Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M401396200 on April 26, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28320-28329, July 2, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
279/27/28320    most recent
M401396200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Basu, N. K.
Right arrow Articles by Owens, I. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Basu, N. K.
Right arrow Articles by Owens, I. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Gastrointestinally Distributed UDP-glucuronosyltransferase 1A10, Which Metabolizes Estrogens and Nonsteroidal Anti-inflammatory Drugs, Depends upon Phosphorylation*

Nikhil K. Basu{ddagger}, Shigeki Kubota{ddagger}, Meselhy R. Meselhy§, Marco Ciotti{ddagger}, Bhabadeb Chowdhury{ddagger}, Masao Hartori§, and Ida S. Owens{ddagger}

From the {ddagger}Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892 and the §Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan

Received for publication, February 9, 2004 , and in revised form, April 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Among gastrointestinal distributed isozymes encoded at the UGT1 locus, UDP-glucuronosyltransferase 1A10 (UGT1A10) metabolizes a number of important chemicals. Similar to broad conversion of phytoestrogens (Basu, N. K., Ciotti, M., Hwang, M. S., Kole, L., Mitra, P. S., Cho, J. W., and Owens, I. S. (2004) J. Biol. Chem. 279, 1429–1441), UGT1A10 metabolized estrogens and their derivatives, whereas UGT1A1, -1A3, -1A7, and -1A8 differentially exhibited reduced activity toward the same. UGT1A10 compared with UGT1A7, -1A8, and -1A3 generally exhibited high activity toward acidic nonsteroidal anti-inflammatory drugs and natural benzaldehyde derivatives, while UGT1A3 metabolized most efficiently aromatic transcinnamic acids known to be generated from flavonoid glycosides by microflora in the lower gastrointestinal tract. Finally UGT1A10, -1A7, -1A8, and -1A3 converted plant-based salicylic acids; methylsalicylic acid was transformed at high levels, and acetylsalicylic (aspirin) and salicylic acid were transformed at moderate to low levels. Atypically UGT1A10 transformed estrogens between pH 6 and 8 but acidic structures preferentially at pH 6.4. Furthermore evidence indicates UGT1A10 expressed in COS-1 cells depends upon phosphorylation; UGT1A10 versus its single, double, and triple mutants at three predicted protein kinase C phosphorylation sites incorporated [33P]-orthophosphate and showed a progressive decrease with no detectable label or activity for the triple T73A/T202A/S432G-1A10 mutant. Single and double mutants revealed either null/full activity or null/additive activity, respectively. Additionally UGT1A10-expressing cultures glucuronidated 17{beta}-[14C]estradiol, whereas cultures containing null mutants at protein kinase C sites showed no estrogen conversion. Importantly UGT1A10 in cells supported 10-fold higher glucuronidation of 17{beta}-estradiol than UGT1A1. In summary, our results suggest gastrointestinally distributed UGT1A10 is important for detoxifying estrogens/phytoestrogens and aromatic acids with complementary activity by UGT1A7, -1A8, -1A3, and/or -1A1 evidently dependent upon phosphorylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many aromatic chemicals are taken into the body unwittingly as components of plant-based diets and as therapeutic agents. Although there are several different categories of chemicals in dietary plants, the flavonoids are by far the largest group and include quasiactive phytoestrogens, which often mimic endogenous estrogens due to structural similarities (1). Additionally there are non-flavonoid-type estrogenic chemicals in plants, e.g. coumestrol and zearalenone, which are reported to have toxic effects in the body (2). While it is known that phytoestrogens can have beneficial tumor-suppressing activity (3) and other effects (4), it is not generally known that the chemicals can also be destructive to animal reproductive systems as shown by the collapse and emaciation of the reproductive and urinary systems of livestock that grazed on clovers and crops with high phytoestrogen content (2). Additionally the phytoestrogens genistein, daidzein, and equol as well as the estrogenic flavonoid precursors (5) chalcones have been shown to have potent effects on cardiac contractility (6). While modest accumulations of phytoestrogens may be inconsequential or beneficial, absorption of high levels of phytoestrogens (isoflavones) can lead to adverse and serious disruption of systems and individual enzymes that could possibly initiate disease(s).

Many ingested chemicals also have the potential to interact with each other or with conventional therapeutic agents. The plant antistress agent, salicylic acid, is ingested unwittingly (7) and has the potential to interact with nonsteroidal anti-inflammatory drugs (NSAIDs).1 Because both salicylic acid and NSAIDs depress the proinflammatory cyclooxygenase enzyme, the combination of these two antiplatelet chemicals enhances the risks of bleeding (7). To the extent that our plant-based diet is a source of large quantities of these exogenous chemicals with many structural variations, such conditions, no doubt, have forced animal systems to limit absorption or to adapt to both natural and synthetic chemicals. Notably a study detected less than 1% uptake of an ingested flavonoid in the blood compared with 98% metabolism and binding to protein in the body when 40-fold less of the same chemical was administered intravenously (8). The results suggest that the chemical was not absorbed when administered via the gastrointestinal (GI) tract or was metabolized and excreted.

Also a variety of simple phenols are generated in the GI system from plant constituents (9, 10). Flavonoid glycosides reach the lower GI tract where the microflora both hydrolyze the glycosides and further metabolize free flavonoids to form a range of aromatic chemicals (9). The metabolites include various aromatic acids and aldehydes as break-down products. Hence absorption of ingested chemicals and those generated in the GI tract determine chemical exposure. As these lipophiles are both simple and polyaromatic containing hydroxyl and/or carboxyl group(s), they are potential substrates of UDP-glucuronosyltransferase (UGT) isozymes, which convert chemicals to glucuronides to enhance water solubility and excretion from cells. To that end, the cluster UGT1A1, -1A7, -1A8, -1A9, and -1A10 of the human UGT1-encoded isozymes (11) has recently been shown to be differentially distributed throughout the GI tract but strategically located in the mucosal layer of tissues (12). The observation that the Gunn rat model (UGT1–/–) is 100-fold more sensitive to acetaminophen than the wild-type Wistar rat (13) indicates glucuronidation is protective against chemical toxins. Because UGT1A10, among the isozyme cluster, is effective in metabolizing all categories of phytoestrogens (12), it suggested that the isozyme would also metabolize structurally similar endogenous estrogens. Furthermore the high abundance of the isozyme in the lower GI tract led to speculation that it may play a role in converting aromatic acids formed in that region by microflora and ingested therapeutic acidic drugs.

In this study, we show that UGT1A10 glucuronidated three different categories of chemicals including estrogens, plant-based cinnamic acid and aromatic acid structures, nonsteroidal anti-inflammatory drugs, and salicylic acid derivatives. While UGT1A10 was the primary metabolizer, studies showed differential overlapping activity with UGT1A1, -1A3, -1A7, and -1A8. The isozyme exhibited different pH optima for catalysis, and it showed a dependence on phosphorylation that is linked to protein kinase C (PKC). Additionally UGT1A10-transfected COS-1 cell cultures glucuronidated 17{beta}-[14C]estradiol unlike those transfected with its null mutants at PKC phosphorylation sites.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—All substrates were obtained from Sigma, Fluka (Milwaukee, WI), Aldrich, or Wako Pure Chemical Industries (Richmond, VA).

Preparation of Microsomes—Human tissue adjacent to that used for in situ analysis (12) was snap frozen and stored at –80 °C until microsomes were prepared (14). Microsomes were resuspended in phosphate-buffered saline and stored at –80 °C until UGT was analyzed.

Source of UGT1A1, -1A7, -1A8, and -1A10 Expression Units—The pSVL-based UGT1A1 expression unit was described previously (15). UGT1A7, -1A8, and -1A10 were constructed as described previously (12). Similarly UGT1A3 and -1A5 were constructed using the UGT1A4 cDNA (15) as template, instead of UGT1A9 cDNA, and an appropriate genomic subclone as described previously (12).

Transfection of UGT1 cDNA Expression Units into COS-1 Cells—All cDNA expression units were transfected into COS-1 cells using DEAE-dextran as the carrier (15).

Western Blot Analysis of UGT cDNA-dependent Expression—To establish the relative amounts of each UGT protein, an antibody (16) prepared by Veritas (Rockville, MD) was raised in rabbit against the common 245-amino acid carboxyl peptide present in each UGT1A-encoded isozyme and used for Western blot analysis (16). pSVL-based cDNA-dependent expression of UGT1A1, -1A3, -1A7, -1A8, or -1A10 protein in COS-1 cells was analyzed 72 h after transfection; 100 µg of cellular protein was prepared and electrophoresed in a 7.5 or 15% polyacrylamide-SDS gel and electrotransblotted onto nitrocellulose membranes that were processed as described previously (16). Proteins were visualized by immunoreaction with rabbit anti-UGT1A and horseradish peroxidase-conjugated goat anti-rabbit second antibody according to the ECL protocol (Amersham Biosciences) using exposed x-ray films for development and quantitation.

Assay for Glucuronidation of Chemicals by UGT1A1, -1A3, -1A7, -1A8, and -1A10 —The modified glucuronidating assay system has been described previously (17, 18). The common donor substrate UDP-[14C]glucuronic acid (1.41 mM, 1.4 µCi/µmol) was used in all in vitro reactions with an unlabeled acceptor/aglycone substrate. Similar to reactions for Km determinations, pH profiles reflect product generated in 2 h at 37 °C with 300 µg of protein from pSVL(UGT)-transfected COS-1 cells. Linear glucuronidation reactions for substrate screens were conducted at both pH 6.4 and 7.6 over 4 h and defined as glucuronide accumulated/4 h as described previously (19). All chemicals were solubilized in fresh Me2SO.

Mutagenesis of Predicted PKC Phosphorylation Sites in UGT1A10 — Site-directed mutagenesis at amino acid 73, 202, or 432 in UGT1A10 was carried out as described previously (18). Oligos for converting the common PKC site S432G in UGT1A10 were as follows: sense 214C, 5'-ctgcttggtcacccgatgaccc-3', and antisense 463C, 5'-ggcgcatgatgttctccttgtaacctttg-3' for fragment 1; sense 435C, 5'-caaaggttacaaggagaacatcatgcgcc-3', and antisense BamH1Stop, 5'-cccggatccacccacttctcaatgggtctt-3' for fragment 2. For T73A mutation of UGT1A10, primers were as follows: sense Xho1A10S, 5'-ccctcgagggagctgctggctcgggct-3', and antisense 73A10AS, 5'-cgaggttgagtaagtcttcactgcgca-3' for fragment 1; for its overlapping unit, the sense oligo was the complement of 73A10AS, and the antisense oligo was PXAS6 (18). Finally, for T202A mutation for UGT1A10, primers were as follows: sense Xho1A10S and antisense 202A10AS, 5'-ccatactctctccttgaaagccatggcatc-3'); its overlapping fragment was synthesized using the complement of 202A10AS as sense and PXAS6 as antisense.

[33P]Orthophosphate Labeling of UGT1A10 in COS-1 Cells—Sixty hours after transfection with a pSVL-based construct containing either UGT1A10, single mutant (T73A-, T202A-, or S432G-1A10), double mutant (T73A/T202A-, T73A/S432G-, or T202A/S432G-1A10), or triple mutant (T73A/T202A/S432G-1A10), labeling of each was compared with UGT1A10 and control cells. Cells were conditioned in phosphate-free medium for 12 h and also serum-free conditions for the last 3 h before exposure to [33P]orthophosphate (5.0 mCi/ml) for 8 h as described previously (20, 21). With all manipulations carried out at 0–4 °C, attached cells were washed five times with phosphate-buffered saline; solubilized in 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1.0 mM EDTA in 25 mM Tris borate, 137 mM NaCl (TBS), pH 7.4 (buffer A); passed through a 26-gauge needle to reduce viscosity; and microcentrifuged in tubes to remove insoluble cellular debris. Supernatants were recovered, protein estimations were carried out, and equal amounts of protein were incubated with anti-UGT1A for 1 h before addition of protein A-Sepharose equilibrated in buffer A to allow immunocomplexes to form for 12 h with sample rotation (21). UGT immunocomplexes bound to protein A-Sepharose were washed four times with buffer A for 1 h, washed overnight with buffer A containing 1 M KCl, and finally washed once each with TBS and water. After samples were boiled in SDS sample buffer to detach protein, each sample was centrifuged through a microSpin column (Amersham Biosciences) to remove beads. Duplicate sets of radiolabeled UGT1A10 and mutants were electrophoresed in an SDS-7.5% polyacrylamide gel; one gel was processed for exposure to x-ray film, and the duplicate gel was analyzed by Western blot as described above.

17{beta}-[14C]Estradiol Glucuronidation by COS-1 Cells Transfected with UGT1A10, Its Mutants at PKC Phosphorylation Sites, or UGT1A1— Forty-eight hours after transfection with pSVL-based UGT1A1, UGT1A10, or T73A-, T202A-, or S432G-1A10 mutants, COS-1 cells were conditioned in Dulbecco's modified Eagle's medium containing 4% charcoal-stripped (22) fetal bovine serum for 24 h before adding 20 or 40 µM 17{beta}-[14C]estradiol (2.5 µCi/40 µM) in fresh Dulbecco's modified Eagle's medium/charcoal-stripped fetal bovine serum. Also a concentration range of 17{beta}-estradiol was studied. After 3 h, 500 µl of medium in triplicates were diluted 1:2 with cold ethanol and centrifuged to remove protein. Recovered supernatant was spun dry and resuspended in phosphate-buffered saline. One-half of each sample was treated with buffer or 50 units of {beta}-glucuronidase in 50 µM phosphate buffer, pH 6.8, for 2 h at 37 °C. Samples were vacuum-dried, resuspended in 70% ethanol, and applied to TLC plates for elution as described previously (18) for quantitation (19).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Glucuronidation of Epigallocatechin 3-O-Gallate, 17{beta}-Estradiol, and Flurbiprofen by Microsomes Isolated from Human GI Tissues—To gain insight into the function of the UGT1A-encoded isozymes, we previously compared their pattern of tissue distribution and cellular location by Northern blot analysis and in situ hybridization of messenger RNAs (12). We found that UGT1A10 is primarily distributed in the mucosal layer of the GI tract below the stomach (12) with substantial amounts in other tissues. We also used microsomes prepared from tissue adjoining to that used for in situ hybridization in conjunction with the highly specific UGT1A10 substrate epigallocatechin 3-O-gallate (ECG) (see Ref. 12) to assess the relative level of UGT1A10 activity in GI tissues. (Determination of substrate specificity was based on a screen of 40 chemicals using recombinant UGT1A1, -1A7, -1A8, -1A9, and -1A10 (12) at both pH 6.4 and 7.6. Under similar conditions and for a given substrate, isozyme specificity was determined as the ratio of an isozyme activity to the sum of all isozyme activities.) In this study with GI microsomes, we compared ECG with two other UGT1A10 substrates, flurbiprofen (pH 6.4) and 17{beta}-estradiol (pH 7.0), which were 83 and 89% specific, respectively. UGT1A10 appeared to be present in duodenum:ileum:colon:stomach:esophagus at relative levels of 9:8:3:2:1 (Fig. 1). While the isozyme was most abundant in small and large intestines, followed by stomach, and it showed greater turnover of 17{beta}-estradiol than ECG and flurbiprofen, it appeared to be detectable in esophageal microsomes by substrate analysis. Although UGT1A8 is detectable only in the stomach by Northern blot analysis (12), glucuronidation studies with phloretin at pH 7.0 and in situ hybridization with specific probes have demonstrated moderately high to high levels in esophagus, duodenum, ileum, and colon (12). Except for stomach, UGT1A8 was concentrated in a highly erratic pattern in limited regions of all GI tissues. UGT1A7, on the other hand, was highest in esophagus and at low to intermediate levels in nearly every tissue with the highest level in thyroid and adrenal glands (12). While Northern blot detected UGT1A3 only in liver,2 it has been shown that the isozyme, like UGT1A8 (12), can be found in GI tissues following reverse transcription-PCR amplification of mRNA (23). In a recent study with bilirubin as UGT1A1-specific substrate (12), we found that UGT1A1 was distributed in duodenum, ileum, and stomach at relative levels of approximately 10:4:1 with activities expressed as pmol/h/mg of protein.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 1.
Glucuronidation of epigallocatechin 3-O-gallate, 17{beta}-estradiol, and flurbiprofen by human microsomes from GI tissues. Normal human GI tissues were collected (12) and stored at –80 °C until microsomes were prepared (14), and glucuronidation of ECG, 17{beta}-estradiol, and flurbiprofen at 200 µM each was carried out in 2-h incubations at 37 °C as described under "Experimental Procedures." The images represent autoradiograms of the TLC plate that resolved the [14C]glucuronides. The product is the mean ± S.E. A total of two esophagi, three stomachs, three duodena, two ilea, and four colons were analyzed thrice in triplicates (12). gluc, glucuronide; prot, protein.

 

Western Blot Analysis of UGT1A1, -1A3, -1A7, -1A8, and -1A10 Expressed in COS-1 Cells—To normalize the relative levels of UGT proteins, we carried out Western blot analysis (Fig. 2) with the UGT1A common end antibody (16), and the relative levels of UGT1A1, -1A3, -1A7, -1A8, and -1A10 synthesized in COS-1 cells were determined by scanning blots with a UMAX system and quantifying with Adobe Photoshop software. After normalizing relative protein levels, specific activities were calculated and are shown.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 2.
Western blot analysis to establish relative levels of UGT1A1, -1A3, -1A7, -1A8, and -1A10 used to generate activities shown in Tables I, II, and III. Levels were determined as described under "Experimental Procedures." Sufficient protein was generated to allow the completion of the study. Experiments were repeated thrice in triplicates.

 
pH Optimization and Km Determination for Glucuronidation of 17{beta}-Estradiol and Flurbiprofen by UGT1A10—Since our earlier studies with UGT1A-encoded isozymes demonstrated optimal conditions for glucuronidation depended, to a large extent, upon the acceptor substrate (12), we optimized conditions for the type of chemical. UGT1A10 metabolized 17{beta}-estradiol with a pH optimum between 6 and 8, and it metabolized flurbiprofen with a narrower pH range between 6 and 6.4 (Fig. 3, A and C); typical pH 7.6 assay conditions did not show glucuronidation of the latter aglycone. The pH profile for glucuronidation of ECG (not shown) was essentially the same as for flurbiprofen.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3.
pH optimization and Km determination of 17{beta}-estradiol and flurbiprofen glucuronidation by UGT1A10. A and C, pH profiles were carried out with sodium phosphate from pH 5 to 7 and with triethanolamine from pH 7.0 to 9.0 (12) with 200 µM substrate in 2-h incubations at 37 °C as described under "Experimental Procedures." B and D, similarly Km values were established using 300 µg of cellular protein in a 2-h incubation for glucuronidation of 17{beta}-estradiol (pH 7.0) and flurbiprofen (pH 6.4). Assays were repeated thrice in triplicates; standard errors ranged from ±1 to 3%.

 
Since increasing concentrations of phloretin and eugenol up to 200 µM stimulated UGT1A10 activity and higher levels showed progressive inhibition that culminated at 100% by 800 µM (12), we examined effects of 17{beta}-estradiol or flurbiprofen concentration on activity. Concentrations of 17{beta}-estradiol and flurbiprofen from 200 to 400 µM stimulated activity, which was sustained up to 800 µM. The Km value for both 17{beta}-estradiol and flurbiprofen was 64 µM (Fig. 3, B and D). Complete inhibition of UGT1A10 by 800 µM phloretin or eugenol (12), but not by high concentrations of 17{beta}-estradiol and flurbiprofen, indicates inhibition is substrate-dependent.

Because published studies (24, 25) indicate UGT1A1 also glucuronidates 17{beta}-estradiol, we compared its metabolism by UGT1A10 to that by UGT1A1. Increasing the concentration of 17{beta}-estradiol from 2 to 20 µM caused a 2-fold increase in UGT1A10 activity as seen in Fig. 3B, and further increases in concentrations up to 100 µM (Fig. 4) showed almost a linear increase in activity. To the contrary, 20 µM 17{beta}-estradiol showed no detectable conversion by UGT1A1, but 40 µM elicited a substantial UGT1A1 activity that was barely enhanced further by increases up to 100 µM (Fig. 4). Unlike its limited concentration effects on UGT1A1 conversion, 17{beta}-estradiol supported a high rate of metabolism by UGT1A10 between 2 and 200 µM (Figs. 3B and 4). At 40 µM, UGT1A10 showed >10-fold greater activity than UGT1A1 (Fig. 4).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4.
Comparison of in vitro glucuronidation of 17{beta}-estradiol by UGT1A1 and UGT1A10. Glucuronidation of 17{beta}-estradiol was carried out at pH 6.4 in 2-h incubations at 37 °C as described under "Experimental Procedures." prot, protein.

 
Glucuronidation of Estrogens, Plant-derived Aromatic Acids, and Nonsteroid Anti-inflammatory Drugs by UGT1A10, -1A1, -1A3, -1A7, and -1A8 —In an earlier study (12), we observed that UGT1A10 metabolized two different types of phytoestrogens nearly equally at both pH 6.4 and 7.6. Furthermore we found that UGT1A4, -1A5, -1A6, and -1A9 and UGT2B7 and -2B15 did not show a significant activity toward the primary estrogens (data not shown) (26), although recombinant UGT2B7 was reported previously to have detectable activity when microsomes were analyzed (24). Since phytoestrogens and mammalian estrogens often exhibit similar actions and show competitive interactions via the estrogen receptor (1,6), we compared the metabolism of endogenous estrogens by UGT1A10 to that by UGT1A8, -1A7, -1A3, and -1A1 using equal specific protein (Fig. 2). Eight of nine estrogens, including their derivatives, were excellent substrates for UGT1A10 at both pH 6.4 and 7.6 (Table I). UGT1A10, -1A8, -1A7, and -1A3 showed a 5- to >10-fold preference for the predictably more hydrophobic 2-CH3O(methoxy)-estradiol than 2-OH(hydroxyl)-estradiol; similarly, this enzyme set exhibited from 6- to 17-fold greater preference for 4-OH-estrone than 2-OH-estrone. UGT1A10 showed exceptional and equally high activity toward three estriol isomers, which contained three different versions of 16- and 17-hydroxyls that project in versus out of the plane of the core steroid structure (see Table I); the turnovers of the estriols were only 23–30% less than that for estrone and 2-CH3O-estradiol, which gave the highest activities for steroids. UGT1A8 showed more than 4-fold preference for the 16,17-estriol than for 17-estriol. Interestingly the two least effective estrogenic substrates, 2-OH-estradiol and 2-OH-estrone, for UGT1A10 were converted at the highest level by UGT1A1, which was 2.5–4.5-fold higher than by UGT1A10. Hence the two isozymes showed complementary activity toward the 2-OH estrogens, and overall UGT1A10 was superior to UGT1A1, -1A8, -1A7, and -1A3 in metabolizing estrogens.


View this table:
[in this window]
[in a new window]
  		TABLE I
Glucuronidation of steroids by human UGTs

Nine estrogens or their derivatives were screened for glucuronidation by the UGTs identified using a 200 µM concentration of each chemical at pH 6.4 and 7.6 with 300 µg of UGT-transfected COS-1 cell protein. Reactions were incubated 4 h at 37 °C and were processed for quantitation as described under "Experimental Procedures." While UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 showed no detectable glucuronidation of 17{beta}-estradiol or other estrogens, UGT1A9 showed detectable conversion (pmol/mg of protein/4 h) of 2-OH estrone (30 and 15 at pH 6.4 and 7.6, respectively), 4-OH estrone (180/520 at pH 6.4/7.6), and 2-methoxyestradiol (30/210 at pH 6.4/7.6).

 
With few exceptions, all isozymes metabolized the estrogens and their derivatives nearly equally under both pH conditions, suggesting a broad pH optimum as seen in Fig. 3A for 17{beta}-estradiol. UGT1A9 inefficiently metabolized certain estrogen derivatives at either pH 6.4 or 7.6 (legend of Table I).

In a previous report (12), UGT1A10 was shown to glucuronidate 32 of 40 chemicals with a 1.5–10-fold preference for pH 6.4 optimum. Substrates included anthraquinones and related chemicals, flavonoids/isoflavones (phytoestrogens) and related chemicals, polycyclic aromatic hydrocarbon and simple phenols, and lactone-containing phytoestrogens. In contrast, UGT1A10 converted seven of eight estrogens (this study) and six of seven phytoestrogens (12) nearly equally at pH 6.4 and 7.6; only estrogenic 2-OH-estradiol and phytoestrogenic daidzein showed a pH 6.4 preference. Among the 40 chemicals (12), UGT1A10 metabolized isoflavones or phytoestrogens the most efficiently; UGT1A7 had comparably high turnover for the lactone-containing phytoestrogens (coumestrol and zearalenone) and the isoflavan equol. Among estrogenic compounds, UGT1A3 effectively conjugated only 2-CH3O-estradiol.

Because UGT1A10 exhibits a high activity toward many dietary agents, including phytoestrogens (12), and it is distributed primarily in GI mucosa from duodenum through colon, we carried out substrate analysis on carboxyl-containing chemicals known to form by microflora in the lower GI tract from flavonoids found in high levels in our plant-based diet (9) or to be ingested as dietary constituents or as therapeutic agents such as NSAIDs. Studies with such substrates, which included salicylates, transcinnamic acids, and {beta}-phenylpyruvic through diphenyl/biphenyl acids as well as 14 therapeutic NSAIDs (Tables II and III) show UGT1A10 exhibited the major activity, whereas UGT1A7, -1A8, and -1A3 exhibited significant activity. For plant-based salicylates, methylsalicylate was the most effective substrate in this study for UGT1A10; UGT1A7 and -1A8 were approximately one-half as effective, and UGT1A3 exhibited 3% the level of UGT1A10 activity. Acetylsalicylic acid (aspirin) was best converted by UGT1A10, but there was little conversion of salicylic acid by any isozyme. Among metabolites (aldehydes and acids) (Table II) originating from plants, UGT1A10 conversion of aldehyde-containing vanillins was second to methylsalicylate showing 50% the level of conversion; UGT1A8 and -1A7 also showed high activity toward the vanillins yielding 30–50% that of UGT1A10 at both pH values. Oxidation of the aldehyde group in 4-OH,3-CH3O-benzoic acid (vanillic acid) dramatically reduced turnover by UGT1A8, -1A10, and -1A8 (Table II) showing only 6–37% the activity for the best aldehyde analog.


View this table:
[in this window]
[in a new window]
  		TABLE II
Glucuronidation of aromatic acids and aldehydes by human UGTs

Sixteen aromatic acids, which are either therapeutic NSAIDs, derived directly from plants, or catabolites from flavonoid glycosides produced by intestinal microflora were analyzed for glucuronidation by the UGTs shown as described in the legend to Table I. Also glucuronidation of nonacidic aromatic acetaminophen and synthetic clofibric acid was compared. Each substrate was analyzed at 200 µM.

 


View this table:
[in this window]
[in a new window]
  		TABLE III
Glucuronidation of nonsteroidal anti-inflammatory drugs and hydroxylated aromatic hydrocarbons by human UGTs

Eight therapeutic aromatic acids were analyzed for glucuronidation by the UGTs identified as described in the legend to Table I; each substrate was analyzed at 200 µM. Also results were compared to those for biphenyl acetic acid and anthralin.

 
Also UGT1A10 expressed the highest conversion of transcinnamic acids, including caffeic and ferulic acids, at pH 6.4. For the pyruvic, propionic, and isovaleric phenyl acids (Table II), UGT1A3 showed ~2-fold greater activity than other isozymes. Diphenyl and biphenyl acetic acids, with free rotation of phenyl groups versus fixed groups, were metabolized nearly equally by the isozymes examined.

Results with therapeutic polyaromatic acids, NSAIDs (Tables II and III), show that UGT1A10 converted mefenamic acid, diflunisal, flurbiprofen, fenoprofen, ibuprofen, and indomethacin at levels comparable to the vanillins and certain estrogens but with pH 6.4 preference. Overall UGT1A3 metabolized the NSAIDs at a moderate level with pH 6.4 preference except for indoprofen.

It is notable that the nonacidic but aromatic acetaminophen is not a substrate for either of these isoforms or a poor substrate for UGT1A3 and UGT1A10. There are other significant observations concerning glucuronidation of these chemicals. While UGT1A1 metabolized certain estrogens, it did not metabolize any of the aromatic acids in this study. This is notable since this isozyme forms ester-linked glucuronides via the propionic acid groups of the bilirubin IX{alpha} molecule (15).

[33P]Orthophosphate Labeling of UGT1A10 with Effects of Mutations at Predicted PKC Phosphorylation Sites and on in Vitro Glucuronidation—Because exposure of colon cells to the dietary constituent curcumin at 50 µM rapidly inhibited UGT activity for test substrates eugenol, phloretin, capsaicin, ECG, and bilirubin (12, 21), we carried out experiments to assess the substrate range of inhibition. Earlier results showed that calphostin-C, a highly specific PKC inhibitor, irreversibly down-regulated glucuronidation suggesting UGT proteins undergo phosphorylation. Furthermore we carried out computer searches for consensus sequences for phosphorylation and uncovered four to five predicted PKC phosphorylation sites in each protein (21). Consistent with these findings and under conditions of equal protein (Fig. 5, top panel), the radiogram (Fig. 5, bottom panel) shows UGT1A10 incorporated [33P]phosphate that when compared with predicted PKC site mutants for single positions (T73A, T202A, and S432G) and for double positions (T73A/T202A, T73A/S432G, and T202A/S432G) showed progressively less label and that for triple positions (T73A/T202A/S432G) exhibited no detectable labeling or activity. The graded effect based on number of sites remaining, not position, suggests each predicted PKC site is utilized for phosphorylation evidently by PKC isozyme(s) (21). Furthermore calphostin-C, the PKC-specific inhibitor, inhibited 17{beta}-estradiol activity without detectable labeling in UGT1A10 as previously demonstrated for UGT1A1 (21). Glucuronidation of 17{beta}-estradiol by other mutants was comparable to that shown in Fig. 6 and is discussed below. Complete inactivation of the triple mutant but full 17{beta}-estradiol activity for wild-type UGT1A10 indicates activity is dependent upon phosphorylation.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 5.
Labeling of recombinant UGT1A10 and its mutants with [33P]orthophosphate. Cells were either mock-transfected or transfected with UGT1A10 or its single mutants (S432G, T73A, and T202A), three double mutants (T73A/T202A, T73A/S432G, and T202A/S432G), or the triple mutant (T73A/T202A/S432G). Conditioned transfected COS-1 cells, grown in 35-mm plates, were exposed to [33P]orthophosphate (5.0 mCi/ml)-containing medium for 8 h as described under "Experimental Procedures." With all samples containing equal cellular protein, duplicate sets of unlabeled and radiolabeled samples for control, UGT1A10, and mutants were immunocomplexed and processed in an SDS-7.5% PAGE system as described under "Experimental Procedures." The radiolabeled gel was fixed and exposed to x-ray film for 48 h (bottom panel), and the unlabeled gel was subjected to Western blot using anti-UGT (top panel). Glucuronidating activity was analyzed with 200 µM 17{beta}-estradiol in a 2-h incubation at 37 °C. The experiment was repeated thrice. ND denotes not detectable. prot, protein; WT, wild type.

 



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 6.
Effects of various mutations at predicted PKC phosphorylation sites in UGT1A10 on activity toward different substrates. A, Western blot analysis with anti-UGT1A was carried out with 10% SDS-PAGE as described under "Experimental Procedures." B, glucuronidation assays were carried out using equal amounts of specific protein from COS-1 cells transfected with constructs having either single, double, or triple mutations at PKC sites in UGT1A10. Single mutants were T73A, T202A, and S432G; double mutants were T73A/T202A, T73A/S432G, and T202A/S432G; and the triple mutant was T73A/T202A/S432G. Flurbiprofen was incubated for 3 h at 37 °C, while 17{beta}-estradiol and genistein (a phytoestrogen) were incubated for 2 h. Each chemical was at 200 µM in the reactions. Experiments were repeated thrice in triplicates; standard errors ranged from ±2 to 4%. WT, wild type; prot, protein.

 
It is also evident that a major radiolabeled species and a barely detectable one are present in the nascent UGT1A10 evidently reflecting ongoing protein maturation due to glycosylation at three predicted glycosylation sites as seen previously for other UGTs (26). A minor band at the top is not distinguishable by Western blot analysis.

In Vitro Glucuronidation by COS-1 Cells Transfected with Wild Type or UGT1A10 Mutants—Since UGT1A10 exhibited high activity toward estrogens (Table I), phytoestrogens (12), and NSAIDs (Tables II and III), we examined the effect of independent double and triple mutations on the capacity of UGT1A10 to glucuronidate 17{beta}-estradiol, genistein, and flurbiprofen. The isozyme exhibited the greatest activity toward the phytoestrogen genistein followed by 17{beta}-estradiol and flurbiprofen (Fig. 6). Its Km for genistein (not shown) is similar to that for 17{beta}-estradiol and flurbiprofen (Fig. 3). Furthermore the T73A and T202A single mutants and their double mutant T73A/T202A exhibited null activity for each substrate. The S432G mutant caused an increase in activity for each of the substrates, and its double mutant with either T73A or T202A showed partial or additive activity with genistein and 17{beta}-estradiol but barely detectable activity with flurbiprofen. Finally the triple mutant showed null activity for each of the test substrates (Fig. 6). There was no detectable difference in activity between S432G-1A10 and S432A-1A10.3

Glucuronidation of 17{beta}-[14C]Estradiol by COS-1 Cells Transfected with Wild Type or UGT1A10 Mutants—To determine whether phosphorylation of a recombinant isozyme controls glucuronidation in cells, we compared conversion of 17{beta}-[14C]-estradiol by control COS-1 cells with UGT1A10-, T73A-1A10-, T202A-1A10-, or S432G-1A10-transfected cells. Before cell culture results are presented, we provide evidence that 17{beta}-[14C]estradiol glucuronide produced in vitro is resolved by the TLC system (Fig. 7A). Lanes from left to right show migration of 17{beta}-[14C]estradiol (17{beta}-est), effect of control cell protein on migration of 17{beta}-est, resolution of 17{beta}-est and 17{beta}-[14C]estgluc, and hydrolysis of 17{beta}-est-gluc by {beta}-glucuronidase (Fig. 7A). Results in Fig. 7B show UGT1A10 and its S432G mutant formed comparable levels of 17{beta}-est-gluc, which was sensitive to {beta}-glucuronidase (lower panel). To the contrary, cells transfected with either T73A- or T202A-1A10 mutant failed to generate the glucuronide. The results demonstrate that wild-type UGT1A10 and its mutants at PKC sites exhibited glucuronidation patterns in cell culture similar to those in in vitro assays.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 7.
Glucuronidation of 17{beta}-estradiol by UGT1A10 and its mutants is similar under in vivo and in vitro conditions (A–C), and a comparison of glucuronidation of 17{beta}-estradiol by UGT1A10- or UGT1A1-transfected COS-1 cell culture is shown (D). A, replica of the TLC system used to separate 17{beta}-[14C]est from its glucuronide. Lanes from left to right, migration of 17{beta}-[14C]est, effect of control COS-1 cell homogenate on 17{beta}-[14C]est, production of 17{beta}-[14C]est-gluc by homogenates containing UGT1A10, and loss of 17{beta}-[14C]est-gluc formed by UGT1A10 following {beta}-glucuronidase treatment. B, production of 17{beta}-[14C]est-gluc by COS-1 cell cultures transfected with UGT1A10, S432G-1A10, T73A-1A10, T202A-1A10, or control. Three hours after exposure to 40 µM 17{beta}-[14C]est, 500 µl of culture medium were prepared for TLC analysis as described under "Experimental Procedures." Before samples were applied to plates, one-half of certain samples was treated with {beta}-glucuronidase (lower panel) as described under "Experimental Procedures." C, in vitro production of 17{beta}-est-gluc and genistein glucuronide using duplicate cultures to those in B. In vitro glucuronidation of 17{beta}-est or genistein used [14C]UDP-glucuronic acid as co-substrate and is shown after normalization with the Western blots (top panel) as described under "Experimental Procedures." D, comparison of 17{beta}-[14C]est glucuronidation (Counts) by UGT1A1- and UGT1A10-transfected COS-1 cell cultures. 17{beta}-[14C]Estradiol was added to COS-1 cell cultures expressing UGT1A1 or UGT1A10 and incubated for 3 h, and 500 µl were processed and quantitated as described above in B. Western blots (designated Protein) of each UGT in duplicate cultures were carried out, scanned, and used to normalize counts as described under "Experimental Procedures." {beta}-G'dase, {beta}-glucuronidase.

 
With duplicate cultures to those that synthesized 17{beta}-[14C]estradiol glucuronide (Fig. 7B), we demonstrate again that in vitro results with 17{beta}-estradiol and genistein combined with UDP-[14C]glucuronic acid replicated findings in cell culture (Fig. 7C). A scan of the Western blot (top panel) was used to normalize specific protein levels.

Relative Effectiveness of UGT1A10-Versus UGT1A1-transfected COS-1 Cells toward Glucuronidation of 17{beta}-[14C]Estradiol—Despite the fact UGT1A10 (pH 6.4) showed more than 13-fold higher activity toward 17{beta}-estradiol than UGT1A1 (Table I and Fig. 4), UGT1A10 has presumably a high Km (estradiol) value as shown in Fig. 3 making it unclear which isozyme is likely to glucuronidate the estrogen under in vivo conditions. Furthermore the ineffectiveness of 20 µM or lower 17{beta}-estradiol on UGT1A1 activity but substantial stimulation by 40 µM complicated expectations for in vivo glucuronidation by UGT1A1. Hence we carried out glucuronidation of 20 and 40 µM 17{beta}-[14C]estradiol in COS-1 cells transfected with UGT1A1 or -1A10 (Fig. 7D). After normalization for transfection efficiency, 17{beta}-[14C]estradiol (20 or 40 µM) showed >10-fold greater metabolism by UGT1A10 than by UGT1A1 (Fig. 7D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Among the isozymes encoded at the UGT1A complex locus, UGT1A10, distributed primarily in the GI tract (12) with significant levels in heart, placenta, spleen, pancreas, lymph node, adrenal, and uterus, expressed more than 10- and 100-fold higher activity toward the primary estrogens 17{beta}-estradiol and estrone, respectively, than UGT1A1 (Table I). Moreover UGT1A10 metabolized all estrogen derivatives tested at 16- to more than 10,000-fold higher rates than UGT1A1 except for 2,3-catechol estrogens. With an inverse pattern, UGT1A1 metabolized 2-OH-estradiol and 2-OH-estrone at rates 2- and 4.5-fold higher, respectively, than UGT1A10. Overall this study shows that UGT1A10 metabolized estrogens at a higher level than other UGT1A isozymes and with an atypically broad pH range compared with its high pH 6.4 preference observed for a screen of 40 test chemicals (12). It is notable that UGT2B7 metabolized many estrogen derivatives (26–28) but not estrone or 17{beta}-estradiol (data not shown here) (26), although UGT2B7 did show activity using microsomes isolated from a different expression system (24). The ineffectiveness of UGT1A1 at converting 17{beta}-estradiol compared with the high level of conversion by UGT1A10 in cell culture suggests it is most likely responsible for glucuronidating endogenous estrogens despite its high Km value. Thus, UGT1A10 appears to be unique in carrying out significant transformation of the primary estrogen 17{beta}-estradiol.

Interestingly we discovered earlier that UGT1A10 also metabolizes several categories of phytoestrogens (12): the isoflavone type (daidzein, genistein, formononetin, and biochanin A), an isoflavan type (equol), lactone-containing compounds (coumestrol and zearalenone), and estrogenic flavonoid precursors (5) (phloretin and others (12)) with a broad pH range and at a level comparable to the estrogens described in this study. Among four different categories of chemicals (12) and after eugenol, phytoestrogens were the most highly preferred class of substrates by UGT1A10. Phytoestrogens, like catechol estrogens (26, 27), are estrogenic due to structural similarities, which allow competition at the level of the estrogen receptor (1, 5). As UGT1A10 metabolized 17{beta}-estradiol in vitro at low concentrations (2–20 µM) to a high extent, unlike UGT1A1, it is likely that UGT1A10 is adapted to convert both estrogens and dietary phytoestrogens despite its Km (64 µM) but broad pH range (Fig. 3). The devastation that phytoestrogens can inflict on the reproductive systems of livestock that consume large quantities of clovers with high phytoestrogen content (2) underscores the potential damage these agents can impose via interaction with the estrogen receptor and the need to limit their GI absorption. Predictably GI distributed UGT1A10, which is the most effective at converting these dietary phytoestrogens in vitro, is adapted to allow humans to limit their absorption to reduce the risks of reproductive failures.

A striking feature of ingested test flavonoids has been demonstrated by a lack of absorption even when taken in high levels (8, 9). The possibility exists that the chemicals undergo catabolism in the lower GI tract by microflora (9) as suggested by many aromatic acids derived from flavonoids found in urine of treated animals (9). Our examination of flavonoid- and catechin-derived catabolic acids uncovered their capacity to undergo glucuronidation. Unexpectedly UGT1A3, detectable in GI tissue by reverse transcription-PCR (23) and at moderate levels in liver by Northern blot analysis,2 showed significant glucuronidation of simple aromatic acids, such as pyruvic, propionic, isovaleric, cinnamic (caffeic and ferulic), and benzoic (vanillic) acids, as well as a benzaldehyde-containing metabolite of flavonoids produced by microflora in the lower gut (10). The two benzaldehydes were metabolized at least an order of magnitude better by UGT1A10, -1A8, or -1A7 than by UGT1A3. Generally the evidence shows that acid-containing flavonoid metabolites are most effectively metabolized by UGT1A10 and -1A3.

Additionally we compared the more complex carboxyl-containing therapeutic NSAIDs with the simple plant-derived acids. While UGT1A10 was superior to the other isozymes in converting NSAIDs, as demonstrated by mefenamic acid and diflunisal (Table III), UGT1A8, -1A7, and -1A3 also avidly transformed mefenamic acid. Diclofenac was preferred by UGT1A3. UGT1A10 and -1A7 transformed the related acidic structure furosemide. In summary, UGT1A10 appears to be the primary isozyme for converting both simple and polycyclic aromatic acids. Despite the essentially unique capacity of UGT1A1 to metabolize the endogenous acid bilirubin (15), it was completely ineffective with exogenous acids in this study. Although catalysis by UGT1A3 did not reach the level observed for UGT1A7, -1A8 and -1A10, it is possible that over time its conversions under in vivo conditions have an impact.

Because biphenyl acetic acid, mefenamic acid, flurbiprofen, fenoprofen, ibuprofen, and indomethacin and other test chemicals in this study have acidic groups but lack a hydroxyl group for glucuronide formation and were exceptional substrates for UGT1A10, the results demonstrate that UGT1A10 is likely the major catalyst for forming carboxyl-linked glucuronides and not UGT1A3. Also the high activity with methylsalicylate and poor activity with salicylate indicates the absence of intramolecular hydrogen bonding between hydroxyl and carboxyl substituent groups has a highly positive effect on metabolism by UGT1A10. The inability to form intramolecular hydrogen bonding in the highly effective substrates, aldehyde-containing vanillin and o-vanillin compared with ferulic and vanillic acid (Table II), is further evidence such bonding has a negative impact on conjugation (29).

Labeling of UGT1A10 expressed in COS-1 cells with [33P]orthophosphate, which was not detected with the T73A/T202A/S432G-1A10 triple PKC mutant, confirms that the isozyme undergoes phosphorylation most likely by PKC. Furthermore the progressive decrease in labeling of single and double mutants suggests each predicted PKC site is phosphorylated (Fig. 5). Because the S432G mutant exhibited increased and apparently wild-type activity and T73A and T202A mutants were completely inactive, one would expect S432G/T73A and S432G/T202A double mutants to be null. It is interesting, however, that the two double mutants show additive activity, indicating that S432G is different and not the equivalent of wild type. Furthermore additive activity suggests phosphate groups are utilized in a hierarchical and/or combinatorial manner in controlling activity (21). Also the complete loss of activity of both T73A and T202A demonstrates that phosphorylation is required and that both threonines are critical for activity (Figs. 5 and 6). PKC site mutants in the amino terminus of UGT1A10 and UGT1A1 proved to have null activity, whereas mutants at the site located in the most carboxyl position (Ser-432/Ser-435) caused either an increase (this study) or decrease in activity (21). One can conclude that phosphorylation at specific sites (Thr-73 and Thr-202) is critical for activity; structural studies will be required to establish site-specific effects. Furthermore the fact that cultures of COS-1 cells transfected with the wild type or the active S432G mutant, but not the inactive mutants, glucuronidated 17{beta}-[14C]estradiol is evidence that phosphorylation of UGT controls in vivo glucuronidation.

While a natural polymorphism, T202I in UGT1A10, has been reported (30) that exhibited partial activity for 17{beta}-estradiol and not null activity as for our T202A mutant, it is not known what role phosphothreonine 202 plays in enzyme activity and how isoleucine 202 is able to support partial glucuronidation of 17{beta}-estradiol. Structural studies of a UGT or UGT1A10 are required to understand the role phosphothreonine/serine plays in catalysis. Partial activity by T202I-UGT1A10 is similar to our observation that T73A-UGT1A9 has 20% activity with propofol, whereas T202A or T202G and T73G mutants are null. Also our test with 17{beta}-estradiol and S432G-versus S432A-1A10 mutants showed no detectable difference.3

This study and our earlier results (12) support the conclusion that UGT1A isozymes have overlapping activities that collectively expand detoxification by having and using flexible properties of different pH optima, which depend upon acceptor substrate. Since mucosal tissues of the GI tract that harbor different UGT isozymes face wide variations in chemical structures and chemical gradients in the GI tract, it is likely that the differential properties represent adaptations that enhance the detoxification process. As UGT1A10 is located primarily in GI mucosa from duodenum through colon (12) positioned to restrict GI absorption of damaging chemicals, this study represents the first demonstration that UGT1A10 is pivotal for detoxifying estrogens/phytoestrogens and aromatic acids with evident dependence upon phosphorylation. Why this critical function of limiting chemical absorption of potentially damaging dietary constituents toward the reproductive system and other endogenous targets is dependent upon phosphorylation warrants further investigations. Unfortunately many medicinal drugs, such as NSAIDs, also encounter premature conversion by these isozymes, compromising therapeutic efficacy. The requirement for phosphorylation allows a target for down-regulation to enhance absorption of orally administered therapeutic drugs, which undergo significant glucuronidation, as recently suggested via transient inhibition of UGT activity by curcumin treatment (12).


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: NICHD, National Institutes of Health, Bldg. 10, Rm. 9S-241, Bethesda, MD 20892. Tel.: 301-496-6091; Fax: 301-480-8042; E-mail: owensi{at}mail.nih.gov.

1 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; UGT, UDP-glucuronosyltransferase; GI, gastrointestinal; PKC, protein kinase C; 17{beta}-est, 17{beta}-estradiol; 17{beta}-est-gluc, 17{beta}-estradiol glucuronide; ECG, epigallocatechin 3-O-gallate. Back

2 N. K. Basu, S. Kubota, M. Ciotti, and I. S. Owens, unpublished data. Back

3 N. K. Basu, M. Koarova, T. Saha, J. Rivera, and I. S. Owens, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jayanta Mukhopadhyay for critical comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rickard, D. J., Monroe, D. G., Ruesink, T. J., Khosla, S., Riggs, B. L., and Spelsberg, T. C. (2003) J. Cell. Biochem. 89, 633–646[CrossRef][Medline] [Order article via Infotrieve]
  2. Seawright, A. A. (1995) Nat. Toxins 3, 227–232[Medline] [Order article via Infotrieve]
  3. Lamartiniere, C. A., Cotroneo, M. S., Fritz, W. A., Wang, J., Mentor-Marcel, R., and Elgavish, A. (2002) J. Nutr. 132, 552–558
  4. Fitzpatrick, L. A. (1999) Mayo Clin. Proc. 74, 601–607[Abstract]
  5. Frigo, D. E., Duong, B. N. Melnik, L. I., Schief, L. S., Collins-Burow, B. M., Pace, D. K., McLachlan, J. A., and Burow, M. E. (2002) J. Nutr. 132, 1848–1853[Abstract/Free Full Text]
  6. Liew, R., Williams, J. K., Collins, P., and MacLeod, K. T. (2003) J. Pharmacol. Exp. Ther. 304, 985–993[Abstract/Free Full Text]
  7. Adebe, W. (2002) J. Clin. Pharm. Ther. 27, 391–401[CrossRef][Medline] [Order article via Infotrieve]
  8. Gugler, R., Leschik, M., and Dengler, H. J. (1975) Eur. J. Clin. Pharmacol. 9, 229–234[CrossRef][Medline] [Order article via Infotrieve]
  9. Kühnau, J. (1976) World Rev. Nutr. Diet. 24, 117–191[Medline] [Order article via Infotrieve]
  10. Brown, J. P. (1980) Mutat. Res. 75, 243–277[CrossRef][Medline] [Order article via Infotrieve]
  11. Gong, Q.-H., Cho, J. W., Huang, T., Potter, C., Gholami, N., Basu, N. K., Kubota, S., Carvalho, S., Pennington, M. W., Owens, I. S., and Popescu, N. (2001) Pharmacogenetics 11, 357–368[CrossRef][Medline] [Order article via Infotrieve]
  12. Basu, N. K., Ciotti, M., Hwang, M. S., Kole, L., Mitra, P. S., Cho, J. W., and Owens, I. S. (2004) J. Biol. Chem. 279, 1429–1441[Abstract/Free Full Text]
  13. de Morais, S. M., and Wells, P. G. (1989) Hepatology 10, 163–167[Medline] [Order article via Infotrieve]
  14. Bock, K. W., Bock-Hennig, B. S., Münzel, P. A., Brandenburg, J. O., Köhle, C. T., Soars, M. G., Riley, R. J., Burchell, B., von Richter, O., Eichelbaum, M. F., Swedmark, S., and Orzechowski, A. (2002) Biochem. Pharmacol. 63, 1683–1690[CrossRef][Medline] [Order article via Infotrieve]
  15. Ritter, J. K., Crawford, J. M., and Owens, I. S. (1991) J. Biol. Chem. 266, 1043–1047[Abstract/Free Full Text]
  16. Ciotti, M., Basu, N., Brangi, M., and Owens, I. S. (1999) Biochem. Biophys. Res. Commun. 260, 199–202[CrossRef][Medline] [Order article via Infotrieve]
  17. Ritter, J. K., Yeatman, M. T., Kaiser, C., Gridelli, B., and Owens, I. S. (1993) J. Biol. Chem. 268, 23573–23579[Abstract/Free Full Text]
  18. Ciotti, M., and Owens, I. S. (1996) Biochemistry 35, 10119–10124[CrossRef][Medline] [Order article via Infotrieve]
  19. Ciotti, M., Yeatman, M. T., Sokol, R. J., and Owens, I. S. (1995) J. Biol. Chem. 270, 3284–3291[Abstract/Free Full Text]
  20. Akhand, A. A., Pu, M., Senga, T., Kato, M., Suzuki, H., Miyata, T., Hamaguchi, M., and Nakashima, I. (1999) J. Biol. Chem. 274, 25821–25826[Abstract/Free Full Text]
  21. Basu, N. K., Kole, L., and Owens, I. S. (2003) Biochem. Biophys. Res. Commun. 303, 98–104[CrossRef][Medline] [Order article via Infotrieve]
  22. Liew, R., MacLeod, K. T., and Collins, P. (2003) FASEB J. 17, 1307–1309[Abstract/Free Full Text]
  23. Strassburg, C. P., Kneip, S., Topp, J., Obermayer-Straub, P., Barut, A., Tukey, R. H., and Manns, M. P. (2000) J. Biol. Chem. 275, 36164–36171[Abstract/Free Full Text]
  24. Gall, W. E., Zawanda, G., Mojarrabi, B., Tephly, T. R., Green, M. D., Coffman, B. L., Mackenzie, P. I., and Radominska-Pandya, A. (1999) J. Steroid Biochem. Mol. Biol. 70, 101–108[CrossRef][Medline] [Order article via Infotrieve]
  25. Williams, J. A., Ring, B. J., Cantrell, V. E., Campanale, K., Jones, D. R., Hall, S. D., and Wrighton, S. A. (2002) Drug Metab. Dispos. 30, 1266–1273[Abstract/Free Full Text]
  26. Ritter, J. K., Sheen, Y. Y., and Owens, I. S. (1990) J. Biol. Chem. 265, 7900–7906[Abstract/Free Full Text]
  27. Ritter, J. K., Chen, F., Sheen, Y. Y., Lubet, R. A., and Owens, I. S. (1992) Biochemistry 31, 3409–4314[CrossRef][Medline] [Order article via Infotrieve]
  28. Cheng, Z., Rios, G. R., King, C. D., Green, M. D., Mojarrabi, B., Mackenzie, P. I., and Tephly, T. R. (1998) Toxicol. Sci. 45, 52–57[Abstract/Free Full Text]
  29. Coitti, M., Marrone, A., Potter, C., and Owens, I. S. (1997) Pharmacogenetics 7, 485–495[Medline] [Order article via Infotrieve]
  30. Juno, H., Saeki, M., Tanaka-Kagawa, T., Hanioka, N., Saito, Y, Ozawa, S., Ando, M., Shirao, K., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., and Sawada, J. (2003) Drug Metab. Dispos. 31, 528–532[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
N. K. Basu, L. Kole, M. Basu, K. Chakraborty, P. S. Mitra, and I. S. Owens
The Major Chemical-detoxifying System of UDP-glucuronosyltransferases Requires Regulated Phosphorylation Supported by Protein Kinase C
J. Biol. Chem., August 22, 2008; 283(34): 23048 - 23061.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
K. K. Miles, F. K. Kessler, P. C. Smith, and J. K. Ritter
Characterization of Rat Intestinal Microsomal UDP-Glucuronosyltransferase Activity toward Mycophenolic Acid
Drug Metab. Dispos., September 1, 2006; 34(9): 1632 - 1639.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
R. W. Dellinger, J.-L. Fang, G. Chen, R. Weinberg, and P. Lazarus
IMPORTANCE OF UDP-GLUCURONOSYLTRANSFERASE 1A10 (UGT1A10) IN THE DETOXIFICATION OF POLYCYCLIC AROMATIC HYDROCARBONS: DECREASED GLUCURONIDATIVE ACTIVITY OF THE UGT1A10139LYS ISOFORM
Drug Metab. Dispos., June 1, 2006; 34(6): 943 - 949.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
L. M. Butler, Y. Duguay, R. C. Millikan, R. Sinha, J.-F. Gagne, R. S. Sandler, and C. Guillemette
Joint Effects between UDP-Glucuronosyltransferase 1A7 Genotype and Dietary Carcinogen Exposure on Risk of Colon Cancer
Cancer Epidemiol. Biomarkers Prev., July 1, 2005; 14(7): 1626 - 1632.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
G. E. Kuehl, J. W. Lampe, J. D. Potter, and J. Bigler
GLUCURONIDATION OF NONSTEROIDAL ANTI-INFLAMMATORY DRUGS: IDENTIFYING THE ENZYMES RESPONSIBLE IN HUMAN LIVER MICROSOMES
Drug Metab. Dispos., July 1, 2005; 33(7): 1027 - 1035.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. K. Basu, M. Kovarova, A. Garza, S. Kubota, T. Saha, P. S. Mitra, R. Banerjee, J. Rivera, and I. S. Owens
Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity
PNAS, May 3, 2005; 102(18): 6285 - 6290.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
279/27/28320    most recent
M401396200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Basu, N. K.
Right arrow Articles by Owens, I. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Basu, N. K.
Right arrow Articles by Owens, I. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement