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Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, Chicago, Illinois (J.G., A.L., H.C., Y.L., R.B.v.B.); and School of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii (J.M.P.)
(Received April 10, 2008; Accepted July 23, 2008)
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Abstract |
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,β-unsaturated ketone, are under investigation as cancer chemoprevention agents. Isoliquiritigenin (2,4,4'-trihydroxychalcone) (see structure in Fig. 1) is a flavonoid and chalcone found in licorice (Glycyrrhiza uralensis), shallot (Allium ascalonicum), Sinofranchetia chinensis, soybeans (Glycine max L.), and Dalbergia odorifera (Kape et al., 1992
; Kong et al., 2000; Pan et al., 2000; Ramadan et al., 2000; Cao et al., 2004). Antioxidant (Vaya et al., 1997), anti-inflammatory (Chan et al., 1998), and estrogenic properties (Tamir et al., 2001; Maggiolini et al., 2002) have been reported for isoliquiritigenin. Anticancer activities of isoliquiritigenin include the induction of cell cycle arrest and up-regulation of p21 expression in lung cancer cells (Ii et al., 2004), suppression of pulmonary metastasis of mouse renal cell carcinoma (Yamazaki et al., 2002), induction of apoptosis in human gastric cancer cells (Ma et al., 2001), and inhibition of colon and mammary carcinogenesis in rodent models (Wattenberg et al., 1994; Baba et al., 2002). Another mechanism of chemoprevention by isoliquiritigenin is induction of phase 2 enzymes that protect cells against reactive, toxic, and potentially carcinogenic species (Ross et al., 2000; Cuendet et al., 2006).
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). Among these, the monooxygenated metabolites sulfuretin and butein have been reported previously to have antiproliferation and antimutagenic effects (Iwashita et al., 2000; Park et al., 2004). Although phase 1 metabolites often retain biological or pharmacological activity, phase 2 conjugates are usually less active or inactive and are excreted rapidly in urine or bile. Some of the phase 2 enzymes responsible for forming conjugates of xenobiotic compounds include UDP glucuronosyltransferase (UGT), sulfotransferase, N-acetyl transferase, and glutathione S-transferase. In particular, UGTs are quantitatively the most important phase 2 enzymes involved in the inactivation and elimination of anticancer drugs and most polyphenolic natural products (Yu et al., 2002; Wynalda et al., 2003; Hariparsad et al., 2006). The aims of this study were to identify the major phase 2 metabolites of isoliquiritigenin formed in vitro by human hepatocytes and hepatic microsomal enzymes, and to identify the enzymes responsible for the formation of the major conjugates. In addition, the in vitro hepatic clearance of isoliquiritigenin was estimated based on these in vitro data as an indication of the significance of phase 2 metabolism to the elimination of isoliquiritigenin.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Pooled HLMs, human kidney microsomes, human intestine microsomes, and cryopreserved human hepatocytes were purchased from In Vitro Technologies (Baltimore, MD). The cytochrome P450 (P450) content of the microsomes was 0.17 nmol of P450/mg of protein. cDNA-expressed human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 (5 mg of protein/ml), as well as a human UGT control preparation, were purchased from BD Gentest (Woburn, MA). Microcon centrifugal filters containing regenerated cellulose membranes with a 30,000 molecular weight cutoff were purchased from Millipore (Bedford, MA). The glucuronidation activity of each UGT enzyme was confirmed by assay using trifluoperazine (UGT1A4), eugenol (UGT2B17), and 7-hydroxy-4-trifluoromethylcoumarine (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, and UGT2B15) as substrates according to the protocol provided by BD Gentest.
Phase 2 Metabolism by Human Hepatocytes. Cryopreserved human hepatocytes were thawed according to the supplier's instructions, and approximately 1 x 106 cells in a 1-ml suspension were incubated with isoliquiritigenin (10 µM) per well of a six-well plate. Control experiments were identical except for the substitution of heat-inactivated hepatocytes. The plate was placed in an incubator at 37°C with 5% CO2 and 90% relative humidity and gently shaken at 50 rpm for 4 h. Incubations were terminated by addition of 3 ml of ice-cold methanol/acetonitrile (1:1, v/v). The cell suspensions were centrifuged, and aliquots of the supernatants were analyzed directly using liquid chromatography/tandem mass spectrometry (LC/MS/MS).
Deconjugation by Glucuronidase and Sulfatase. A 200-µl aliquot of the hepatocyte incubation was evaporated to dryness and reconstituted in 200 µlof ammonium acetate buffer (10 mM, pH 5.0) containing sulfatase (20 units) or a combination of β-glucuronidase (400 units) and sulfatase (40 units). The deconjugation reactions were carried out at 37°C for 4 h and terminated by the addition of methanol/acetonitrile as described above. After centrifugation, supernatants of each sample were analyzed using LC/MS/MS. Samples that were treated identically except for the addition of hydrolytic enzymes were used as controls.
Glucuronidation of Isoliquiritigenin by HLMs, Human Intestine Microsomes, and Human Kidney Microsomes. Glucuronidation was carried out as described previously (Fisher et al., 2000) with some modification. Briefly, 0.5 mg of HLMs, human intestine microsomes, or human kidney microsomes, 25 µg/ml alamethicin, and 146 µl of 0.1 mM phosphate buffer, pH 7.4, were mixed and placed on ice for 15 min. Next, MgCl2 (8 mM), 5 mM saccharic acid, and 10 µM isoliquiritigenin were added to the mixture and preincubated at 37°C for 3 min. Reactions were initiated by adding UDP-glucuronic acid (UDPGA) (5 mM final concentration) in a total volume of 200 µl. After 20 min, reactions were terminated by the addition of 0.8 ml of ice-cold methanol/acetonitrile (1:1, v/v). After centrifugation to remove the precipitated proteins, the supernatant was evaporated to dryness in vacuo. Each residue was reconstituted in 150 µl of HPLC mobile phase immediately before analysis using LC/MS/MS and HPLC/UV. All the incubations were carried out at least three times.
To determine the amount of isoliquiritigenin that noncovalently bound to the microsomes, isoliquiritigenin (1 µM) was incubated in 200 µl of phosphate buffer at 37°C for 30 min with HLMs at protein concentrations used for the respective metabolic incubations. The sample was then filtered at 12,000g using a Microcon centrifugal filter device (Millipore) for 30 min to remove the solvent. The filter membrane was washed three times with water to eliminate unbound isoliquiritigenin. Isoliquiritigenin bound to the microsomes was released by washing the filter with 90% methanol. After evaporation of the methanol from the filtrate, the residue was reconstituted in 200 µl of 50% methanol and analyzed for isoliquiritigenin using LC/MS/MS. Control incubations were identical except for the omission of HLMs. Additional control incubations to assess nonspecific binding were carried out using denatured HLMs that were prepared by heating in boiling water.
Glucuronidation by UGTs. The UGTs responsible for the glucuronidation of isoliquiritigenin were identified using cDNA-expressed human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 (5 mg of protein/ml). All the incubations were carried out according to the protocol recommended by the supplier (BD Gentest). In each sample, 0.5 mg of protein/ml of UGT, 25 µg/ml alamethicin, and 146 µl of 0.1 mM phosphate buffer, pH 7.4, were mixed and placed on ice for 15 min. Next, MgCl2 (8 mM), 5 mM saccharic acid, and 10 µM isoliquiritigenin were added to the mixture and preincubated at 37°C for 3 min. Reactions were initiated by adding UDPGA (5 mM final concentration) in a total volume of 200 µl, and the incubations were carried out and terminated as described above for the microsomal incubations before analysis using LC/MS/MS. As a negative control, incubations were carried out that were identical except that the microsomes containing recombinant human UGT were replaced by microsomes prepared only from wild-type baculovirus-infected cells. All the incubations were carried out at least three times, and the mean values were compared using one-way analysis of variance with Tukey's test, p 
0.01.
Kinetics of Isoliquiritigenin Glucuronidation. Before the kinetics assays, the linearity of formation of the isoliquiritigenin glucuronides was investigated by incubating isoliquiritigenin (10 µM) with HLMs (1.0 mg/ml) up to 50 min and measuring the glucuronides using LC/UV/MS as described below. The formation of all the isoliquiritigenin monoglucuronides was linear up to 30 min (data not shown). The linearity of glucuronidation of isoliquiritigenin was also evaluated with respect to protein concentration of HLMs or recombinant UGTs. Based on these preliminary results (data not shown), kinetics studies of the formation of the two most abundant isoliquiritigenin glucuronides (MG4 and MG5) were carried out using HLMs, UGT1A1, UGT1A9, or UGT1A10 at 0.1 mg of protein/ml, 0.1 mg of protein/ml, 0.25 mg of protein/ml, or 0.25 mg/ml, respectively. The concentration of isoliquiritigenin in the incubations ranged from 0.1 to 100 and 0.5 to 100 µM for assays using HLMs and UGT isoforms, respectively. The incubation temperature was 37°C, and the incubation time was 20 min.
Kinetic parameters were estimated from the fitted curves using SigmaPlot 9.0 software (Systat Software Inc., San Jose, CA). The Michaelis-Menten equation (eq. 1), substrate inhibition (a process in which metabolic rate decreases at high substrate concentration; eq. 2), or the Hill equation (eq. 3) was used to describe the kinetics of isoliquiritigenin glucuronidation as follows:
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In these equations, V is the rate of the reaction, Vmax is the maximum velocity, Km is the Michaelis constant, [S] is the substrate concentration, Ksi is the constant describing the substrate inhibition interaction, S50 is the substrate concentration producing 50% of Vmax (analogous to the Km), and n is the Hill coefficient.
Metabolic Stability. In metabolic stability experiments, isoliquiritigenin (1 µM) was incubated with HLMs (1 mg of protein/ml) according to the method described above. The total incubation volume was 800 µl. Aliquots (100 µl each) were removed at 0, 5, 10, 20, 30, 40, or 50 min, and the reaction was terminated by addition of 400 µl of ice-cold methanol/acetonitrile (1:1, v/v). After centrifugation to remove the precipitated proteins, the supernatant was evaporated to dryness in vacuo. Each residue was reconstituted in 100 µl of HPLC mobile phase containing eriodictyol (1.0 µM) as an internal standard immediately before the measurement of residual isoliquiritigenin using LC/MS/MS.
LC/UV/MS and LC/MS/MS. Reversed-phase HPLC separations were carried out using a Zorbax (Waters, Milford, MA) SB 2.1 x 100 mm C18 column (3.5-µm particle size) connected to a Waters 2690 HPLC system. A linear gradient was used at 0.2 ml/min from 0.1% formic acid in water to methanol as follows: 20 to 70% methanol over 25 min, and then 70 to 95% methanol over the next 5 min. The column was re-equilibrated for 7 min between injections. The column temperature was 30°C, and the autosampler was maintained at 4°C. Accurate mass and product ion tandem mass spectra were obtained using a Micromass (Manchester, UK) Q-TOF-2 hybrid quadrupole/time-of-flight mass spectrometer with negative ion electrospray. The ion source parameters included a capillary voltage of 2.5 kV, a cone voltage of 30 V, a source block temperature of 120°C, and a drying gas temperature of 320°C. Tandem mass spectra were acquired at a collision energy of 25 eV using argon as the collision gas at a pressure of 2.0 x 10–5 mBar.
Quantitation of isoliquiritigenin and its monoglucuronides was carried out using LC/MS/MS with negative ion electrospray and selected reaction monitoring on an Applied Biosystems (Foster City, CA) API 4000 triple quadrupole mass spectrometer. The HPLC system consisted of Shimadzu (Columbia, MD) LC-10ADvp pumps, an LC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland), and a Zorbax SB 2.1 x 100 mm C18 column (3.5-µm particle size). Separations were carried out using a 20-min linear gradient from 20 to 70% methanol with a cosolvent of 0.1% formic acid in water followed by a 5-min gradient from 70 to 90% methanol at a flow rate of 0.2 ml/min. The column and autosampler were maintained at room temperature. Nitrogen was used for nebulization and the drying and collision gas. The collision energy was –30 eV for isoliquiritigenin and –20 eV for eriodictyol, and the dwell time was 500 ms/ion. During selected reaction monitoring, isoliquiritigenin, isoliquiritigenin monoglucuronides, and eriodictyol (internal standard) were measured using the ion transitions of m/z 255 to 119, m/z 431 to 255, and m/z 287 to 151, respectively. Alternatively, LC/UV/MS was used with selected ion monitoring of m/z 255 and 431 for isoliquiritigenin and isoliquiritigenin monoglucuronide, respectively, on an Agilent (Palo Alto, CA) G1946A quadrupole mass spectrometer and 1100 HPLC system. Ultraviolet spectra were acquired over the range 210 to 400 nm using an Agilent photodiode array detector.
Because the maximum UV absorbance of the isoliquiritigenin monoglucuronides (MG4 and MG5) is close to that of isoliquiritigenin, the quantitative analysis of these metabolites was carried out using HPLC/UV. Assuming that isoliquiritigenin and its glucuronides have similar 
values, UV spectra were recorded from 364 to 380 nm (encompassing the UV maxima), and standard curves were prepared using isoliquiritigenin at concentrations from 0.5 to 100 µM.
Calculation of Intrinsic Clearance. If isoliquiritigenin is eliminated primarily as phase 2 conjugates, then metabolic stability studies based on the rate of disappearance of isoliquiritigenin in a microsomal model that includes glucuronidation can be used to estimate the in vitro hepatic intrinsic clearance. Therefore, the estimate of intrinsic clearance of isoliquiritigenin was based on substrate disappearance during 50-min incubations with pooled HLMs. The results were converted to the percentage of substrate remaining, using t = 0 as 100%. The slope of the linear regression analysis of the plot of log percentage remaining versus incubation time relationships (–k) was used in the conversion to in vitro half-life using the equation t1/2 =–0.693/k. The intrinsic clearance (CLint', ml/min/kg) was calculated from the following equation:
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). The fraction unbound in the incubation mixture (fui) was calculated by using eq. 5 as follows:
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), were not observed after incubation with human hepatocytes.
The product ion tandem mass spectra of MG1 and MG2 were identical, and the tandem mass spectra of MG3 through MG5 were identical as shown in Fig. 3. The base peak of the tandem mass spectra of MG3, MG4, and MG5 was observed at m/z 255 and corresponded to loss of dehydrated glucuronic acid, [M-H-Gluc]–. Ions of lower abundance were detected at m/z 175 and 113, corresponding to dehydrated glucuronate and a fragment ion of the glucuronic acid moiety, respectively (Nikolic et al., 2006). In addition to the aglycon ion of m/z 255 corresponding to the mass of deprotonated isoliquiritigenin, fragment ions were observed at m/z 135 and 119, corresponding to the A-ring and B-ring of isoliquiritigenin, respectively (see structure of isoliquiritigenin and designations of the A-ring and B-ring in Fig. 1). The product ions in the tandem mass spectra of MG1 and MG2 were identical, although the relative abundances of these ions differed from those of MG3 through MG5 (Fig. 3). Because conjugation of isoliquiritigenin at any of three phenolic oxygens could produce only three monoglucuronides, two of these glucuronides detected during LC/MS analysis (Fig. 2) might be conjugates of liquiritigenin, a flavanone derivative of isoliquiritigenin, which was found previously in incubations of HLMs with isoliquiritigenin (Guo et al., 2008).
Although isoliquiritigenin conjugates in the incubation with human hepatocytes could be identified as monoglucuronides based on LC/MS/MS and hydrolysis by β-glucuronidase, the position of glucuronidation of isoliquiritigenin could not be determined based on these data. Therefore, UV spectroscopy was used to determine the site of substitution on this flavonoid according to published guidelines (Markham, 1980; Otake et al., 2002; Alonso-Salces et al., 2004). A chalcone, isoliquiritigenin contains two aromatic rings connected by an ,β-unsaturated ketone. UV spectra of chalcones exhibit two major absorption peaks in the region 240 to 400 nm: Band I (300–380 nm) and Band II (240–280 nm), which are associated with the B-ring and A-ring, respectively (Markham, 1980). This conjugated system can be energetically stabilized by delocalization of unpaired electrons from the 4-OH oxygen into the aromatic ring. In addition, the formation of an intramolecular hydrogen bond between the 2'-OH hydrogen and the oxygen of the neighboring ketone can also stabilize this conjugated system. Therefore, glucuronidation at either the 4-OH or 2'-OH position would interrupt this stability, resulting in a UV band shift relative to unconjugated isoliquiritigenin. However, this shift would be most pronounced for glucuronidation at the 4-OH position.
During LC/UV/MS and LC/MS/MS analyses, a particular metabolite (such as MG5) produced identical retention times, tandem mass spectra, and UV spectra whether it was generated using human hepatocytes or HLMs (with added UDPGA). One set of the UV-visible spectra of the five glucuronides MG1 through MG5 is shown in Fig. 4. The UV spectrum of isoliquiritigenin exhibited three specific bands: Band Ib (300 nm), Band Ia (372 nm), and Band II (240 nm), with Band Ia being the most intense (Fig. 4A). MG1 and MG2 (Fig. 4, B and C) were easily distinguished from isoliquiritigenin by their UV spectra because Band II (270 nm) was the most intense and Band I was absent. Therefore, MG1 and MG2 lacked conjugation between the A-ring and B-ring and were no longer chalcones (see structures in Fig. 1). Instead, MG1 and MG2 were monoglucuronides of liquiritigenin that were formed by conjugation at either the 4-OH or 4'-OH position. However, which metabolite, liquiritigenin 4-O-glucuronide or liquiritigenin 4'-O-glucuronide, corresponded specifically to MG1 and MG2 could not be determined from these data.
Shown in Fig. 4F, the UV spectrum of MG5 was identical to that of isoliquiritigenin, which indicated that MG5 is isoliquiritigenin 4'-O-glucuronide because conjugation at the 4'-OH position will not affect its UV absorption (Markham, 1980). The UV spectrum of MG3 (Fig. 4D) showed a Band I (352 nm) with a large hypsochromic shift (20 nm) and lower intensity than that of isoliquiritigenin associated with glucuronidation at the 4-OH position (Markham, 1980). Therefore, MG3 was identified as isoliquiritigenin 4-O-glucuronide. The UV spectrum of MG4 exhibited a smaller hypsochromic shift (10 nm) of Band I, associated with glucuronidation at the 2'-OH position (Fig. 4E). Therefore, MG4 was determined to be isoliquiritigenin 2'-O-glucuronide.
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). The results of these assays are shown in Fig. 5. UGT1A9 was the most significant enzyme contributing to the formation of MG3, isoliquiritigenin 4-O-glucoronide. Among the 12 UGT enzymes assayed, UGT1A1 and UGT1A10 were the most active in forming MG4, and UGT1A10 showed the greatest catalytic activity. UGT1A8 and UGT1A9 displayed some activity for M4 formation but only 
10% as much as UGT1A10 (Fig. 5). MG5 formation was catalyzed most efficiently by UGT1A1 and UGT1A9, with UGT1A3, UGT1A7, UGT1A8, and UGT1A10 showing weak to moderate activity. Both UGT1A9 and UGT1A10 catalyzed the formation of MG4 and MG5 from isoliquiritigenin but in different ratios corresponding to 0.06 (MG4/MG5) for UGT1A9 and 2.9 for UGT1A10. In addition, UGT1A9 but not UGT1A10 catalyzed the glucuronidation of MG3. The kinetics of formation of the two most abundant glucuronides of isoliquiritigenin, MG4 and MG5, were also investigated using the recombinant UGTs UGT1A1, UGT1A9, and UGT1A10, which were found to be the most efficient for this process. These results are shown in Fig. 6, A and B, and Table 1. The formation of MG4 and MG5 by UGT1A1 fits the substrate inhibition model with R2 values of 0.982 and 0.996, respectively. In particular, the Eadie-Hofstee plot (v/S ratio on the x-axis and v on the y-axis) showed a hook pattern in the upper quadrant (see Fig. 6, A and B). Although the Km and Vmax values for MG4 and MG5 formation were comparable, the Ksi for MG4 (10.89 ± 2.92 µM) was much lower than that of MG5 (189.1 ± 36.0 µM) (Table 1).
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The formation of MG4 and MG5 by UGT1A10 fit the Michaelis-Menten equation (R2 = 0.998 and 0.996) (Fig. 6, E and F). Despite the similarity of Km values, the Vmax for the formation of MG4 was approximately 3-fold greater than that of MG5. This resulted in a specificity constant Vmax/Km for the formation of MG4 by UGT1A10 that was 3-fold greater than that of MG5 (see Table 1). The biphasic Eadie-Hofstee plots indicated that more than one enzyme or active site was involved in the generation of MG4 and MG5 by UGT1A10.
The kinetics constants for the formation of MG4 and MG5 by pooled HLMs are shown in Fig. 7. Like the formation of these metabolites by UGT1A1, these data fit the substrate inhibition model with R2 values of 0.994 and 0.996, respectively (see Fig. 7). The apparent Km and Vmax values for the formation of MG4 were 2.84 ± 0.42 µM and 0.25 ± 0.01 nmol/min/mg protein, respectively, whereas those for the formation of MG5 were 8.42 ± 1.21 µM and 1.47 ± 1.21 nmol/min/mg protein, respectively. The specificity constant (Vmax/Km) for the formation of MG5 by HLMs was approximately 1-fold higher than that of MG4, indicating that MG5 was formed more efficiently than MG4 by HLMs.
Prediction of in Vitro Hepatic Clearance of Isoliquiritigenin Using HLMs. Next, the metabolic stability of isoliquiritigenin in the presence of HLMs and UDPGA was monitored, and the percentage of isoliquiritigenin remaining versus incubation time is shown in Fig. 7. Isoliquiritigenin was rapidly eliminated through glucuronidation, decreasing 98.7% in 50 min. The elimination rate constant (k) was 0.0344, and the in vitro t1/2 for isoliquiritigenin in pooled HLMs was 20.1 min. By comparison, the contribution of P450 enzymes to the metabolism of isoliquiritigenin was much less significant because the t1/2 for substrate depletion in phase 1 reactions was 141 min (data not shown).
Finally, the binding of isoliquiritigenin to microsomal proteins was measured using ultrafiltration, and the fraction unbound in the incubation mixture (fui) was estimated to be 0.85 according to eq. 5. Based on eq. 4 and in vitro measurements, the in vitro hepatic intrinsic clearance of isoliquiritigenin was calculated to be 36.4 ml/min/kg. This value is consistent with rapid hepatic elimination.
Formation of Isoliquiritigenin Glucuronides by Microsomes from Human Liver, Kidney, and Intestine. After incubation of isoliquiritigenin with HLMs, human kidney microsomes, and human intestine microsomes, isoliquiritigenin glucuronides were detected using LC/MS as shown in Fig. 9. MG1, MG4, and MG5 were detected in all three incubations. MG2 was formed only by the liver microsomes and intestine microsomes, and MG3 was formed only by liver microsomes and kidney microsomes. The relative yields of the most abundant glucuronides, MG4 and MG5, differed between incubations. HLMs produced more than 2-fold more MG5 than MG4, whereas the intestine microsomes formed 4-fold more MG4 than MG5. In the incubation with human kidney microsomes, the yield of MG5 was 14-fold more than that of MG4.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Ruefer et al., 2005). The preference for conjugation at the 4'-position instead of the 2'-position was probably because of the stabilization of the 2'-OH group by intramolecular hydrogen bond formation with the oxygen of the ketone or because of the specific orientation of isoliquiritigenin in the active sites of corresponding UGTs.
The experiments with cDNA-expressed UGTs showed that the glucuronidation of isoliquiritigenin is catalyzed primarily by UGT1A1, UGT1A9, and UGT1A10. UGT1A9 formed MG5 and MG3 in preference to MG4. However, UGT1A10 catalyzed glucuronidation most efficiently at the 2'-OH position to form MG4, followed by conjugation at the 4'-OH position to form MG5. The specificity constant for the formation of MG4 by UGT1A10 was 3-fold greater than that for MG5 (Table 1). These results indicate regioselective conjugation by these UGT enzymes. The formation of MG5, the most abundant isoliquiritigenin glucuronide, was catalyzed by several UGTs but predominantly by UGT1A1 and UGT1A9 (Fig. 5). UGT1A1 has been reported to display significant genetic polymorphism (Fisher et al., 2001). For example, the UGT1A1*28 polymorphic allele leads to reduced enzyme expression and is associated with Gilbert's syndrome (Burchell, 2003). Because UGT1A1 can contribute significantly to the formation of both MG5 and MG4, decreased expression of this enzyme might prolong the half-life of isoliquiritigenin in vivo. Furthermore, the half-life of isoliquiritigenin would be affected by any xenobiotics that inhibited UGT1A1 or altered its expression.
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UGT1A9 is expressed in the liver, colon, and kidney (Tukey and Strassburg, 2001). Because UGT1A9 mRNA has been reported to be higher in human kidney than in liver (McGurk et al., 1998) and UGT1A1 is not expressed in kidney (Fisher et al., 2001), UGT1A9 in human kidney should contribute significantly to the extrahepatic clearance of isoliquiritigenin. This prediction is supported by the observation that MG5 was the most abundant isoliquiritigenin glucuronide formed by human kidney microsomes (Fig. 8).
The atypical kinetic plot of MG5 formation by UGT1A9 (Fig. 6) suggests autoactivation, which has been reported for estradiol 3-glucuronidation by UGT1A1 and acetaminophen glucuronidation by UGT1A6 (Fisher et al., 2000). A potential explanation for autoactivation is that UGT enzymes exist as homo-oligomers or heterooligomers, and binding of one substrate molecule to the active site of UGT1A9 facilitates the binding of a second substrate molecule to another UGT1A9 enzyme (Ghosh et al., 2001; Radominska-Pandya et al., 2005). Homodimers of human recombinant UGT1A9 have been detected by chemical cross-linking and coimmunopurification (Kurkela et al., 2003). The existence of homodimers might also explain the biphasic Eadie-Hofstee plot of MG4 and MG5 formation by UGT1A10 (Fig. 6).
Recombinant UGT1A1 and UGT1A10 efficiently catalyzed the formation of MG4 (Fig. 5). Not only expressed in liver, UGT1A1 and UGT1A10 have been detected in human intestine and contribute to the first-pass metabolism of p.o. ingested xenobiotics (Tukey and Strassburg, 2001; Kemp et al., 2002). The high yield of MG4 from incubations of isoliquiritigenin with human intestine microsomes suggests that intestinal first-pass glucuronidation of isoliquiritigenin might be significant (Fig. 9).
Although human intestine microsomes and kidney microsomes catalyzed the glucuronidation of isoliquiritigenin, the liver might still be the major organ involved in the glucuronidation of this chemopreventive agent. To estimate the metabolic efficiency of isoliquiritigenin glucuronidation in liver, the in vitro intrinsic clearance of isoliquiritigenin was obtained using the substrate depletion method (Soars et al., 2002). This assay complemented the kinetics data for UGT1A1, UGT1A9, and UGT1A10, which were found to include substrate inhibition, autoactivation, and Michaelis-Menten kinetics, respectively, for the glucuronidation of isoliquiritigenin. Our results indicate that glucuronidation represents a major metabolic pathway of isoliquiritigenin that should result in its rapid metabolism and elimination.
Although recombinant human UGTs expressed in baculovirus-infected cells are useful tools for the identification and evaluation of isoforms involved in the metabolism of xenobiotics, recent immunoblot analyses using anti-human UGT antibodies have revealed that the levels of microsomal UGTs expressed in the insect cells are not identical (Kato et al., 2008). For example, the amounts of microsomal UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 expressed in the insect cells were 1.6, 0.9, 1.2, 1.4, 1.3, 1.1, 1.1, and 1.2 nmol/mg protein, respectively. Therefore, it should be noted that using UGTs expressed in baculovirus-infected cells for kinetic experiments without normalization to the level of UGT expression in each system might affect the accuracy of the results.
In summary, three isoliquiritigenin glucuronides were identified, MG3, MG4, and MG5, corresponding to conjugation at the 4-OH, 2'-OH, and 4'-OH positions, respectively. In addition, the cyclization product liquiritigenin was also formed and glucuronidated on the remaining A-ring or B-ring hydroxyl groups. The most abundant metabolite, MG5, was formed most efficiently by UGT1A1 and UGT1A9; MG4 was formed primarily by UGT1A1 and UGT1A10; and MG3 was formed preferentially by UGT1A9. The existence of genetic polymorphisms of UGT1A1 with low enzyme activity implies that, in certain individuals, isoliquiritigenin might be eliminated more slowly and have enhanced chemopreventive activity. Because the 4'-OH group was the primary site for glucuronidation, modification of isoliquiritigenin at this position might enhance its bioavailability, prolong its half-life, and enhance its pharmacological activity.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: isoliquiritigenin, 2,4,4'-trihydroxychalcone; HLM, human liver microsome; UGT, UDP-glucuronosyltransferase; HPLC, high-performance liquid chromatography; HPLC/UV, high-performance liquid chromatography with UV absorbance detection; LC/MS, liquid chromatography/mass spectrometry; P450, cytochrome P450; LC/MS/MS, liquid chromatography/tandem mass spectrometry; UDPGA, UDP-glucuronic acid; MG4, isoliquiritigenin 2'-O-glucuronide; MG5, isoliquiritigenin 4'-O-glucuronide; MG1, liquiritigenin 4- or 4'-O-glucuronide; MG2, liquiritigenin 4 or 4'-O-glucuronide; MG3, isoliquiritigenin 4-O-glucuronide.
Address correspondence to: Richard B. van Breemen, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 S. Wood St., Chicago, IL 60612-7231. E-mail: breemen{at}uic.edu
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