when the presence of one transported substrate on the opposite, or trans, side of the membrane increases the flux-labeled substrate. Trans-inhibition can also occur when an agent presented on the opposite side of the membrane reduces flux of the labeled substrate, e.g., by binding tightly to the carrier and reducing the effective number of carriers available to mediate labeled substrate transport. As shown in Fig. 5A, the efflux of [3H]PAH significantly increased by the addition of 1 mM unlabeled PAH in the incubation medium. Similarly, 1 µM steviol significantly trans-stimulated [3H]PAH efflux, whereas at higher concentrations (10 µM–1 mM), trans-stimulation was not seen. In fact, 100 µM and 1 mM steviol significantly trans-inhibited [3H]PAH efflux. This result was comparable to that seen when 1 mM probenecid was applied. Human OAT3-mediated [3H]PAH was also significantly trans-stimulated when 1 mM unlabeled PAH was added to the external buffer. External addition of 1 µM steviol also increased the efflux of [3H]PAH. However, this effect was not observed at 10 and 100 µM steviol, and at 1 mM, steviol (like probenecid) trans-inhibited [3H]PAH (Fig. 5B).
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Electrophysiology. Since human, rat, and flounder OAT1 have all been shown to be electrogenic, exchanging one monovalent organic anion for one divalent dicarboxylate (Burckhardt et al., 2000; Aslamkhan et al., 2003), it should be possible to directly assess whether stevioside or steviol were being transported via electrophysiology. To do this, voltage-clamp experiments using fOat1-expressing oocytes were performed. As noted above, flounder OAT1 shows a greater electrical signal than rat or human OAT1. Since the signal resulting from transport of stevioside (if any) and steviol might be modest, flounder Oat1 was chosen for these experiments. As shown in Fig. 6A, at a holding membrane potential of –80 mV, addition of 100 µM PAH induced an inward current of –61.9 ± 2.4 nA. Addition of 100 µM steviol evoked membrane inward current of –11.7 ± 1.0 nA, indicating that steviol is translocated by fOat1, whereas inward current was not observed upon addition of 100 µM stevioside (Fig. 6A). As shown in Fig. 6B, the magnitude of the inward current induced by steviol was concentration-dependent. Control water-injected oocytes did not show inward current upon addition of PAH and steviol (data not shown). To determine whether steviol translocation is in fact slower than PAH, we assessed the effect of low (1 µM) and high (100 µM) steviol concentrations on the PAH-induced current. Coapplication of 1 µM steviol with 60 µM PAH (K50 for fOat1 PAH current) (Burckhardt et al., 2000) resulted in a slightly larger current than the application of 60 µM PAH alone (103.9 ± 1.2% of control PAH current; Fig. 6C). Moreover, coapplication of 100 µM steviol with 60 µM PAH resulted in a greatly reduced current (47.3 ± 5.4% of control PAH current; Fig. 6C), paralleling its effect in the cis-inhibition experiments described above (Figs. 2A and 3A). Thus, it appears that although steviol is a substrate for fOat1, its rate of translocation is markedly less than that of PAH.
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). Indeed, stevioside seems to have little or no acute toxicity (Matsui et al., 1996; Toskulkao et al., 1997). Likewise, chronic stevioside consumption is believed to pose little human risk based on findings such as those of Yamada et al. (1985) that showed oral consumption of stevioside in amounts as high as 550 mg/kg b.wt./day (i.e., more than 200 times its likely maximum intake in man of about 2 mg/kg b.wt./day) (Xili et al., 1992) for 2 years, had no toxic or carcinogenic effects in the rat. However, pharmacological effects are suggested by other studies. For example, stevioside administration was reported to reduce blood pressure and blood glucose levels in humans (Chan et al., 1998; Jeppesen et al., 2000, 2002, 2003). Furthermore, its aglycone metabolite, steviol, was reported to be mutagenic and bactericidal in Salmonella typhimurium TM677 (Perzzuto et al., 1985). Thus, the spectrum of its biological effects and potential for adverse interactions with other pharmacological reagents remains uncertain.
Stevioside absorption, distribution, and metabolism have been evaluated in experimental animals. Absorption of stevioside by everted sacs of rat intestine and Caco-2 cell monolayers was very poor, whereas its aglycone, steviol, was rapidly absorbed (Geuns et al., 2003; Koyama et al., 2003b). Since stevioside can be degraded to steviol by intestinal microflora from various animal species, including man (Cardoso et al., 1996; Hutapea et al., 1997; Geuns, 2003; Koyama et al., 2003a), it has been suggested that following stevioside consumption only steviol is absorbed. Furthermore, it appears that stevioside shows nearly complete metabolic conversion to steviol in vivo. Thus, the "acceptable daily intake" of 7.9 mg/kg b.wt./day stevioside suggested by Xili et al. (1992) would yield a maximum plasma concentration of approximately 0.2 mM steviol, assuming that oral stevioside would be fully converted to steviol and fully absorbed by the gut in vivo. A recent in vivo study in rats gave plasma values in the same range. Peak plasma steviol concentration was approximately 18 µM 2 h after single oral administration of stevia extract (equimolar dose of 45 mg/kg b.wt. steviol), and 15 min after oral administration of steviol itself (45 mg/kg b.wt.), plasma steviol reached a peak of 57.4 µM (Koyama et al., 2003a). Based on these estimates, we have used concentrations of 0.05 to1 mM for stevioside and 0.01 to 1 mM steviol to bracket the likely exposure experienced by man.
In addition, stevioside and its metabolites are very rapidly eliminated from the body. For example, following intravenous injection of [131I]stevioside into the rat, plasma radioactivity decreased rapidly to only 6% of injected dose in the first 60 min. The excreted label was equally found in urine and feces (Cardoso et al., 1996). Moreover, in the rat, stevioside renal clearance was higher than inulin clearance, suggesting active tubular secretion of stevioside (or its aglycone) by renal tubular epithelium (Melis, 1992). Similarly, the excretion of steviol-17-[14C] was largely found in urine of bile duct ligated rats after both oral and intracecal administration (Wingard et al., 1980). In keeping with the possibility of tubular transport and secretion, steviol was shown to inhibit PAH uptake in rat renal cortical slices (Toskulkao et al., 1994).
The two basolateral organic anion/dicarboxylate exchangers, OAT1 and OAT3, drive the critical energy-dependent uphill step in organic drug and xenobiotic secretion and, thus, control the renal elimination of endogenous and exogenous anionic drugs and metabolites from the body (Hosoyamada et al., 1999; Cha et al., 2001; Sweet et al., 2003). Together, the rapid renal clearance of stevioside/steviol from the body and the ability of steviol to inhibit PAH transport suggest that one or both agents may use these transporters as a route for the excretion into urine, reducing their systemic concentration and potential for toxicity. However, it should be noted that if they are substrates for these carriers, they may also demonstrate significant renal accumulation and increased potential for proximal tubular damage. They may also alter the handling of other organic anion substrates by these systems, changing their retention and potential for adverse effects. Hence, it is essential to gain a molecular understanding of the mechanisms by which both stevioside and steviol are transported by the kidney. The recent studies of Jutabha et al. (2000) suggest that inhibition of organic anion transport also occurs at the tubular level. They found that 0.7 mM stevioside modestly reduced PAH transport, whereas only 10 µM steviol was needed to significantly inhibit transepithelial PAH transport by the rabbit renal proximal tubule (Chatsudthipong and Jutabha, 2001). However, those studies did not assess the interactions of stevioside and steviol with specific basolateral OATs. In the studies reported above, we have demonstrated that stevioside itself did not inhibit either hOAT1 or hOAT3 (Fig. 2, A and B; Fig. 3, A and B). Likewise, our electrophysiological analysis showed that stevioside did not produce membrane currents in oocytes expressing fOat1 (Fig. 6A), suggesting that stevioside had no interaction with either hOAT1 and hOAT3 and thus, that these OATs are unlikely to mediate basolateral uptake of stevioside. However, this finding is in contrast with previous in vivo studies that found urinary stevioside clearance to be greater than that of inulin (Melis, 1992; Cardoso et al., 1996). In light of the present data using the cloned transporters, two explanations for the earlier data are possible. First, it is possible that the stevioside used in the earlier studies contained other stevioside derivatives or that some stevioside metabolism occurred in vivo after its injection. Alternatively, another transport process may be responsible for these in vivo findings. For example, very recently a novel organic anion transporter from human kidney (OATP4C1) has been cloned and characterized (Mikkaichi et al., 2004). This basolateral transporter appears to handle organic anions, as well as digoxin and ouabain, large neutral cardiac glycosides. Hence, OATP4C1 may play a possible role for transport stevioside. However, this possibility needs to be explored.
In contrast, the aglycone stevioside metabolite, steviol, gave very different results. Steviol very effectively inhibited hOAT1- and hOAT3-mediated transport in a dose-dependent manner (Fig. 2, A and B; Fig. 3, A and B). The inhibition of OAT1 produced by steviol was as great as that produced by equal concentrations of unlabeled PAH and furosemide (Fig. 2A), suggesting that the affinity of steviol approximates that of these compounds for hOAT1. Indeed, it was nearly as effective against hOAT1 as probenecid. Against hOAT3, it was a more effective inhibitor than PAH, but it was much less effective than estrone sulfate, bumetanide, or probenecid (Fig. 2B), all of which are known to have a high affinity for hOAT3 (Wright and Dantzler, 2004). However, these cis-inhibition experiments only indicate that steviol has a high affinity for basolateral OAT binding and leave open the question of whether or not steviol is itself translocated by the cloned OATs. To address this possibility, we took advantage of the observation that, like hOAT1 and rOat1, fOat1 shows a 1:1 coupling of organic anion and dicarboxylate and shows an inward positive current upon exposure to transported substrates (Burckhardt et al., 2000; Aslamkhan et al., 2003, 2004). Moreover, that current is more robust than that seen with the mammalian isoforms, in effect amplifying the signal associated with substrate transport. Using this system, we were able to demonstrate current movements upon exposure of fOat1-expressing oocytes to steviol. As shown in Fig. 6, A and B, both 1 and 100 µM steviol induced an inward current in fOat1-expressing oocytes. Additionally, consistent with its transport by hOAT1 and hOAT3, we observed that 1 µM steviol trans-stimulated the efflux of [3H]PAH mediated by both hOAT1 and hOAT3 (Fig. 5, A and B). However, at higher concentrations steviol trans-inhibited [3H]PAH efflux, similar to the effect of probenecid which binds very tightly to the carrier. The reason for differences in the effects of low (1 µM) and high (100 µM) steviol concentrations on PAH efflux is not clear, but may indicate that steviol translocation has a low turnover rate. Thus, at high concentrations it may mask the binding site and, like probenecid, produce trans-inhibition. Indeed, that rate of steviol translocation (i.e., current induction) is clearly lower than that of PAH (Fig. 6, A–C). This finding is similar to that of Groves et al. (1994), who showed that the tightly bound substrate, tetrapentylammonium, completely blocked TEA efflux from isolated rabbit proximal tubules. These investigators proposed that tetrapentylammonium binds to the organic cation transporter and markedly slows carrier turnover rate (Groves et al., 1994; Bednarczyk et al., 2003).
In conclusion, natural sweetener stevioside is not transported via basolateral organic anion transporters. In contrast, both hOAT1 and hOAT3 transport steviol, the aglycone metabolite stevioside, indicating that transport of steviol by these carriers may be responsible for the renal secretion of stevioside-derived radioactivity seen previously in vivo. Furthermore, steviol has a high affinity for both hOAT1 and hOAT3 and, therefore, has significant potential to diminish the renal clearance of anionic drugs and their metabolites.
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Footnotes |
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ABBREVIATIONS: PAH, para-aminohippurate; hOAT, human organic anion transporter; ES, estrone sulfate; fOat1, renal organic anion transporter from winter flounder; OR-2, oocyte ringer 2; DMSO, dimethyl sulfoxide.
Address correspondence to: Dr. John B. Pritchard, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, 111 TW Alexander Dr., Research Triangle Park, NC 27709. E-mail: pritcha3{at}niehs.nih.gov
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) or presence of 50 µM steviol (
) for 30 min. Each point represents mean ± S.E. of three separate experiments.
