Sequential Metabolism of Sesamin by Cytochrome P450 and UDP- Glucuronosyltransferase in Human Liver

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1 /11/ $25.00 DRUG METABOLISM AND DISPOSITION Vol. 39, No. 9 Copyright 2011 by The American Society for Pharmacology and Experimental Therapeutics 39875/ DMD 39: , 2011 Printed in U.S.A. Sequential Metabolism of Sesamin by Cytochrome P450 and UDP- Glucuronosyltransferase in Human Liver Kaori Yasuda, Shinichi Ikushiro, Masaki Kamakura, Eiji Munetsuna, Miho Ohta, and Toshiyuki Sakaki Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan (K.Y., S.I., M.K., E.M., T.S.); and Development Nourishment Department, Soai University, Osaka, Japan (M.O.) Received March 30, 2011; accepted May 27, 2011 ABSTRACT: Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified Introduction Sesamin is a major lignan in sesame, and its biological effects such as cholesterol and lipid-lowering (Hirose et al., 1991; Ogawa et al., 1995; Hirata et al., 1996; Kiso, 2004), anticarcinogenic effects (Hirose et al., 1992; Miyahara et al., 2000), and suppression of hypertension (Miyawaki et al., 2009) have been extensively studied by many researchers. Among them, antioxidant effects (Ikeda et al., 2003; Nakai et al., 2003) are attributed to its metabolites because sesamin itself has few antioxidative properties. Nakai et al. (2003) demonstrated that sesamin was converted to its mono- and dicatechol forms by demethylation of methlenedioxyphenyl (MDP) groups by cytochromes P450 (P450s) in rat livers (Kumagai et al., 1991; Murray, 2000). The resultant catechols had much higher antioxidative activity compared with sesamin, and sesamin dicatechol showed the highest antioxidative activity (Nakai et al., 2003; Miyake et al., 2005). In addition, studies suggested that sesamin This work was supported in part by Ministry of Education, Culture, Sports, Science, and Technology grant. Article, publication date, and citation information can be found at doi: /dmd UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver. monocatechol had the ability to induce enhancement of endotheliumdependent vasorelaxation (Nakano et al., 2006) and neuronal differentiation (Hamada et al., 2009). Thus, the conversion of sesamin to its mono- or dicatechol form is considered to be an important reaction in the production of antioxidants and other bioactive compounds in the human body. Liu et al. (2006) tentatively proposed metabolic pathways of sesamin containing these catechols in vivo (Peñalvo et al., 2005; Liu et al., 2006). The plasma concentrations of sesamin monocatechol and dicatechol in sesamin-administered rats were much higher than that of sesamin itself (Nakano et al., 2006), suggesting biological effects of these catechols in rats. Very little is known about the plasma concentrations of sesamin mono- and dicatechols in humans. Moazzami et al. (2007) reported the presence of glucuronide of sesamin monocatechol in human urine, but other metabolites such as dicatechol were not detected. Recently, we examined the sesamin catecholization by P450s in human liver microsomes (Yasuda et al., 2010). Sesamin monocatechol was detected as a metabolite of sesamin, but the dicatechol form was not detected. Our results are consistent with those of Moazzami et al. (2007) and suggest a species-based difference in sesamin metabolism by P450 between humans and rats. To understand the biological significance of the sesamin metabolites, especially their antioxidative ABBREVIATIONS: MDP, methylenedioxyphenyl; P450, cytochrome P450; UGT, UDP-glucuronosyltransferase; Glc-UA, glucuronic acid; COMT, catechol O-methyl transferase; MBI, mechanism-based inhibition; HPLC, high-performance liquid chromatography; SAM, S-adenosyl methionine; AZT, 3 -azido-3 -deoxythymidine; TFA, trifluoroacetic acid; CL int, intrinsic clearance. 1538

2 METABOLISM OF SESAMIN IN HUMAN LIVER 1539 properties, it is essential to understand what controls the ratio of conversion of sesamin to its mono- or dicatechol or glucuronide forms. In addition, it is important to identify the P450 and UGT isoforms that are responsible for sesamin metabolism from the viewpoint of drug-sesamin interaction. Our previous study demonstrated that the most essential P450 isoform for sesamin metabolism is CYP2C9 and secondly CYP1A2 in human liver. We also found a weak mechanism-based inhibition (MBI) of CYP2C9 by sesamin (Yasuda et al., 2010) and other MDP compounds (Nakajima et al., 1999; Murray, 2000; Chatterjee and Franklin, 2003; Usia et al., 2005). Sesamin monocatechol still has another MDP group; thus, it is possible that monocatechol is also a mechanism-based inhibitor of CYP2C9 or other P450s. In this study, we focused on the metabolism of sesamin that occurs after its monocatecholization to reveal overall metabolic pathways of sesamin in humans. We identified the UGT isoform responsible for sesamin glucuronidation. We also observed methylation of sesamin monocatechol by catechol O-methyl transferase (COMT). Finally, we describe a species-based difference between humans and rats in sesamin metabolism. Materials and Methods Materials. Sesamin was purchased from Sigma-Aldrich (St. Louis, MO). NADPH and NADH were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). UDP-GlcUA, Escherichia coli -glucuronidase, and S-adenosyl methionine were purchased from Sigma-Aldrich. Human single-donor liver microsomes (HG43, HH47, HH18, HH74, HH77, HG95, HH715, HH581, HG3, and HH741), a 50-donor human liver microsome pool, human liver cytosol pool, human small intestinal microsome pool, male Sprague-Dawley rat liver microsomes and cytosol, and recombinant human UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) expressed in baculovirus-infected insect cells were purchased from BD Gentest (Woburn, MA). The recombinant Saccharomyces cerevisiae AH22 cells expressing human CYP2C19 with the vector pgyr were kindly provided by Sumitomo Chemical Co., Ltd. (Osaka, Japan) (Yasuda et al., 2010). Sesamin dicatechol obtained by using the rat liver S9 fraction was used as an authentic standard. All other chemicals were purchased from standard commercial sources of the highest quality available. Preparation of by Recombinant Yeast Cells Expressing Human CYP2C19. Our previous study revealed that sesamin was efficiently converted to its monocatechol form by whole-cell fraction of recombinant S. cerevisiae cells expressing human CYP2C19 (Yasuda et al., 2010). Thus, we used these cells for preparing sesamin monocatechol. The recombinant yeast cells were cultivated in 50 ml of synthetic minimal medium containing 2% glucose, 0.67% yeast nitrogen base without amino acids, and 20 mg/l L-histidine at 30 C for 24 h. The culture was harvested by centrifugation at 5000g at 4 C for 10 min. The cell pellet was suspended in 5 ml of 100 mm potassium phosphate buffer (ph 7.4) containing 4% glucose and 100 M sesamin and incubated at 37 C under shaking conditions. Glucose was added again to 4% of the final concentration at 24 h after addition of sesamin. After 48 h of incubation, the metabolites were extracted with four volumes of chloroform/methanol (3:1, v/v). The organic phase was recovered and dried in a vacuum evaporator centrifuge (Sakuma Seisakusyo, Tokyo, Japan). The resultant residue was solubilized with methanol and applied to HPLC under the conditions described under HPLC Analysis of Metabolites of Sesamin, Sesamin Monocatechol, and AZT. The peak at a retention time of 25.3 min, which was identified as sesamin monocatechol in our previous study (Yasuda et al., 2010), was recovered and dried and then dissolved in dimethyl sulfoxide to be used as a substrate as follows. Metabolism of by P450 or UGT, or COMT in the Liver, Small Intestinal Microsomes, or Liver Cytosol. In P450-dependent catecholization of sesamin monocatechol, the reaction mixture containing 0.5 mg protein/ml of the liver or small intestinal microsomes, 1 to 50 M sesamin monocatechol, and 1 mm NADPH in 100 mm potassium phosphate buffer (ph 7.4) was incubated for 10 to 30 min at 37 C, and the metabolite was analyzed as described below. In UGT-dependent glucuronidation of sesamin monocatechol, the reaction mixture containing 0.5 mg protein/ml of the liver or small intestinal microsomes, 1 to 50 M sesamin monocatechol, 2 mm UDP-GlcUA, and 1 mm MgCl 2 in 100 mm potassium phosphate buffer (ph 7.4) was incubated for 15 to 30 min at 37 C, and the metabolite was analyzed by HPLC as described under HPLC Analysis of Metabolites of Sesamin,, and AZT. To confirm that the metabolite was the glucuronide, the aliquot of the reaction mixture was further incubated for 60 min at 37 C in the presence of 0.1 mg/ml -glucuronidase in 20 mm potassium phosphate buffer (ph 7.4). In COMT-dependent methylation, the reaction mixture containing 0.5 mg protein/ml of the liver cytosol, 1 to 50 M sesamin monocatechol, 200 M SAM, 2 mm MgCl 2, and 1 mm dithiothreitol in 100 mm potassium phosphate buffer (ph 7.4) was incubated for 5 to 20 min at 37 C, and then the metabolites were analyzed by HPLC as described under HPLC Analysis of Metabolites of Sesamin,, and AZT. Metabolism of Sesamin by P450 and UGT in Human Liver Microsomes or Small Intestinal Microsomes in the Presence of NADPH and UDP- GlcUA. The reaction mixture containing 0.5 mg protein/ml human liver microsomes or small intestinal microsomes, 1 to 50 M sesamin, 1 mm NADPH, 1 mm UDP-GlcUA, and 1 mm MgCl 2 in 100 mm potassium phosphate buffer (ph 7.4) was incubated for 0 to 60 min at 37 C, and then the metabolites were analyzed as described under HPLC Analysis of Metabolites of Sesamin,, and AZT. Metabolism of by Recombinant Human UGTs. The reaction mixture containing 0.1 mg protein/ml recombinant human UGT (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, or 2B17) microsomes, 10 M sesamin monocatechol, 2 mm UDP-GlcUA, and 1 mm MgCl 2 in 100 mm potassium phosphate buffer (ph 7.4) was incubated at 37 C for 30 min, and then the metabolites were analyzed by HPLC as described under HPLC Analysis of Metabolites of Sesamin,, and AZT. Metabolism of 3 -Azido-3 -Deoxythymidine in Human Liver Microsomes. The reaction mixture containing 0.5 mg protein/ml human liver microsomes, 1 mm 3 -azido-3 -deoxythymidine (AZT), 2 mm UDP-GlcUA, and 1 mm MgCl 2 in 100 mm potassium phosphate buffer (ph 7.4) was incubated for 15 min at 37 C, and then the metabolites were analyzed as described under HPLC Analysis of Metabolites of Sesamin,, and AZT. Inhibition of AZT Glucuronidation in Human Liver Microsomes by. AZT glucuronidation reactions in a 50-donor human liver microsome pool were performed by the same methods described under Metabolism of 3 -Azido-3 -Deoxythymidine in Human Liver Microsomes in the presence of 0 to 100 M sesamin monocatechol. Metabolites were analyzed by HPLC as described under HPLC Analysis of Metabolites of Sesamin, Sesamin Monocatechol, and AZT. HPLC Analysis of Metabolites of Sesamin,, and AZT. Each reaction was terminated by addition of an equal volume of ice-cold methanol. After centrifugation at 14,500g for 15 min, the supernatant was applied to HPLC under the following conditions: column, YMC-pack ODS-AM ( mm) (YMC, Inc., Wilmington, NC); UV detection, 280 nm; flow rate, 1.0 ml/min; column temperature, 40 C; and linear gradients of 10 to 90% methanol aqueous solution containing 0.05% trifluoroacetic acid (TFA) for 30 min and 90 to 100% methanol containing 0.05% TFA for 5 min for metabolites of sesamin and sesamin monocatechol. On the other hand, AZT metabolites were analyzed under the following conditions: column, YMC-pack ODS-AM ( mm) (YMC, Inc., Wilmington, NC); UV detection, 267 nm; flow rate, 1.0 ml/min; column temperature, 40 C; and linear gradients of 10 to 65% methanol aqueous solution containing 0.05% TFA for 20 min. Liquid Chromatography-Mass Spectrometric Analysis of Methylated Metabolite of. The metabolites of sesamin monocatechol in the presence of SAM were isolated by HPLC and subjected to mass spectrometric analysis using a Finnigan LCQ Advantage Mix (Thermo Fisher Scientific, Waltham, MA) with atmospheric pressure chemical ionization, positive mode. The conditions of liquid chromatography were as follows: column, reverse-phase ODS column (2 150 mm, Develosil ODS-HG-3; Nomura Chemical Co., Ltd., Aichi, Japan); mobile phase, acetonitrile/methanol/water 3:4:3; flow rate, 0.2 ml/min; and UV detection, 280 nm. Kinetic Analysis. The kinetic studies for P450-dependent oxidation, UGTdependent glucuronidation, and COMT-dependent methylation were per-

3 1540 YASUDA ET AL. A B C A 280 A 280 A 280 M (min) A=0.005 A=0.002 M3 M2 M4 M5 A=0.002 M2 human (min) FIG. 1. HPLC profiles of sesamin monocatechol and its oxidated (A) or glucuronidated (B) or methylated (C) metabolites. Human liver microsomes (Aa) or rat liver microsomes (Ab) were incubated with NADPH for 30 min. Human liver microsomes (Ba, Bb) or rat liver microsomes (Bc, Bd) were incubated with UDP-GlcUA for 30 min (Ba, Bc), and they were further incubated with -glucuronidase for 60 min (Bb, Bd). Human liver microsomes (Ca) or rat liver microsomes (Cb) were incubated with S-adenosyl methionine for 20 min (human liver microsomes) or for 5 min (rat liver microsomes). The concentration of each reaction was 10 M. formed using each of the microsomal and cytosolic fractions. In determining the kinetic parameters, sesamin monocatechol or sesamin concentration varied from0to50 M. The kinetic parameters, K m and V max, were calculated by nonlinear regression analysis using KaleidaGraph (Synergy Software, Reading, PA). The equation was applied for Michaelis-Menten kinetics. Results HPLC Analysis of the Metabolites of. Figure 1A shows the HPLC profiles of sesamin monocatechol and its metabolite in human and rat liver microsomes in the presence of NADPH. One major metabolite (M1) was observed at a retention time of 17.5 min in human and rat. Cochromatography of M1 with the authentic sesamin dicatechol strongly suggested that M1 was sesamin dicatechol. It should be noted that the conversion ratio in rat liver microsomes was much higher than that in human liver microsomes. Figure 1B shows the HPLC profiles of sesamin monocatechol and its metabolite in human and rat liver microsomes in the presence of UDP-GlcUA. Only one major metabolite (M2) was observed at a retention time of 23.4 min in human microsomes (Fig. 1Ba). In rats, another metabolite in addition to M2 was detected at a retention time of 22.4 min (M3) (Fig. 1Bc). M2 and M3 were converted to sesamin monocatechol upon incubation with -glucuronidase (Fig. 1B, b and rat human human + -gluc rat rat + -gluc (min) human rat a b c d a b a b d), suggesting that M2 and M3 were glucuronide isomers of sesamin monocatechol. Figure 1C shows the HPLC profiles of sesamin monocatechol and its metabolites in human and rat liver cytosol in the presence of SAM. Two major metabolites were detected at retention times of 26.9 min (M4) and 27.4 min (M5) in human and rat liver cytosol. To identify the chemical structures of the metabolites, we collected M4 and M5 in the HPLC effluents and subjected them to mass spectrometric analysis. Relative intensities (percentage) of major ion fragments of the authentic standard of sesamin monocatechol and its metabolites (M4 and M5) were as follows: sesamin monocatechol, m/z 307 (M H- 2H 2 O), 4%; m/z 325 (M H-H 2 O), 100%; m/z 343 (M H) 43%. M4: m/z 321 (M H-2H 2 O), 8%; m/z 339 (M H-H 2 O), 100%; m/z 357 (M H) 50%. M5: m/z 321 (M H-2H 2 O), 8%; m/z 339 (M H-H 2 O), 100%; m/z 357 (M H) 46%. These results strongly suggest that M4 and M5 are methylated isomers of sesamin monocatechol. We conclude that sesamin monocatechol can be converted to sesamin dicatechol by P450s, glucuronide forms by UGTs, and methylated forms by COMT (Fig. 2). Among these enzymes, P450s and UGTs are localized in microsomal membranes, whereas COMT is localized in the cytosol. Kinetic Analysis of Oxidation, Glucuronidation, and Methylation. Table 1 shows the kinetic parameters for sesamin monocatechol glucuronidation and oxidation in liver microsomes and methylation in liver cytosol. In human samples, the V max value for UGT-dependent glucuronidation of sesamin monocatechol (4.35 nmol/mg protein/min) was 18 times higher than that of P450-dependent oxidation of sesamin monocatechol (0.24 nmol/mg protein/min). UGT and P450 activities had almost the same K m values. Thus, the V max /K m value for glucuronidation was 18 times higher than that for oxidation. It is noted that P450 and UGT reactions depend on NADPH and UDP-glucuronic acid, respectively. Thus, their activities cannot be compared directly. However, if enough amounts of NADPH and UDP-glucuronic acid are present under physiological conditions, both activities could be compared. Our results indicate that sesamin monocatechol is predominantly converted to the glucuronide form rather than the dicatechol form in human liver microsomes. It is noted that the V max /K m value for the oxidation of sesamin monocatechol is 9 times lower than that of sesamin (Table 1), which indicates that sesamin monocatechol is a poor substrate for human liver P450s compared with sesamin itself. The kinetic parameters of COMT for the methylation of sesamin monocatechol in human liver cytosol were determined. Because P450 and UGT reactions occur in microsomes, and the COMT reaction occurs in the cytosol, the kinetic parameters of COMT cannot be compared with those of the P450 and UGT reactions. However, it is clear that methylation by COMT is also an important reaction for the metabolism of sesamin monocatechol in human liver. We also examined the same activities in rat liver microsomal and cytosolic fractions to reveal species-based differences between rats and humans in the metabolism of sesamin and sesamin monocatechol (Table 1). To our surprise, a drastic difference was observed between human and rat liver microsomes. In rat liver microsomes, P450- dependent catecholization activity toward sesamin and sesamin monocatechol is predominant over glucuronidation (Table 1). In contrast, the V max /K m value of COMT-dependent methylation in the cytosolic fraction of rat liver was not so different from that of human liver. The observed differences in the metabolism of sesamin suggest that the potential health benefits of sesamin also differ between rats and humans. Metabolism of Sesamin in the Presence of NADPH and UDP- GlcUA by Human Liver Microsomes or Human Small Intestinal

4 METABOLISM OF SESAMIN IN HUMAN LIVER 1541 Sesamin CYP2C9 (Yasuda et al. (2010)) CYP2C9 (This study) or UGT Glucuronide (M2, M3) COMT Sesamin Dicatechol (M1) or Methylate (M4, M5) FIG. 2. Putative metabolic pathways of sesamin in human liver. Microsomes. Orally administered sesamin is first metabolized in small intestinal epithelial cells, so we examined the metabolism of sesamin in small intestinal microsomes. P450s and UGTs are expressed in human small intestine, thus we are able to compare the metabolism of sesamin in human small intestinal microsomes with the metabolism in liver microsomes. Liver and small intestinal microsomes were incubated with NADPH and UDP-GlcUA, and the metabolites produced were assayed. As shown in Fig. 3Aa, most of the substrate remained intact and the glucuronide form was observed as a single metabolite in small intestinal microsomes. On the contrary, most of the substrate was converted to the metabolites consisting of sesamin monocatechol (7.2%) and its glucuronide (82%) in human TABLE 1 Kinetic analysis of oxidation and glucuronidation of sesamin or sesamin monocatechol in human and rat liver microsomes and methylation in human and rat liver cytosol Each value represents the mean S.D. from three separate experiments. Species Substrate Reaction Fraction V max K m V max /K m Relative V max /K m nmol/mg protein/min M Human Sesamin Cat (P450) mic Sesamin monocatechol Cat (P450) mic Glu (UGT ) mic Met (COMT) cyt Rat Sesamin Cat (P450) mic Sesamin monocatechol Cat (P450) mic Glu (UGT ) mic Met (COMT) cyt Cat, catecholization; Glu, glucuronidation; Met, methylation; mic, microsomal fraction; cyt, cytosolic fraction.

5 1542 YASUDA ET AL. A A 280 B Glucuronide A= (min) Intestine liver Sesamin FIG. 3. HPLC profiles (A) of sesamin metabolites in human small intestinal microsomes (a) and human liver microsomes (b) in the presence of NADPH and UDP-GlcUA for 60 min, and the time courses (B) of the metabolites in human liver microsomes. liver microsomes (Fig. 3Ab). It is noted that the dicatechol produced by P450 was not detected in either microsome. These results suggest that the sequential conversion of sesamin to sesamin monocatechol to sesamin monocatechol glucuronide occurs in liver and small intestinal microsomes. The time course assay of sesamin metabolism in human liver microsomes confirmed the sequential metabolism of sesamin by P450s and UGTs (Fig. 3B). The V max /K m value of P450-dependent sesamin monocatecholization in liver microsomes was 2.2 times higher than that in small intestinal microsomes (Table 2). The V max /K m value of UGTdependent glucuronidation of sesamin monocatechol was similar in the liver and small intestinal microsomes (Table 2). Thus, the drastic difference in sesamin metabolism between the liver and small intestinal microsomes (Fig. 3A) is due to the difference in P450-dependent sesamin catecholization activity. To estimate the in vitro intrinsic clearance (CL int ) of sesamin monocatechol by P450 and UGT, we used the following equation (eq. 1): CL int V max milligram of microsomal protein gram of tissue K m gram of tissue killogram of body weight a b (1) We calculated the CL int values for the glucuronidation of sesamin monocatechol from human liver microsomes (45 mg microsomal protein/g liver and 20 g liver/kg b.wt.) and the human small intestinal microsomes (3 mg microsomal protein/g intestine and 30 g intestine/kg b.wt.) (Soars et al., 2002). The CL int values for monocatecholization in human liver and human intestine were 68 and 3.1, respectively, and the CL int values for glucuronidation were 144 and 16, respectively (Table 2). For monocatecholization and glucuronidation, CL int values in human liver were much higher than those in human small intestine. Metabolism of by the Recombinant Human UGTs. Glucuronidation of sesamin monocatechol is essential for the metabolism of sesamin in the human body (Fig. 3; Table 2). Thus, identification of the UGT isoforms responsible for sesamin glucuronidation is required from the viewpoint of drug-sesamin interaction. We examined the metabolism of sesamin monocatechol by recombinant human UGTs expressed in baculovirus-infected insect cells. Among the 12 UGT isoforms, UGT2B7 and UGT2B17 showed glucuronidation activity toward sesamin monocatechol. The V max /K m of UGT2B7 was 3.4 times higher than that of UGT2B17, whereas both K m values were almost identical (Table 3). Because the levels of UGT2B7 and UGT2B17 in the recombinant microsomes were similar ( nmol/mg protein) as described in our previous report (Uchihashi et al., 2011), the enzymatic activity of UGT2B7 appears to be significantly higher than that of UGT2B17. In addition, UGT2B7 mrna is higher than UGT2B17 mrna in the human liver (Congiu et al., 2002; Court, 2005; Izukawa et al., 2009). Thus, the contribution of UGT2B17 to glucuronidation of sesamin monocatechol in the human liver appears to be small. Correlation between Glucuronidation Activity and UGT2B7-Specific Activity. Kinetic analysis suggests that UGT2B7 plays a central role in the glucuronidation of sesamin monocatechol in the human body. Thus, we examined the correlation between sesamin monocatechol glucuronidation activity and AZT glucuronidation activity, which is known as UGT2B7-specific activity, using single-donor human liver microsomes. There was a 2.1-fold activity range of sesamin monocatechol glucuronidation in human liver microsomes from 10 different individuals (Fig. 4A). Thus, the interindividual difference of sesamin monocatechol glucuronidation was not so large. A good correlation (r 0.80) was observed between sesamin monocatechol glucuronidation activity and AZT glucuronidation activity (Fig. 4B), suggesting that UGT2B7 is the major isoform involved in the glucuronidation of sesamin monocatechol in human liver. Inhibition of UGT2B7-Specific Activity in Human Liver Microsomes by. Figure 5 shows the inhibitory effects of sesamin monocatechol on AZT glucuronidation activity in pooled human liver microsomes. The UGT2B7-specific activity was significantly inhibited by sesamin monocatechol, and the IC 50 value was estimated to be 47.5 M. TABLE 2 Kinetic analysis of catecholization and glucuronidation of sesamin monocatechol in human liver or small intestinal microsomes The V max and K m values represent the mean S.D. from three separate experiments. The CL int values were estimated according to eq. 1 described under Results. Substrate Reaction V max K m V max /K m CL int nmol/mg protein/min M ml min 1 kg 1 Sesamin Cat (P450) Liver a a a 68 Small intestine Sesamin Glu Liver a a 0.16 a 144 Monocatechol (UGT ) Small intestine Cat, catecholization; Glu, glucuronidation. a The values were identical to those in Table 1.

6 METABOLISM OF SESAMIN IN HUMAN LIVER 1543 TABLE 3 Kinetic analysis of sesamin monocatechol glucuronidation by recombinant human UGT2B7 and 2B17 Each value represents the mean S.D. from three separate experiments. UGT V max K m V max /K m nmol/mg protein/min M 2B B Discussion Sesamin, one of the major lignans in sesame, has recently gained attention because of its potential health benefits. Many of the health benefits associated with sesamin may actually be achieved by the action of sesamin metabolites. For example, the antioxidative properties of sesamin seem to be originated from its mono- or dicatechol forms. The plasma concentrations of sesamin mono- and dicatechol (13 and 3 M, respectively) were higher than the concentration of sesamin (0.2 M) in sesamin-administered rats (Nakano et al., 2006). Because the catechol metabolites are present in the plasma, they are expected to perform biological functions in rats. However, very little has been published on the serum levels of such metabolites after sesamin uptake in humans. Peñalvo et al. (2005) reported the conversion of sesamin into enterolactone after uptake of sesame seeds. Fecal fermentation of sesamin strongly suggested that enterolactone was produced by the intestinal microflora. However, Moazzami et al. (2007) detected glucurono- and/or sulfoconjugated forms of sesamin monocatechol as a sole urine metabolite after uptake of commercial FIG. 4. A, glucuronidation activity toward sesamin monocatechol in each of the 10 human single-donor liver microsomes. B, the correlation between glucuronidation activity toward sesamin monocatechol and UGT2B7-specific activity (AZT glucuronidation activity). Each point represents the mean of at least duplicate determination. FIG. 5. The inhibitory effects of sesamin monocatechol on UGT2B7-specific activity (AZT glucuronidation activity) in the human liver microsomes. Each point represents the mean of triplicate determination. sesamin capsules. Thus, the metabolism of sesamin by the intestinal microflora may not be essential for the supplemental use of pure sesamin. Our previous study demonstrated that sesamin was metabolized to its monocatechol form mainly by CYP2C9 in the human liver with a weak MBI of CYP2C9 (Yasuda et al., 2010), which is attributed to the MDP group of sesamin (Nakajima et al., 1999; Murray, 2000; Chatterjee and Franklin, 2003; Usia et al., 2005). Sesamin monocatechol still has one MDP group; thus, it is possible that sesamin monocatechol could result in MBI of CYP2C9. However, the extent of MBI of CYP2C9 by sesamin monocatechol appeared to be much less than the inhibition by sesamin. The reduced inhibition may have resulted because sesamin monocatechol is not a good substrate for CYP2C9 or the other P450 isoforms compared with sesamin itself. When sesamin is administered orally, intestinal P450s may play important roles in the initial metabolism. However, the conversion rate of sesamin to its monocatechol by intestinal P450s is very slow, as shown in Fig. 3A and Table 2. Thus, most sesamin appears to be converted to its monocatechol by P450s in the liver and then converted to its glucuronide by UGTs. In this study, we focused on the metabolism of sesamin monocatechol. As shown in Table 1, the V max /K m value of glucuronidation of sesamin monocatechol was approximately 20 times higher than the oxidation of sesamin monocatechol in human liver microsomes. This result suggests that sesamin monocatechol could be rapidly conjugated by UGTs, whereas oxidation by P450s to yield dicatechol hardly occurs in human liver. Most sesamin was actually metabolized to its glucuronide via sesamin monocatechol in the human liver microsomes. The sulfoconjugated form of sesamin monocatechol was not detected in this study (data not shown). We identified UGT isoforms that may be responsible for the glucuronidation of sesamin monocatechol. First, we examined the conversion of sesamin monocatechol in each of 12 recombinant human UGT isoforms expressed in baculovirus-infected insect cells. Although UGT2B7 and 2B17 showed the activity, UGT2B7 appears to be responsible for glucuronidation of sesamin monocatechol on the basis of their molecular activity and expression levels in the human liver. We conclude that UGT2B7 is the most important UGT isoform on the basis of the following three lines of evidence: 1) our kinetic analysis; 2) the correlation between the glucuronidation activity toward sesamin monocatechol and the UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes (Fig. 4); and 3) the inhibitory effect of sesamin monocatechol on the UGT2B7-specific activity (Fig. 5). However, we could not evaluate the contribution of UGT1A5, 2B10, 2B11, 2B28, 2A1, 2A2, and 2A3 because these isoforms were not commercially available. Thus, the possibility that these UGT isoforms contribute to glucuronidation of sesamin monocatechol cannot be excluded. The correlation coefficient (0.8) between the glucuronidation activity toward sesamin monocatechol and the

7 1544 YASUDA ET AL. CYP2C9 Sesamin CYP2C9 Sesamin Dicatechol P450 Sesamin P450 Sesamin Dicatechol COMT COMT Human Methylate UGT2B7 Glucuronide UGT Rat Methylate Glucuronide FIG. 6. Comparison of metabolism of sesamin between human liver and rat liver. Thickness of arrows roughly represents the strength of the activity on the basis of the relative V max /K m values shown in Table 1. UGT2B7-specific activity (Fig. 4) as well as the incomplete inhibition of UGT2B7-specific activity by sesamin monocatechol (Fig. 5) might indicate some contribution of other UGT isoforms. It is noted that the interindividual difference among 10 human liver microsomes is approximately 2-fold. These results together with our previous results on the metabolism of sesamin by human hepatic P450 suggest a small interindividual difference in sesamin metabolism. Moazzami et al. (2007) demonstrated that only a conjugated form of sesamin monocatechol was detected as a human urinary sesamin metabolite. However, the glucuronides of sesamin mono- and dicatechols were detected in rat urinary metabolites (Liu et al., 2006). These findings strongly suggest the species-based difference in the metabolism of sesamin between humans and rats. As shown in Table 1, the V max /K m value of glucuronidation of sesamin monocatechol is significantly higher in human liver microsomes than in rat liver microsomes. In contrast, the V max /K m value of the catecholization of sesamin monocatechol is much lower in human liver microsomes than in rat liver microsomes. The ratio of glucuronidation to catecholization of sesamin monocatechol is dramatically different between humans and rats. These findings are consistent with the in vivo data by Moazzami et al. (2007). A species-based difference was also observed in the number of metabolites between humans and rats. Two glucuronides designated as M2 and M3, which appear to be isomers, were detected in rat liver microsomes, whereas only one glucuronide was detected in human liver microsomes (Fig. 1). Two metabolites (M4 and M5) were also detected in the metabolism of sesamin monocatechol by COMT in human and rat liver cytosols. The metabolites M4 and M5 (Figs. 1 and 2) appear to be methylated compounds at the meta- or para-position on the basis of the HPLC data of metabolites of sesaminol (Mochizuki et al., 2009), although further studies would be needed to confirm this hypothesis. Figure 6 summarizes the putative pathways of sesamin metabolism in human liver on the basis of our previous report and this study. Three metabolizing enzymes CYP2C9, UGT2B7, and COMT play essential roles in the metabolism of sesamin in human liver. Sesamin monocatechol glucuronide appears to be a major metabolite of sesamin in humans. Because this metabolite has low levels of antioxidant activity, it is possible that sesamin monocatechol glucuronide is an inactive form of sesamin and further processing is necessary to produce a functional molecule. Kawai et al. (2008) recently proposed a novel mechanism for the function of quercetin in humans. When quercetin is taken up into the human body, quercetin is mainly converted to its glucuronide form at the 3 position (Q3GA) by UGTs in the liver. Q3GA in the plasma can be taken up by macrophage cells, probably by a transporter in the plasma membrane, and converted into its active aglycone form by -glucuronidase. This finding could explain why quercetin is effective in atherosclerosis despite the absence of quercetin aglycone in the serum. -Glucuronidase is expressed in most mammalian tissues, thus a similar mechanism might apply to sesamin. It is interesting to note that Kawai et al. (2008) also suggested that not only the aglycone form but also the methylated aglycone form of quercetin by COMT showed the inhibitory effects on the transcription of class A scavenger receptor gene in macrophage cells. In this study, we detected COMT-dependent methylation activity of sesamin monocatechol in the liver cytosol. Thus, we may hypothesize that most sesamin is converted to its glucuronide form in the liver and transported to the target tissues through the blood stream. Sesamin glucuronides can then enter the target cells and be subsequently converted to the aglycone form by -glucuronidase. Finally, a part of the aglycone is methylated by COMT to produce the active molecule. If this hypothesis is correct, then the metabolizing enzymes of sesamin consisting of P450s, UGTs, -glucuronidase, and COMT play essential roles in the biological effects of sesamin. Authorship Contributions Participated in research design: Yasuda, Ikushiro, and Sakaki. 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