Glucuronidation of 1'-Hydroxyestragole (1'-HE) by Human UDP-Glucuronosyltransferases UGT2B7 and UGT1A9

Lalitha V. Iyer1, Mark N. Ho, Walter M. Shinn, Wallace W. Bradford, Mary J. Tanga, Shirley S. Nath and Carol E. Green

1 Biopharmaceutical Division, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025

Received November 18, 2002; accepted February 15, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estragole (4-allyl-1-methoxybenzene) is a naturally occurring food flavoring agent found in basil, fennel, bay leaves, and other spices. Estragole and its metabolite, 1'-hydroxyestragole (1'-HE), are hepatocarcinogens in rodent models. Recent studies from our laboratory have shown that glucuronidation of 1'-HE is a major detoxification pathway for estragole and 1'-HE, accounting for as much as 30% of urinary metabolites of estragole in rodents. Therefore, this study was designed to investigate the glucuronidation of 1'-HE in human liver microsomes in vitro and identify the specific uridine diphosphate glucuronosyltransferase (UGT) isoforms responsible for 1'-HE glucuronidation. The formation of the glucuronide of 1'-HE (1'-HEG) followed atypical kinetics, and the data best fit to a Hill equation, resulting in apparent kinetic parameters of Km = 1.45 mM, Vmax = 164.5 pmoles/min/mg protein, and n = 1.4. There was a significant intersubject variation in 1'-HE glucuronidation in 27 human liver samples, with a CV of 42%. A screen of cDNA expressed UGT isoforms indicated that UGT2B7 (83.94 ± 0.188 pmols/min/mg), UGT1A9 (51.36 ± 0.72 pmoles/min/mg), and UGT2B15 (8.18 ± 0.037 pmoles/min/mg) were responsible for 1'-HEG formation. Glucuronidation of 1'-HE was not detected in cells expressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, and UGT1A10. 1'-HE glucuronidation in 27 individual human liver samples significantly (p < 0.05) correlated with the glucuronidation of other UGT2B7 substrates (morphine and ibuprofen). These results imply that concomitant chronic intake of therapeutic drugs and dietary components that are UGT2B7 and/or UGT1A9 substrates may interfere with estragole metabolism. Our results also have toxicogenetic significance, as UGT2B7 is polymorphic and could potentially result in genetic differences in glucuronidation of 1'-HE and, hence, toxicity of estragole.

Key Words: estragole; 1'-hydroxyestragole; glucuronidation; UDP-glucuronosyltransferase; UGT2B7; UGT1A9.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estragole (4-allyl-1-methoxybenzene) is a naturally occurring flavoring agent found in basil, fennel, anise, bay leaves, and tarragon (De Vincenzi et al., 2000Go). It is used as a constituent of various food flavors, aromatic oils, spices, perfumes, and detergents. There is considerable interest in the safety of estragole and other allylbenzene derivatives, such as safrole, as such compounds have been shown to be hepatocarcinogens in rodent models (Anthony et al., 1987Go; Drinkwater et al., 1976Go). The carcinogenicity of estragole may be related to its metabolism, which involves the formation of several metabolites, some of which are carcinogenic (Fig. 1Go). 1'-Hydroxyestragole (1'-HE) is the proximate metabolite, generated by cytochrome P450 (CYP) enzymes, and it has been shown to be more carcinogenic than estragole (Drinkwater et al., 1976Go). The sulfate conjugate of 1'-HE (1'-sulfoöxyestragole) forms DNA adducts and is considered to be responsible for the hepatocarcinogenic effects of estragole and 1'-HE (Wiseman et al., 1987Go). In addition, 1'-HE is also known to form DNA adducts via formation of 1'-hydroxyestragole-2',3'-oxide (Fig. 1Go), but this metabolite is probably not significant in the carcinogenicity of estragole, as it is known to be rapidly degraded by epoxide hydrolases in vivo (Guenthner and Luo, 2001Go; Luo and Guenthner, 1995Go). 1'-HE also undergoes glucuronide conjugation and the corresponding metabolite is excreted into the urine (Drinkwater et al., 1976Go).



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FIG. 1. Metabolic scheme of estragole, highlighting glucuronidation of 1'-hydroxyestragole (1'-HE) and formation of carcinogenic metabolites.

 
Recent studies in our laboratory have shown that the formation of 1'-hydroxyestragole glucuronide (1'-HEG) is a major pathway of estragole metabolism in rats and mice, which is dose-dependent and accounts for as much as 24% and 33% of the estragole urinary metabolites in rats and mice, respectively (Nath et al., 2001Go, unpublished data). 1'-HE and its glucuronide are also major metabolites formed by human hepatocytes in vitro. By 24 h, about 12.5% of estragole was converted to 1'-HEG by human liver cells (Nath et al., unpublished data). Hence, glucuronidation represents a significant route of detoxification of estragole and 1'-HE in all species studied.

Glucuronidation reactions involve the transfer of glucuronic acid to relatively nonpolar substrates by uridine diphosphate glucuronosyltransferases (UGTs), thereby generating hydrophilic glucuronides that are more easily eliminated (Parkinson, 1996Go). Two distinct families of UGTs have identified: UGT1 and UGT2, each of which has been further classified into individual isozymes (Clarke and Burchell, 1994Go). In humans, the UGT1 locus encodes 13 UGT isoforms (UGT1A1 through UGT1A13p), of which four genes (UGT1A2p, UGT1A11p, UGT1A12p, and UGT1A13p) are pseudogenes (Gong et al., 2001Go). Several human UGT2B isoforms have also been identified, such as UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28 (Levesque et al., 2001Go; Mackenzie et al., 1997Go; Turgeon et al., 2001Go). The current investigation was performed to develop an in vitro glucuronidation assay method for 1'-HE in human liver microsomes and to identify the specific UGT isoform responsible for the glucuronidation of 1'-HE.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Human liver microsomal pool (n = 21) was purchased from BD GentestTM (Woburn, MA). Liver microsomes from 27 individual human donors were prepared from liver specimens obtained from the National Disease Research Interchange (NDRI) and Anatomic Gift Foundation, after approval from the Human Subjects Committee of SRI International. Total protein content of microsomes was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL). UGT SupersomesTM expressing specific isoforms of UGT (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) along with negative control were purchased from BD GentestTM (Woburn, MA). UGT Baculosomes® expressing UGT1A7 were purchased from Panvera Corp (Madison, WI), along with corresponding negative control. Dulbecco’s phosphate-buffered saline (PBS-1X, pH = 7.4) was purchased from GibcoTM Invitrogen Corp, Carlsbad, CA. UDPGA, ß-glucuronidases (from Helix pomatia), morphine sulfate, morphine 3-glucuronide (M3G), morphine 6-glucuronide (M6G), 10,11-dihydrocarbamazepine, D-saccharic acid 1,4-lactone (saccharolactone), ibuprofen, Brij 58, p-nitrophenol (PNP), ethylenediamine tetraacetic acid (EDTA), magnesium chloride (MgCl2), and Triton X-100 were purchased from Sigma-Aldrich Corp, St. Louis, MO.

Synthesis of 1'-HE.
1'-HE was synthesized from 1'-acetoxyestragole, which was prepared by the allylic oxidation of estragole with t-butylperacetate in the presence of cuprous bromide at 100°C. The acetate ester was hydrolyzed with potassium carbonate in methanol at room temperature to give 1'-HE at a 60% overall yield from estragole. The identity of 1'-HE was confirmed using mass spectroscopy in APCI positive ion mode (m/z 147, M-H2O+H, 100% relative intensity), 1H NMR (CDCl3): {delta} 1.41 (1H, br s, -OH), 3.81 (3H, s, -OCH3), 4.30 (2H, m = CH2), 6.24 (1H, m = CH), 6.55 (1H, d, CHOH, J. = 15 Hz), 6.86 (2H, d, arom, J. = 6.6 Hz), 7.32 p.p.m. (2H, d, arom, J. = 6.6 Hz), and 13C NMR (CDCl3):{delta} 55.34, 63.94, 114.01, 126.20, 127.67, 129.41, 130.99, and 159.33 p.p.m.. Purity was determined by HPLC as 99.0% under the following conditions: Brownlee Spheri-5 RP-18, 4.6 x 100 mm column, isocratic mobile phase of 50% methanol/water, 1ml/min flow rate, and UV detection at 254 nm.

1'-HE glucuronidation assay.
Initially, optimal conditions for the in vitro formation of 1'-HEG were determined by varying incubation times (0 to 5 h) and final concentrations of microsomal protein (0 to 4 mg/ml), UDPGA (0 to 15 mM), and substrate (0 to 5 mM). The final conditions used in a typical incubation included the addition of UDPGA (7 mM) to a prewarmed mixture (37°C for 3 min) of microsomes (2 mg/ml) with 1'-HE (800 µM) and MgCl2 (10 mM) in PBS (1X, pH = 7.4) in a total volume of 200 µl. The reaction was stopped with the addition of chilled 0.2% acetic acid in acetonitrile (200 µl) after an incubation period of 4 h in a shaking water-bath (37°C). The samples were then centrifuged at 3,500 r.p.m. for 30 min at room temperature, and the supernatants (80 µl) were injected into a reverse-phase HPLC system with UV detection at 259 nm (Waters Corp, Milford, MA) and Luna C18 (4.6 x 250 mm, 5 µm particle size) column (Phenomenex Inc., Torrance, CA). The mobile phase consisted of 30% of 0.2% acetic acid in acetonitrile and 70% of 0.2% acetic acid in water, used at a flow rate of 1 ml/min. The retention times of 1'-HEG and 1'-HE were ~10 and 14 min, respectively. The formation of 1'-HEG was confirmed by incubation with ß-glucuronidase (3000 U) in 0.2 M sodium acetate buffer (pH = 5.0) for 24 h. Control samples without ß-glucuronidase (buffer alone) were included. Control experiments were performed in presence of a range (0.4 to 10%) of final concentrations of methanol (solvent for 1'-HE) to account for any effects of methanol on 1'-HE glucuronidation.

Morphine glucuronidation assay.
Glucuronidation of morphine to M3G and M6G was studied in microsomes from 27 individual human liver microsomes by a method modified from Innocenti et al.,(2001)Go. Briefly, individual liver microsomal preparations (2 mg/ml) were preincubated with morphine sulfate (0.5 mM) and MgCl2 (5 mM) in PBS-1X (pH = 7.4) for 5 min in a shaking water-bath at 37°C. Final incubation volume was set at 100 µl. The reaction was initiated with the addition of UDPGA (10 mM) and stopped with 400 µl cold ACN after 20 min. The samples were centrifuged at 14,000 r.p.m. for 20 min, after shaking for 20 min in a multitube vortexer (Henry Troemner LLC, Thorofare, NJ). The supernatants were evaporated to dryness under vacuum after addition of 20 µl of internal standard (10,11-dihydrocarbamazepine, 0.5 mg/ml), reconstituted with 100 µl PBS (1X) and centrifuged at 14,000 r.p.m. for 2 min. The resulting supernatant was injected (80 µl) into a reverse-phase HPLC system with fluorescence detection (Waters Corp, Milford, MA) at {lambda}ex = 210 nm and {lambda}em = 340 nm. The mobile phase consisted of 72% 10 mM sodium phosphate and 1 mM lauryl sulfate, pH = 2.1, and 28% acetonitrile used at a flow rate of 1ml/min through a Luna C18 (4.6 x 250 mm, 5 µm) column (Phenomenex Inc., Torrance, CA). Retention times for morphine, M3G, M6G, and internal standard were ~12, 7, 9, and 26 min. Incubations were performed in triplicate for each liver sample, and the rates of formation of M3G and M6G were expressed as pmols/min/mg microsomal protein, using standard curves of M3G and M6G.

Ibuprofen glucuronidation assay.
Individual microsomes (2 mg/ml), preincubated with Brij 58 (0.2 mg/ml) (4°C for 20 min), were incubated with saccharolactone (5 mM), MgCl2 (10 mM), ibuprofen (1 mM), and PBS (1X) (pH = 7.4) for 30 min at 37°C, in a total volume of 200 µl (Magdalou et al., 1990Go; Pritchard et al., 1993Go). The reaction was started with the addition of UDPGA (10 mM) and stopped with 400 µl of 0.1% trifluoro-acetic acid (TFA) in acetonitrile, after which the samples were centrifuged at 3500 r.p.m. for 30 min. The resulting supernatants were injected (100 µl) into a reverse-phase HPLC system with UV detection at 226 nm (Waters Corp., Milford, MA) and a Luna C18 column (5 µm particle size, 4.6 x 250 mm, Phenomenex Inc., Torrance, CA). The mobile phase, consisting of 50% of 0.2% TFA in water and 50% of 0.2% TFA in acetonitrile, was run at a flow rate of 1 ml/min, with resulting retention times of ~6 and 22 min for ibuprofen acyl glucuronide (Ibu-AG) and ibuprofen, respectively.

PNP glucuronidation assay.
PNP glucuronidation was performed as a control for functional UGT activity in the microsomes, as PNP is a substrate for several UGT isoforms (Matsui and Watanabe, 1982Go; Rajaonarison et al., 1991Go; Watanabe et al., 1986Go). A preincubated mix (4°C for 30 min) of human liver microsomes (1 mg/ml) with Triton X-100 (0.025% v/v) was incubated with PNP (360 µM), EDTA (40 µM), MgCl2 (10 mM), and UDPGA (2 mM) in 0.1 M Tris–HCl buffer (pH = 7.4), in a total volume of 500 µl. The reaction was stopped with the addition of 2.8 ml of 0.2 M glycine-NaOH buffer (pH = 10.4). Samples were centrifuged at 2500 r.p.m. for 30 min, and UGT activity in the supernatant was measured colorimetrically from the disappearance of substrate at {lambda} = 400 nm, using a spectrophotometer (Powerwave 200 Microplate Scanning Spectrophotometer, Winooski, UT).

Data analysis.
Each incubation in the various glucuronidation assays was performed in triplicate, and the results were expressed as mean ± SD. The data were fit to Michaelis-Menten (hyperbolic) and Hill (sigmoidal) models and further analyzed using Eadie-Hofstee plots. Standard parameters such as coefficient of determination (R2), Akaike Information Criterion (AIC), standard error of the parameter estimates, and visual inspection of Eadie-Hofstee plots were used to determine the quality of fit to a specific model. The apparent enzyme kinetic parameters of Km, Vmax, and n (for Hill model) for 1'-HE glucuronidation were calculated by nonlinear regression analysis (Enzyme Kinetics ModuleTM 1.1 of Sigma Plot® 2001, version 7.101, SPSS Inc., Chicago, IL). Catalytic efficiency was determined by calculating Vmax/Km ratio. Correlation analyses between glucuronidation of 1'-HE and other UGT2B7 substrates were performed using correlation coefficients (r2) (Primer of Biostatistics, version 4.02 by S.A. Glantz, McGraw Hill, 1996). P values < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formation and Identification of 1'-HEG in Human Liver Microsomes
The chromatograph obtained from a typical 1'-HE assay in pooled human liver microsomes is shown in Figure 2AGo. The retention times for 1'-HE and 1'-HEG were ~ 14 and 10 min, respectively. Control experiments lacking microsomes, UDPGA, and substrate indicated an absence of 1'-HE glucuronidation (not shown). This was further confirmed by treatment with ß-glucuronidases, which resulted in significant deconjugation of the glucuronide, as shown by the major reduction in the 1'-HEG peak in Figure 2BGo. In addition, the UV absorption spectrum (200 to 400 nm) of 1'-HEG indicated a significant resemblance to that obtained with 1'-HE (not shown).



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FIG. 2. HPLC chromatographs from a typical incubation from 1'-HE glucuronidation assay in human liver microsomes. (A) without ß-glucuronidase; (B) with ß-glucuronidase. Retention times for 1'-HEG and 1'-HE are ~ 10 and 14 min, respectively.

 
The effect of varying incubation times, microsomal protein concentrations, and UDPGA concentrations on in vitro glucuronidation of 1'-HE at a substrate concentration of 500 µM is shown in Figure 3A–CGo, respectively. From these results, an incubation time of 4h (Figure 3AGo) was selected for further experiments, along with a final microsomal protein concentration of 2 mg/ml (Figure 3BGo) and UDPGA concentration of 7 mM (Figure 3CGo). Methanol did not significantly affect the glucuronidation of 1'-HE (800 µM) up to a final concentration of 5% (data not shown).



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FIG. 3. Effects of varying incubation conditions on 1'-HE glucuronidation in human liver microsomes. (A) Incubation time; (B) Microsomal protein concentration; (C) UDPGA concentration. Each point represents the mean (± SEM) of three replicates.

 
Determination of Kinetic Constants and InterIndividual Variability in 1'-HE Glucuronidation
The kinetics of 1'-HE glucuronidation in human liver microsomes at a substrate concentration range of 5 µM to 3 mM is shown in Figure 4Go. Two additional substrate concentrations (4 and 5 mM) were also evaluated. However, the concentration of methanol used in these incubations was >=5%, sufficient to significantly decrease the glucuronidation of 1'-HE (1.7- and 2.5-fold lower than Vmax, respectively). Methanol concentration was maintained at <=3% in incubations with the remaining substrate concentrations (<=3 mM). Hence, the data from incubations with the higher substrate concentrations (4 and 5 mM) were not included in the kinetic analysis of the results.



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FIG. 4. Rate of formation of 1'-HEG from 1'-HE in human liver microsomes. Each point represents the mean (± SD) of three replicates. Inset—Eadie-Hofstee plot of the data.

 
The formation of 1'-HEG followed atypical enzyme kinetics, which was best fit to the Hill equation (R2 =.973, AIC = 204.21), resulting in the following parameters: Km = 1.45 ± 0.24 mM, Vmax = 164.52 ± 16.47 pmols/min/mg protein, n=1.4, and Km/Vmax = 0.1134 µl/min/mg. Further analysis of the data resulted in a hooked Eadie-Hofstee plot (inset of Figure 4Go), suggesting activation kinetics or allosterism. The mean (± SD) formation rate of 1'-HEG formation in microsomes from 27 human liver samples was 56.75 (± 23.86) pmols/min/mg protein, with a coefficient of variation (CV) of 42%. The minimal, maximal, and median rate of 1'-HE glucuronidation were 19.83, 131.94, and 48.51 pmols/min/mg protein, respectively.

1'-HE Glucuronidation by cDNA Expressed Human UGT Isoforms
The screening of microsomes from cells expressing specific UGT isoforms for 1'-HE glucuronidation indicated significant 1'-HEG formation (83.94 ± 0.188 pmols/min/mg protein) by the UGT2B7 isoform (Table 1Go). UGT1A9 was also capable of conjugating 1'-HE, with a mean 1'-HEG formation rate of 51.36 ± 0.72 pmols/min/mg protein. Minor 1'-HE glucuronidation was observed in microsomes expressing UGT2B15. The other UGT isoforms screened (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, and UGT1A10) did not exhibit detectable 1'-HE glucuronidating activity (Table 1Go).


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TABLE 1 Formation Rate of 1'-HEG in Human Liver Microsomes and cDNA Expressed UGT Isoforms
 
Correlation Studies with Other UGT Substrates
The association between 1'-HE glucuronidation and morphine 3-glucuronidation, morphine 6-glucuronidation, and ibuprofen glucuronidation in 27 individual human liver samples is illustrated in Figure 5AGo, 5BGo, and 5CGo, respectively. There was a significant correlation between the formation of 1'-HEG and that of M3G (r2 = .74, p < 0.05), M6G (r2 = .71, p < 0.05) and Ibu-AG (r2 = .72, p < 0.05). Morphine 3-glucuronidation and ibuprofen glucuronidation are mediated by UGT2B7 and UGT1A3, while morphine 6-glucuronidation is carried out by UGT2B7 (Coffman et al., 1997Go, 1998Go; Green et al., 1997Go). There was a poor correlation of 1'-HE glucuronidation with PNP glucuronidation (r2 = .28, p = 0.163), which involves several UGT isoforms. In addition, PNP glucuronidation rates were relatively uniform among the 27 liver samples, indicating intact UGT activity in these microsomes (mean ± SD = 8.71 ± 1.29 pmols PNP glucuronidated/min/mg protein, CV = 14%).



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FIG. 5. Correlation of 1'-HE glucuronidation with morphine and ibuprofen glucuronidation in human liver microsomes (n = 27). (A) Morphine 3-glucuronidation; (B) morphine 6-glucuronidation; (C) ibuprofen glucuronidation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This is the first report on the in vitro glucuronidation of 1'-HE in human liver microsomes and the identification of the responsible UGT isoforms. We have determined that 1'-HE, which is the precursor to 1'-sulfoöxyestragole, the active metabolite of estragole believed to be carcinogenic, is conjugated mainly by UGT2B7 using cDNA expressed UGT isoforms and correlation studies with other UGT2B7 substrates. UGT1A9 and UGT2B15 were also found to conjugate 1'-HE (Table 1Go). 1'-HE glucuronidation was undetectable in the cells expressing the other UGT isoforms screened, i.e., UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, and UGT1A10.

Estragole is a component of several food-flavoring substances such as anise, star anise, fennel, basil, bay, tarragon, and marjoram, as well as fragrances, cosmetics, and terpentine oil, thereby resulting in a widespread exposure to humans. It is biotransformed to several metabolites (Figure 1Go), some of which are carcinogenic. Recent studies from our laboratory have shown that the glucuronidation of 1'-HE represents a major detoxification pathway for estragole and 1'-HE (Nath et al., 2001Go, unpublished data). Hence, glucuronidation of 1'-HE may be a significant determinant of the toxicity and carcinogenic potential of estragole.

An assay method was developed to study the conversion of 1'-HE to 1'-HEG in human liver microsomes, by systematically varying each of the main incubation conditions. The formation of 1'-HEG decreased significantly at high substrate concentrations (4 and 5 mM), which is probably due to methanol concentrations >=5%, used to dissolve 1'-HE at these high concentrations. Alternatively, impurities in the substrate might be responsible, although the high purity of the synthesized 1'-HE (99 %) makes this less likely. Product inhibition or a direct effect of 1'-HE on microsomal membrane fluidity or UGT enzyme activity at high concentrations are other possible reasons for this effect.

The atypical kinetic plot of 1'-HE glucuronidation suggests possible auto-activation, which has been reported with CYP3A4 substrates and more recently, with estradiol 3-glucuronidation by UGT1A1 and acetaminophen glucuronidation by UGT1A6 (Fisher et al., 2000Go). To our knowledge, this is the first report of activation kinetics with a UGT2B7 substrate.

The results from incubations of 1'-HE in cells expressing specific UGT isoforms clearly indicate that UGT2B7 and UGT1A9 are responsible for 1'-HE glucuronidation (Table 1Go). Correlation studies were performed with morphine 3- and 6-glucuronidation and ibuprofen glucuronidation, as these reactions are also known to be carried out by UGT2B7 (Coffman et al., 1997Go, 1998Go; Green et al., 1997Go). The significant correlation results with morphine and ibuprofen glucuronidation further substantiate our finding on the role of UGT2B7 on glucuronidation of 1'-HE. Besides, the Km and Vmax values determined in this study are comparable to those reported in other studies (Innocenti et al., 2001Go) for morphine 6-glucuronidation, which is carried out exclusively by UGT2B7 (Green et al., 1998Go).

The finding that UGT2B7 and UGT1A9 are involved in 1'-HE glucuronidation raises important questions on the interaction between estragole and other dietary components and therapeutic drugs. UGT2B7 and UGT1A9 are both expressed in the gastrointestinal and liver tissues (Czernik et al., 2000Go; Strassburg et al., 1998Go) and hence, interactions between dietary estragole and other compounds can occur at both these sites. UGT2B7 is a major human UGT2B isoform that has been shown to conjugate many endogenous compounds and xenobiotics (Radominska-Pandya et al., 2001Go). It is known to conjugate compounds of various therapeutic categories, such as the opioid analgesics (morphine, dihydrocodeine) (Coffman et al., 1997Go; Kirkwood et al., 1998Go), nonsteroidal anti-inflammatory agents (ibuprofen, ketoprofen) (Coffman et al., 1998Go; Jin et al., 1993aGo), benzodiazepines (R-oxazepam) (Court et al., 2002Go), immunosuppressants (cyclosporine, tacrolimus) (Strassburg et al., 2001Go), chemotherapeutic agents (epirubicin) (Innocenti et al., 2001Go) and anti-HIV compounds (zidovudine) (Barbier et al., 2000Go). UGT2B7 is also responsible for the glucuronidation of fatty acids such as linoleic acid, linoleic acid diols, 13-hydroxyoctadecadienoic acid, and 13-oxooctadecadienoic acid (Jude et al., 2001aGo,bGo) and carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Ren et al., 1999Go). UGT1A9 is also involved in the glucuronidation of several compounds such as R-oxazepam (Court et al., 2002Go), acetaminophen (Court et al., 2001Go), flavopiridol (Ramirez et al., 2002Go) and dietary flavonoids (Oliveira and Watson, 2000Go).

The carcinogenicity of 1'-HE is clearly dependent on the balance between formation of the active metabolites, (1'-sulfoöxyestragole) and epoxides, and detoxification by glucuronidation. Our data demonstrate marked interindividual differences in the rate of 1'-HE glucuronidation, which may have toxicogenetic implications. The screen of 1'-HE glucuronidation in liver samples from 27 individuals indicates a significant intersubject variability, with a CV of 42%. UGT2B7 is a polymorphic enzyme, with a histidine to tyrosine substitution at codon 268, resulting in two variant forms—UGT2B7(Y) and UGT2B7(H)—of the enzyme (Bhasker et al., 2000Go; Jin et al., 1993bGo). No significant phenotypic significance has been associated with this polymorphism (Bhasker et al., 2000Go; Gall et al., 1999Go). However, there is recent evidence of a single nucleotide polymorphism (C to T) at position 161 in the UGT2B7 promoter, which is in complete linkage disequilibrium with the polymorphism in codon 268 and is associated with low glucuronidation rates of morphine in humans (Sawyer et al., 2002Go). Furthermore, UGT2B7 expression is regulated by transcription factors such as hepatic nuclear factor-1a (HNF1a) and octamer transcription factor-1 (Oct-1) (Ishii et al., 2000Go; Toide et al., 2002Go) and hence, genetic and environmental factors and concomitant medications affecting HNF1a and Oct-1 may influence glucuronidation of 1'-HE by UGT2B7.

To date, no polymorphisms have been reported for UGT1A9 (Gagne et al., 2002Go). The lack of a role of UGT1A1 in 1'-HEG formation suggests that 1'-HE glucuronidation will be unaffected in Gilbert’s syndrome, a common genetic hyperbilirubinemic syndrome that is associated with reduced UGT1A1 activity and is prevalent up to about 20% in Caucasian subjects (Bosma et al., 1995Go; Monaghan et al., 1996Go).

The presence of polymorphic UGT2B7 with low enzyme activity implies that certain individuals could be "low glucuronidators" of 1'-HE and may have a greater susceptibility to estragole and 1'-HE induced carcinogenesis, as its metabolism may be shunted toward formation of the carcinogenic metabolites (Fig. 1Go). Environmental and dietary factors and concomitant chronic drug administration can influence the regulation of UGT2B7 expression. Our future studies will address this issue of genetic influences on estragole metabolism and carcinogenesis, as well as interactions of estragole with other dietary components, natural compounds, and therapeutic drugs.


    NOTES
 
Preliminary results of this study were presented at the American Association for Cancer Research (AACR) meeting, April 2003.

1 To whom correspondence should be addressed. Tel: (650) 859-2232. Fax: (650) 859-2889. E-mail: lalitha.iyer{at}sri.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anthony, A., Caldwell, J., Hutt, A. J., and Smith, R. L. (1987). Metabolism of estragole in rat and mouse and influence of dose size on excretion of the proximate carcinogen 1'-hydroxyestragole. Food Chem. Toxicol. 25, 799–806.[CrossRef][ISI][Medline]

Barbier, O., Turgeon, D., Girard, C., Green, M. D., Tephly, T. R., Hum, D. W., and Belanger, A. (2000). 3'-azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP- glucuronosyltransferase 2B7 (UGT2B7). Drug Metab. Dispos. 28, 497–502.[Abstract/Free Full Text]

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