Cancer Research The Future of Cancer Research: Science and Patient Impact
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thibaudeau, J.
Right arrow Articles by Guillemette, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thibaudeau, J.
Right arrow Articles by Guillemette, C.
[Cancer Research 66, 125-133, January 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Characterization of Common UGT1A8, UGT1A9, and UGT2B7 Variants with Different Capacities to Inactivate Mutagenic 4-Hydroxylated Metabolites of Estradiol and Estrone

Jean Thibaudeau1, Johanie Lépine1, Jelena Tojcic1, Yannick Duguay1, Georges Pelletier2, Marie Plante3, Jacques Brisson4, Bernard Têtu5, Simon Jacob6, Louis Perusse7, Alain Bélanger2 and Chantal Guillemette1

1 Canada Research Chair in Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUQ Research Center, Faculty of Pharmacy, 2 Oncology and Molecular Endocrinology Research Center, CHUQ Research Center, Faculty of Medicine, 3 Gynecologic Oncology Service, Hôtel-Dieu de Québec, Faculty of Medicine, 4 Population Health Research Unit, Hôpital Saint-Sacrement du CHA de Québec and Faculty of Medicine, 5 Department of Pathology, Hôtel-Dieu de Québec, Faculty of Medicine, 6 Anatomic-Pathological and Cytological Laboratory, Hôpital Saint-Sacrement, Faculty of Medicine and 7 Division of Kinesiology, Department of Social and Preventive Medicine, Faculty of Medicine, Laval University, Quebec, Canada

Requests for reprints: Chantal Guillemette, Canada Research Chair in Pharmacogenomics, Pharmacogenomics Laboratory, CHUQ Research Center, T3-67, 2705 Boulevard Laurier, Quebec, Canada G1V 4G2. Phone: 418-654-2296; Fax: 418-654-2761; E-mail: chantal.guillemette{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The oxidative metabolism of estrone (E1) and estradiol (E2) to form carcinogenic 4-hydroxy-catecholestrogens (4-OHCE) is associated with uterine and breast carcinogenesis. In this study, we conducted functional analyses of genetic variants in the UDP-glucuronosyltransferase UGT1A8, UGT1A9, and UGT2B7 enzymes primarily involved in the inactivation of 4-OHCEs. Compared with UGT2B7*2 (H268Y), UGT2B7*1 exhibited a 2-fold lower efficiency (intrinsic clearance) at conjugating 4-hydroxyestrone and 4-hydroxyestradiol at positions 3 and 4 caused by altered capacities (Vmax) and affinities (Km). The –79 G>A promoter variation, characterizing the UGT2B7*2g haplotype, leads to a 50% reduction of transcription (P < 0.001) in human endometrial carcinoma-1B cells. Furthermore, a >12-fold decreased intrinsic clearance of the *1 proteins was induced by selected amino acid substitutions in UGT1A8 (*3 C277Y) and UGT1A9 (*3 M33T). Frequencies of the low-activity alleles in Caucasians were 45% for UGT2B7*1, 5% for the –79A promoter variant, 1.2% for UGT1A8*3, and 2.2% for UGT1A9*3. Supporting a protective role in two organs sensitive to 4-OHCE–induced damages, the expression of UGT enzymes was shown by immunohistochemistry in normal breast and endometrial tissues and confirmed by Western blotting in a subset of samples. Altogether, findings suggest that specific polymorphisms in UGT genes may modulate the exposure to carcinogenic metabolites of E2 and potentially lead to an altered risk of breast and endometrial cancers in women carrying the variant alleles. (Cancer Res 2006; 66(1): 125-33)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast and endometrial cancers have been associated with higher exposure to endogenous or exogenous estrogens (1, 2). In addition to estrogens from the circulation, the uterine and mammary gland tissues possess the required enzymes for estrogen synthesis from circulating C19 steroid precursors. Besides, the contribution of ovarian and in situ synthesis varies during aging and the later source of estrogens becomes more significant at menopause. It is also quite evident that in situ estrogen concentrations are also dependent on local metabolism. Transformation of estrogens to hydroxylated metabolites, i.e., catecholestrogens, has recently received attention because of their association with breast and uterine tumorigenesis (3–5).

Estrogens are substrates of cytochromes P450 CYP1A1/1A2 and CYP1B1, that generates predominantly 2- and 4-hydroxy-catecholestrogens (2- and 4-OHCE) derivatives, respectively (6). These metabolic pathways lead to the formation of metabolites with distinctive biological activities compared with parent estradiol (E2) and estrone (E1) in a number of tissues including uterus and mammary gland (7, 8). Both 2-OHCEs and 4-OHCEs may be subsequently converted to corresponding semiquinone and quinone forms and then undergo a CYP450/peroxidase catalyzed free radicals generating metabolic redox cycling. These quinones and free radicals can mediate DNA adducts and cellular oxidation, leading to possible mutagenesis (9–12). Quinone intermediates of 4-OHCEs are particularly carcinogenic because they form unstable DNA adducts and induce tumor formation in animal models (13, 14). In opposition, 2-OHCEs generate stable DNA adducts and they possess antiproliferative and antiangiogenic properties once transformed to methoxy derivatives, mostly 2-methoxyestradiol, by the action of catechol-O-methyltransferase (15).

One of the major pathways of conjugation for E2 and its oxidized and methoxylated metabolites is through UDP-glucuronosyltransferases (UGT), which catalyze the covalent addition of glucuronic acid (UDPGA). In our recent study, we showed that glucuronidation of E2 and E1, as well as their catecholestrogen and methoxyestrogen metabolites, is mostly catalyzed by six UGTs, i.e., UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 (16). The study of Lepine et al. further showed that among these, UGT1A8, 1A9, and 2B7 are highly efficient toward the carcinogenic 4-OHCEs (16).

Limited attention was given to the expression of estrogen-conjugating UGT enzymes in steroid target tissues, but the available data indicate their presence in breast and uterine tissues (16–18). In turn, the significant amounts of estrogen-glucuronides in the breast cyst fluid supports a local inactivation of estrogens by UGT enzymes (19, 20). In support of a role of the glucuronidation pathway in the metabolism of estrogens in target cells is the consistent association of UGT polymorphisms with altered circulating hormones and modified risk of endometrial, breast, and ovarian cancers (18, 21–24). Altogether, these results suggest that the metabolic pathway mediated by UGT enzymes is implicated in estrogen inactivation and that the activity of these enzymes may play a critical role in the modulation of in situ estrogen exposure.

Given the central role of UGT1A8, UGT1A9, and UGT2B7 in the metabolism of 4-OHCEs, we studied the influence of polymorphisms in these genes to identify those which modify enzyme catalytic activity and the level of expression. Four nonsynonymous polymorphisms of UGT1A8 and UGT1A9 exons 1 have been associated with changes in phenotypes (Fig. 1). Three alleles of UGT1A9, *1, *2 (C3Y), and *3 (M33T), and three alleles of UGT1A8, *1, *2 (A173G), and *3 (C277Y), are present in Caucasian and African-American populations (25, 26). For UGT2B7, only one nonsynonymous polymorphism was found, UGT2B7*2 (H268Y; refs. 27, 28). Functional studies revealed that some forms of UGT proteins present modified activity against a variety of xenobiotics but their effects on estrogen glucuronidation has never been investigated (25–29). In addition, a functional variation in the UGT2B7 promoter is located at position –79 G>A and is in linkage disequilibrium with the codon 268 variation and is referred as haplotype III, UGT2B7*2g (ref. 30; Fig. 1). This promoter polymorphism was associated with a significant decrease of transcriptional activity in hepatoma and colon cells, two sites of expression of the UGT2B7 enzyme, whereas its functional effect in estrogen-dependent cells remains to be established (30). We initially investigated the functional effect of common polymorphisms by in vitro enzymatic assays with 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) using individual overexpressed UGT1A8, UGT1A9, and UGT2B7 allozymes to determine the maximal velocity (Vmax), the affinity (Km), and catalytic efficiencies (CLint). We also assessed the transcriptional activity of the UGT2B7 –79G>A promoter polymorphism in human endometrial carcinoma (HEC-1B) cells using pGL3 reporter gene constructs. Finally, to gain information into the protective role of UGT against carcinogenic E2 metabolites, we determined the pattern of expression of UGT1A8, UGT1A9, and UGT2B7 enzymes in healthy endometrium and breast samples from postmenopausal women.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of the UGT1A (A) and UGT2B7 (B) genes indicating the locations of the single amino acid substitutions studied. Frequency of each allele is indicated. p, pseudogenes.

 
The main findings of this study are that several forms of UGT enzymes are associated with reduced expression or decreased inactivation of 4-OHE2 and 4-OHE1. In addition, expression analyses in the uterus and breast, two target tissues of 4-OHCE-induced damages, suggest a potential alteration of the lifetime exposure to these genotoxic metabolites in women carrying low-activity alleles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. 4-OHE1 and 4-OHE2 were purchased from Steraloids (Newport, RI). The glucuronides derivatives of 4-OHE1 and 4-OHE2 were obtained as previously described (16). High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, 1-chlorobutane, and iso-amyl-alcohol were purchased from VWR Canlab (Montréal, Quebec, Canada). Ammonium hydroxide was obtained from Fisher Scientific (Nepean, Ontario, Canada). All chemicals were of the highest grade available.

DNA samples and tissue procurement. DNA samples from 258 unrelated Caucasian subjects were obtained from the Quebec Family Study (31). These samples had been anonymized prior to their receipt in our laboratory. All subjects provided written consent for the use of their DNA for experimental purposes, and the present study was reviewed and approved by the Institutional Review Boards (CHUL Research Center and Laval University). Nonmalignant uterine and breast tissues were obtained from postmenopausal women who had not received hormone replacement therapy for at least 3 weeks and who had no menstrual bleeding for at least 1 year. All subjects provided written consent for use of their specimens, and the present study was reviewed and approved by Institutional Review Boards. Fresh tissue from patients were collected by pathologists and deposited in liquid nitrogen within 30 minutes of surgery pending transfer to a freezer at –80°C.

Genotyping and haplotype analyses of UGT1A8. PCR was used to amplify the first exon of the UGT1A8 gene, and primers were designed to amplify overlapping fragments, as previously described for UGT1A9 (26). The primers used for UGT1A8 were: #174F-175R, 5'-ctggaccgggaattcatgga and 5'-gtggctgtagagatcatatgct; #1338F-1322R, 5'-ttcgccaggggaatag and 5'-atttgctctagggggtc. Amplicons were sequenced with an ABI 3700 automated sequencer using Big Dye (Perkin-Elmer, Boston, MA) dye primer chemistry. Sequences were analyzed with Staden preGap4 and Gap4 programs. Haplotypes for UGT1A8 were determined using Phase v2.1 program (26, 32). The linkage between the different polymorphisms was determined with the LDplotter tool program.8

Protein expression analysis assessed by Western blot analysis. An antibody (#519) was raised against amino acids 3 to 118 of UGT1A9 and its characterization revealed that it also recognized UGT1A8 (95% of homology). The specificity of this antibody (1:2,000) was studied by Western blot experiments as previously described (Fig. 2A; ref. 16). The characterization of the polyclonal anti-UGT2B7 (#1809) has already been described (ref. 16; Fig. 2B). UGT1A8, UGT1A9, and UGT2B7 protein expression in five healthy endometrium samples were assessed by Western blot analysis. Microsomes were extracted as follows: 200 mg of frozen tissue were first homogenized using polytron in microsome buffer [20 mmol/L KH2PO4, 20 mmol/L K2HPO4, 20% glycerol, 1 mmol/L DTT (pH 7.0)], sonicated, and then centrifuged at 14,000 x g at 4°C for 20 minutes. The supernatant was centrifuged at 125,000 x g for 60 minutes at 4°C, and finally, the pellet was homogenized in the same microsome buffer. The quantity of protein was determined using the Bradford method (Bio-Rad, Mississauga, Canada) using bovine serum albumin as standard. Ten micrograms of the microsomal proteins were loaded on a 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose (Bio-Rad). The expressions of UGT1A8 and UGT1A9 were monitored with the anti-UGT1A8/1A9 (#519) at a dilution of 1:2,000, whereas UGT2B7 was probed with antibody #1809 at a dilution of 1:2,000 (16). Bands were revealed using enhanced chemiluminescence (Perkin-Elmer, Boston, MA).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 2. Western blot analysis of UGT1A8, UGT1A9, and UGT2B7 expression in healthy endometrium. To determine the specificity of our polyclonal antibodies, microsomal proteins (10 µg) of HK293 cells stably expressing each UGT were separated on a 10% SDS-PAGE gel. Both antibodies were used at a dilution of 1:2,000. Human liver (HL) was used as positive control. A, the anti-UGT1A8/1A9 (#519) binds only to UGT1A8, UGT1A9, and to HL through UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, and 1A10. B, the anti-UGT2B7 (#1809) binds only to UGT2B7 and to HL proteins toward UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28 proteins. C, microsomal fractions (10 µg) of five endometrium samples from postmenopausal women were tested to evaluate the expression of UGT1A8/1A9 and UGT2B7 (#519 and #1809 antibodies, respectively, at a dilution of 1:2,000).

 
Protein expression analysis assessed by immunohistochemistry. Immunohistochemistry analyses were done as previously described (16). Briefly, serial cuts of nonmalignant human uterine and breast tissues from postmenopausal women (n = 5 for each tissue) were incubated with human UGT1A8/1A9 (#519) and UGT2B7 (#1809) antisera diluted 1:1,000 and 1:250, respectively. Sections were washed and incubated with peroxidase-conjugated goat anti-rabbit immunoglobulins (DakoCytomation, Mississauga, Ontario, Canada) diluted 1:200. Endogenous peroxidase activity was eliminated and peroxidase intensity was controlled under the microscope. Sections were counterstained with hematoxylin and the control experiments were done by substituting preimmune rabbit serum.

Enzymatic assays. Prior to enzymatic assays, UGT proteins were stably expressed in HEK-293 cell systems and microsomal fractions were prepared as previously described (16). For a more accurate assessment of the quantitative differences in glucuronidation activity for variant allozymes, HEK293-derived cell lines were characterized for UGT protein expression by Western blot analysis (data not shown) as previously described (16). To establish the functional effect of nonsynonymous polymorphisms of UGT1A8, UGT1A9, and UGT2B7 on 4-OHE1 and 4-OHE2 glucuronidation, we conducted enzymatic assays with microsomal proteins obtained from HEK-293 cells stably overexpressing each variant protein. Enzymatic assays were done with 40 to 60 µg total microsomal proteins in 100 µL reaction volume containing; 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, 5 µg/mL pepstatin, 0.5 µg/mL leupeptin, 10 µg/mL phosphatidylcholine, and 1 mmol/L UDPGA. Reactions were started by adding varying concentrations of 4-OHE1 and 4-OHE2 ranging from 1 to 200 µmol/L and incubating at 37°C. After 3 hours, the assays were stopped by adding 100 µL of ice-cold methanol and than centrifuged at 14,000 x g for 10 minutes in order to remove proteins before mass spectrometry analysis. Relative glucuronidation activities are expressed as pmol/min/UGT level, which was obtained after dividing the absolute activity of UGT (pmol/min/mg) by the protein expression level obtained by Western blot analyses.

Mass spectrometry analysis. Glucuronide formation was assessed by mass spectrometry analyses as previously described (16). Briefly, incubation medium was diluted with 0.1 mL methanol/water (50:50; vol/vol), vortexed, and then transferred into a conical vial for injection into the mass spectrometer. The HPLC and tandem mass spectrometry system consisted of a mass spectrometer (model API 3000; Perkin-Elmer Sciex, Thornhill, Canada) equipped with an electrospray ionization source in the negative ion mode and a HPLC pump plus autosampler (model 2690, Waters, Milford, MA). The mass spectrometer was operated in the multiple reaction monitoring mode.

Enzyme kinetic analysis. Determination of the kinetic variables was done using Sigmaplot 8.02a with Enzyme Kinetic 1.1 (Systat Software, Point Richmond, CA). Best kinetic enzyme model was chosen after visual inspection of the fitted curve, the Eadie-Hofstee plot and the Akaike Information Criterion values. Coherent models were the Michaelis-Menten model [V = (Vmax x S) / (Km + S)], the substrate activation model [Hill equation, V = (Vmax x Sn) / (Kmn + Sn)] and the uncompetitive substrate inhibition model (V = [Vmax x S] / [Km + S x (1 + S/Ks)]), where Vmax is the maximal velocity, Km is the substrate concentration at half-maximal velocity, "n" is the degree of curve sigmoidicity, and Ks is an inhibition constant. Based on these models, the intrinsic clearance (CLint) estimation values were calculated using the following equations: Michealis-Menten and substrate inhibition, CLint = Vmax/Km; substrate activation, CLmax = (Vmax/Km) x ([n – 1] / [n x (n – 1)1/n]) (33, 34). Statistical analysis was done with a two-tailed Student's t test.

Cell culture and transfection of UGT2B7 promoters. HEC-1B cells were obtained from American Type Culture Collection (Manassas, VA) and cultured as recommended by the supplier in a humidified 5% carbon dioxide incubator at 37°C. Cells were seeded 24 hours before transfection assays in a 24-well plate with 60,000 cells per well (corresponding to 50% of confluence). Plasmids were obtained as previously described (30). Transfections of 1,000 ng plasmid constructs with pRLnull as an internal transfection efficiency control were carried out in MEM using 4 µL of ExGen 500 transfection reagent (MBI Fermentas, Burlington, Ontario, Canada). Supplemented serum was added after 3 hours. Cells were harvested 24 hours after transfection and promoter activity was monitored using the Dual-Luciferase Reporter Assay System as stated by the manufacturer (Promega, Madison, WI). Luciferase activity was measured in a 96-well plate on a LB96V microplate luminometer (EG&G Berthold, Bad Wildbad, Germany) with 30 µL of cell lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enzymatic activities of UGT allozymes. Estrogens are potentially conjugated at the 2-, 3-, 4- or 17-hydroxyl positions. Our previous work showed that glucuronidation occurs predominantly at the 3- and 4-hydroxyl positions of 4-OHCEs (16). Therefore, both positions were investigated.

UGT1A8. The codon 277 cysteine for a tyrosine *3 variation of UGT1A8 had a dramatic effect on the catalytic activity of the enzyme towards the 4-hydroxylated estrogens. Compared with the UGT1A8*1 enzyme, the velocity of conjugation of 4-OHE1 was reduced at position 3 by 30-fold (8 versus 246 pmol/min/UGT level; P < 0.001; Fig. 3) whereas a 13-fold decreased velocity was also observed for the conjugation of 4-OHE1 at position 4 (158 versus 2,113 pmol/min/UGT level; P < 0.001). Despite better affinities (Km) of the UGT1A8*3 allozyme for the conjugation of 4-OHE1 at both positions 3 and 4 (29 and 35 versus 290 and 162 µmol/L, respectively), this variant had an intrinsic clearance diminished by at least 2- to 3-fold (P < 0.05) compared with the UGT1A8*1 protein. For the conjugation of 4-OHE2, the C277Y *3 variation also had a remarkable effect on the velocity of the enzyme for the conjugation of the 3-hydroxyl (4 versus 163 pmol/min/UGT level; P < 0.01) and the 4-hydroxyl (265 versus 7,730 pmol/min/UGT level; P < 0.01; Fig. 3). Similarly to 4-OHE1, the Km values were also enhanced for the UGT1A8*3 allozyme compared with the UGT1A8*1 at both positions 3 and 4 (20 and 63 µmol/L versus 103 and 133 µmol/L, respectively). As a result, the CLint values were significantly reduced by 8- to 13-fold (3-hydroxyl, 0.2 versus 1.6 µL/min/mg; P < 0.01; 4-hydroxyl, 4.2 versus 56.5 µL/min/mg; P < 0.05). In contrast, the change of UGT1A8 codon 173 alanine to a glycine (UGT1A8*2) had no influence on enzymatic activity using 4-OHE1 or 4-OHE2 (Table 1).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Kinetic profiles for the glucuronidation of 4-OHE1 (A and C) and 4-OHE2 (B and D) at position 4 by UGT1A8 (A and B) and UGT1A9 (C and D). Microsomes, produced from HEK-293 cells as described in Materials and Methods, were incubated for 60 minutes at 37°C with concentrations of 4-OHE1 or 4-OHE2 ranging from 1 to 200 µmol/L. Quantification of estrogen-glucuronides was done by HPLC tandem mass spectrometry and absolute glucuronidation activities were divided by UGT protein expression assessed by Western blot analysis. Points, mean of two independent experiments done in duplicate; bars, ± SEM.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Kinetic variables for the glucuronidation of 4-OHE1 and 4-OHE2 by UGT1A8, UGT1A9, and UGT2B7

 
UGT1A9. The codon 3 cysteine to a tyrosine change, characterizing the *2 allele of UGT1A9, had no significant effect on enzyme kinetics compared with UGT1A9*1, both for the glucuronidation at positions 3 and 4 of 4-OHE1 and 4-OHE2 (Fig. 3). However, compared with the UGT1A9*1 protein, the *3 allozyme with a threonine instead of a methionine at codon 33, had a Vmax almost 4-fold lower for the conjugation of 4-OHE1 at position 3 (18 versus 70 pmol/min/UGT level; P < 0.05) and 6.5-fold lower for the conjugation of 4-OHE1 at position 4 (63 versus 412 pmol/min/UGT level; P < 0.05). The Km values for both UGT1A9*1 and *3 proteins were similar, except for the conjugation at position 3 of 4-OHE1 that was increased by 3.5-fold (392 versus 111 µmol/L; P < 0.05). Intrinsic clearances were significantly decreased by >9- and >12-fold (P < 0.05) for the glucuronidation of 4-OHE1 at positions 3 and 4 by the *3 protein, respectively. In parallel, the effect of the UGT1A9*3 (M33T) variation on the glucuronidation of 4-OHE2 was exclusively within the capacity of the enzyme. Vmax values for the conjugation at positions 3 and 4 were significantly reduced by the presence of a threonine at codon 33 (8 and 422 versus 32 and 3,135 pmol/min/UGT level, respectively; P < 0.01). This leads to a significant decrease of intrinsic clearances by 4-fold at position 3 (0.1 versus 0.4 µL/min/mg; P <0.01) and by almost 6-fold at position 4 (8.2 versus 47.4 µL/min/mg; P < 0.01; Table 1).

UGT2B7. The UGT2B7*1 enzyme with a histidine at codon 268 had almost a 3-fold lower intrinsic clearance for 4-OHE1 at position 4 (163.6 µL/min/mg; P < 0.01) compared with the UGT2B7*2 allozyme (475.0 µL/min/mg), characterized by a tyrosine at codon 268 (Fig. 4). The altered clearance is the result of a change in the affinity of the variant *2 enzyme, which was decreased by almost 2-fold for the glucuronidation at position 4 (25 versus 45 µmol/L; P < 0.05). The velocity was also increased by a factor of 1.6 compared with UGT2B7*1 (11,637 versus 7,317 pmol/min/UGT level). Similarly, UGT2B7*2 Y268 is more efficient at conjugating 4-OHE2 at both positions compared with the UGT2B7*1 allozyme (P < 0.05; Fig. 4). In this case, a higher Vmax for the conjugation rate of 4-OHE2 at both positions 3 and 4 for UGT2B7*2 Y268 is observed compared with UGT2B7*1 H268 (94 and 4,398 versus 58 and 2,426 pmol/min/UGT level, respectively; Table 1).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Kinetic profiles of UGT2B7 and transcriptional activity of UGT2B7 promoter. A and B, kinetic profiles for the glucuronidation of 4-OHE1 (A) and 4-OHE2 (B) at position 4 by UGT2B7. Microsomes, produced from HEK-293 cells as described in Materials and Methods, were incubated for 60 minutes at 37°C with concentrations of 4-OHE1 or 4-OHE2 ranging from 1 to 200 µmol/L. Quantification of estrogen-glucuronides was done by HPLC tandem mass spectrometry and absolute glucuronidation activities were divided by UGT protein expression assessed by Western blot analysis. Points, mean of two independent experiments done in duplicate; bars, ± SE. C, transcriptional activities of UGT2B7 promoter constructs in HEC-1B endometrial cells. Promoter constructs of UGT2B7 covering the –1,362/34 and the –190/34 regions (relative to the transcription starting site) were transfected in HEC-1B endometrial cells and promoter activities were measured by a luciferase reporter plasmid assay. *, P < 0.05; ***, P < 0.001.

 
To assess the effect of the promoter –79 G>A variant transcriptional activity, we did transient cell transfection using luciferase reporter promoter constructs covering the –190/34 and –1,362/34 UGT2B7 promoter regions (positions are relative to the transcriptional start site; ref. 30). HEC-1B cells were used as a cellular model. Transcription levels of the –79A variant plasmids were significantly reduced by 50% (P < 0.001) and by 30% (P < 0.05) using short or long promoter constructs, respectively (Fig. 4).

Linkage disequilibrium in the UGT1 gene. The importance of understanding linkage disequilibrium in candidate loci is becoming increasingly evident, and future epidemiologic studies should consider the haplotype of UGT1A8 and UGT1A9 because of the genomic structure of the UGT1A gene. Indeed, the entire UGT1 family is derived from a single gene locus (UGT1A) which is composed of 17 exons. To synthesize the final protein, 1 of 13 different exon 1 sequences on the locus is associated with four downstream exons common to all UGT1A isoforms (Fig. 1; refs. 35, 36). It is thus likely that a linkage disequilibrium might be present between UGT1A8 and UGT1A9. However, our data obtained in 516 chromosomes indicate a prevalence of 1.2% and 2.2% for UGT1A8*3 and UGT1A9*3 alleles, respectively, and no significant linkage between both loci (R2= 0.0003, D'= 1.00).



View larger version (99K):
[in this window]
[in a new window]
  		Figure 5. Expression of UGT1A8, UGT1A9, and UGT2B7 in healthy endometrium and breast tissues from postmenopausal women by immunohistochemistry. A and C, immunolabeling of breast for UGT1A8/1A9 and UGT2B7 is observed in the cytoplasm of ductal epithelial cells (arrows). B and D, immunolabeling of endometrium for UGT1A8/1A9 and UGT2B7 is observed in the cytoplasm of epithelial cells of endometrial glands (G) and those covering the uterine cavity (C). Some stromal cells are also labelled (arrows). When the nonimmune serum was used, no staining could be detected (data not shown). Original magnification is x300.

 
Expression of UGT1A8, UGT1A9, and UGT2B7 in estrogen-responsive tissues. Immunohistochemistry of UGTs was done in normal breast and endometrial tissues obtained from five postmenopausal women. Representative results are shown in Fig. 5. In breast tissues, immunostaining of UGT1A8/1A9 and UGT2B7 is observed in the cytoplasm of ductal epithelial cells (Fig. 5A-C). For the uterus, immunostaining of UGT1A8/1A9 and UGT2B7 was detected in the cytoplasm of both epithelial cells of endometrial glands and those lining the uterine cavity and some stromal cells were also labeled (Fig. 5B-D). In the myometrium, smooth muscle cells had weak immunolabeling (data not shown). When the nonimmune serum was used, no staining could be detected (data not shown). Western blot analyses using microsomal preparations of endometrial tissues also confirmed the expression of UGT1A8/1A9 and UGT2B7 proteins (Fig. 2C). Because the antibody (#519) used in the immunohistochemistry experiments recognizes both UGT1A8/1A9 proteins, we confirmed mRNA expression of individual genes by specific reverse transcription-PCR in these patients. Both UGT1A8 and UGT1A9 transcripts were detected in samples from postmenopausal women (n = 5) although with variability between samples (data not shown), despite the limited number of samples analyzed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One potential mechanism of estrogen carcinogenesis in breast and endometrial tissues involves the formation of catechol metabolites, especially the 4-oxidized metabolites of E2 and E1. Several authors have reported that these estrogen metabolites induced genotoxicity and participate in tumor initiation (10, 11, 13). The elimination and inactivation of these metabolites has thus far received little attention, and the observation of the high specificity of UGT enzymes for these substrates attracted our interest (16). UGT represents a metabolic pathway that participates in blocking the production of reactive oxygen species induced by the catecholestrogen-redox cycle and the production of DNA adducts by enhancing their elimination and preventing their accumulation in tissues. The present study assessed the influence of common polymorphisms in the glucuronidation pathway for the inactivation of genotoxic endogenous catecholestrogens. We present the evaluation of the kinetic properties of UGT1A8, UGT1A9, and UGT2B7 variants towards 4-OHCE inactivation and the identification of UGT enzymes with low enzymatic activity. In addition, the biological relevance of UGT enzymes is further supported by their expression in the breast and the uterus, two organs sensitive to 4-OHCE-induced damage supporting the protective role of this metabolic pathway.

We previously showed that, out of all known human UGTs, UGT2B7, UGT1A8, and UGT1A9 are mainly involved in the glucuronidation of 4-OHE2 and 4-OHE1 (16). Particularly, UGT2B7 represents the most efficient UGT for the glucuronidation of 4-OHE1 with a 25-fold higher intrinsic clearance for the formation of the predominant glucuronide at position 4, compared with UGT1A8 and UGT1A9, respectively (163.6 versus 7.1 and 5.7 µL/min/mg). However, all three enzymes share similar intrinsic clearances for the glucuronidation of 4-OHE2 at position 4 (49.0, 56.5, and 47.4 µL/min/mg; Table 1). In this study, we show a significant influence of variants UGT1A8*3, UGT1A9*3, and UGT2B7*2 on the formation of 4-OHE1 and 4-OHE2 glucuronides. Two of these mutations in the UGT1A8 and UGT1A9 genes (the *3 alleles) severely reduce enzyme function, whereas the UGT2B7*2 variation is associated with a significant 2-fold increase in clearance.

The A173G (*2) and C277Y (*3) polymorphisms in UGT1A8 are the only amino acid substitutions described for this gene (25). A previous functional study revealed that the capacity of the UGT1A8*2 protein in transient transfections is similar to the *1 for various molecules (25). From the data presented here, a modest effect of the UGT1A8*2 is also predicted because this protein shares similar catalytic properties (Km, Vmax, and CLint) with UGT1A8*1. In contrast, the near loss of activity for UGT1A8*3 was previously reported for a number of molecules suggesting an effect independent of the nature of the substrate (25, 29). For 4-OHCEs, a drastic reduction of activity for the *3 allozyme was observed at positions 3 and 4. Unexpectedly, the affinity of UGT1A8*3 was significantly enhanced by the presence of a tyrosine at codon 277 compared with the UGT1A8*1 protein with a cysteine. On the other hand, due to a drastic decreased velocity (13- to 40-fold) of the UGT1A8*3 protein compared with *1, the intrinsic clearances were between 3- and 13-fold lower for both estrogenic substrates. The UGT1A8*3 polymorphism corresponds to a single base pair mutation at the very highly conserved cysteine to a tyrosine in the substrate-binding region at codon 277. It was postulated that C277 might be involved in a disulfide bridge to another cysteine essential for the protein conformation or catalysis (25).

Similarly, we observed a reduced capacity of the UGT1A9*3 variant to inactivate carcinogenic 4-OHE1 and 4-OHE2 metabolites. This amino acid substitution, located in the substrate-binding domain, was revealed as a loss of function mutation for selected, but not all, substrates (26, 29). Although the activity of UGT1A9*3 was drastically decreased for the anticancer agent SN-38, its activity was maintained for the glucuronidation of another antineoplasic drug flavopiridol and was partially decreased for the immunosuppressive agent mycophenolic acid (26, 29). Thus, it seems that the effect of this amino acid substitution, corresponding to a nonconservative amino acid change, is substrate-dependent. UGT1A8*3 and UGT1A9*3 were found in ~1.2% and 2.2% of the population, respectively, consistent with frequencies found in previous studies (25, 26).

An enhancement of the catalytic efficiency was observed for the amino acid substitution at codon 268 of the UGT2B7 protein. The UGT2B7*2 H268Y allozyme presented a significant increase on both affinity and velocity compared with UGT2B7*1. Previous studies reported similar activities for the glucuronidation of 2-OHCEs and 4-OHCEs (37). Our study has several distinctions compared with previous studies explaining those controversial observations. First, the glucuronide formation was quantified by HPLC coupled with tandem mass spectrometry using authentic standards compared with the nonspecific thin layer chromatographic technique used in this previous study. Our approach has the particularity to quantify glucuronides at both positions 3 and 4. Second, the enzymatic activities were normalized by UGT protein expression in the heterologous system accounting for the variable expression of variant allozymes, which was not the case in this study. The functional analyses reveal a substrate-selective effect because earlier studies showed that the UGT2B7*2 protein has no effect on the in vitro glucuronidation of androsterone, menthol, opioids, propranolol, and epirubicin (27, 38, 39), whereas a reduced activity for lithocholic acid, hyodeoxycholic acid, 17ß-estradiol and 3'-azido-3'-deoxythymidine is observed (40, 41). In addition, we report that the codon 268 amino acid substitution influences the affinity of the protein for the glucuronidation of 4-OHE1 at position 4-hydroxyl suggesting that this variation affects the function and the recognition of the enzyme for selected substrates. Thus, the UGT2B7*1 allele represents a low activity with regard to 4-OHCE glucuronidation and a high activity allele for the conjugation of other molecules.

The functionality of the UGT2B7 –79 G>A promoter polymorphism was also revealed in endometrial cells and leads to a significant decrease of the transcriptional activity of the gene. Consistent with these results, we previously showed that this polymorphism is associated with a 2.5- to 7-fold decrease of transcription in colon and liver cells, respectively (30). Because the –79A variation is in strong linkage disequilibrium with the high-activity UGT2B7*2 (Y268) allele, prediction of the physiologic effect of this haplotype (UGT2B7*2g) remains to be determined. This haplotype occurs in 5% of the population, whereas the frequency of the UGT2B7*2a (–79G and Y268) allele is much higher, accounting for >50% of the Caucasian population (30). Due to the limited number of samples analyzed, and the low frequency of the –79 UGT2B7 variant, its relationship with UGT2B7 expression in breast and uterine tissues could not be assessed in this study.

The biological relevance of the kinetic differences observed needs to be evaluated in relation to enzyme expression in tissues where the 4-OHCEs are formed. The immunohistochemical analyses revealed predominant staining of UGTs in endometrial glandular cells and in epithelial cells covering the uterine cavity and in the ductal epithelial cells of mammary glands. Other studies previously showed a similar expression pattern of CYP1B1, the enzyme responsible for the formation of 4-OHCEs in these tissues (42–44). The expression pattern of 4-OHCEs metabolizing UGTs further supports their role in the local inactivation and modulation of concentrations in E2 and its metabolites in uterine and breast tissues. In addition, whereas the Km values of UGT enzymes for 4-OHCEs are in the micromolar range, other phase I and phase II biotransforming enzymes share similar affinities for estrogens (45–47). In addition, given that higher concentrations of estrogens in tissues, such as the mammary gland, are observed compared with serum concentrations (10- to 50-fold higher), it is plausible that UGT may have a significant physiologic role in uterine and breast tissues (3). Therefore, individuals with low UGT activity alleles might present lower inactivation activities for estrogen metabolites and being exposed to higher concentrations of these mutagenic metabolites. However, the relative contribution of individual UGTs in the tissular conjugation of 4-OHCEs, as determined by their kinetic properties and level of expression, is still unknown and deserves further investigation.

Supporting the hypothesis that glucuronidation modulate estrogen bioavailability within target cells, we recently observed a decreased risk to develop endometrial cancer by 60% to 80% associated with a genetic variation in UGT1A1, depending on the menopausal status (18). The association of a low UGT1A1 activity allele and the protection towards endometrial cancer is explained by the lower expression of UGT1A1 in the endometrium that would reduce the excretion of 2-OHCEs and 2-MeOCEs, the antiproliferative metabolites of E2 (15). Susceptibility to breast cancer associated with the polymorphic UGT1A1 promoter was also shown (21–23). In addition, the results of Gestl and coauthors support the role of UGT on mammary hormonal status because they have shown the formation of 4-OHE1 glucuronides by normal breast tissues (17). These observations were reinforced by the demonstration of the expression of UGT2B7 in the ductal epithelial cells of the mammary glands, a finding confirmed in this study.

In conclusion, this work shows the variable efficiency of specific genetic variants of UGT enzymes expressed in breast and uterine tissues to inactivate the mutagenic metabolites of E2. The pattern of expression for UGT1A8, UGT1A9, and UGT2B7, similar to that of the CYP1B1, involved in the formation of 4-OHCEs, further supports a biological inactivating role for the UGT pathway in favor of the conjugation of 4-OHCEs with glucuronide acid prior to their release into the circulation. This is reinforced by the fact that normal breast tissues are able to form 4-OHCEs glucuronides (17), a finding that was recently confirmed in our preliminary studies for normal endometrial tissues (data not shown). Thus, we propose a possible influence of the reduced capacity to inactivate E2 carcinogenic metabolites by UGT1A8*3, UGT1A9*3, and UGT2B7*1 variant proteins that may compromise the individual's protection against carcinogenic estrogens and estrogen-induced carcinogenesis. Hence, women with specific UGT variants may present a variable lifetime exposure to these mutagenic estrogen metabolites. It is thus hypothesized that an impaired UGT-mediated 4-OHCE inactivation pathway may have an effect on population-based risk estimates of breast and endometrial cancers. Further investigations to determine the influence of UGT1A8, UGT1A9, and UGT2B7 polymorphisms on the risk of cancer in case-control studies are currently ongoing.


   Acknowledgments
 
Grant support: Canadian Institutes of Health Research (CIHR MOP-68964). J. Thibaudeau, J. Lépine, and Y. Duguay are the recipients of graduate studentship awards from the Natural Sciences and Engineering Research Council of Canada, CIHR, and the Canada's Research-Based Pharmaceutical Companies-CIHR, respectively. C. Guillemette is the chairholder of the Canada Research Chair in Pharmacogenomics.

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.

We thank Patrick Caron, Lyne Villeneuve, and Louise Désy for their expertise and technical assistance.


   Footnotes
 
Note: J. Thibaudeau and J. Lépine contributed equally to this work and should be regarded as joint first authors.

For UGT allele nomenclature description, refer to: UDP-Glucuronosyltransferase (UGT) Alleles Nomenclature Home Page, Quebec, Canada, Pharmacogenomics Laboratory, c2005. Available from: http://pha.ulaval.ca/labocg/alleles/alleles.html.

8 https://innateimmunity.net/. Back

Received 8/11/05. Revised 9/23/05. Accepted 10/21/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Key TJ, Appleby PN, Reeves GK, et al. Body mass index, serum sex hormones, and breast cancer risk in postmenopausal women. J Natl Cancer Inst 2003;95:1218–26.[Abstract/Free Full Text]
  2. Kaaks R, Lukanova A, Kurzer MS. Obesity, endogenous hormones, and endometrial cancer risk: a synthetic review. Cancer Epidemiol Biomarkers Prev 2002;11:1531–43.[Abstract/Free Full Text]
  3. Jefcoate CR, Liehr JG, Santen RJ, et al. Tissue-specific synthesis and oxidative metabolism of estrogens. J Natl Cancer Inst Monogr 2000;27:95–112.[Medline]
  4. Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 1998;19:1–27.[Medline]
  5. Raftogianis R, Creveling C, Weinshilboum R, Weisz J. Estrogen metabolism by conjugation. J Natl Cancer Inst Monogr 2000;27:113–24.[Medline]
  6. Lee AJ, Cai MX, Thomas PE, Conney AH, Zhu BT. Characterization of the oxidative metabolites of 17ß-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. Endocrinology 2003;144:3382–98.[Abstract/Free Full Text]
  7. Sasaki M, Kaneuchi M, Fujimoto S, Tanaka Y, Dahiya R. CYP1B1 gene in endometrial cancer. Mol Cell Endocrinol 2003;202:171–6.[Medline]
  8. Iscan M, Klaavuniemi T, Coban T, Kapucuoglu N, Pelkonen O, Raunio H. The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue. Breast Cancer Res Treat 2001;70:47–54.[CrossRef][Medline]
  9. Liehr JG, Ricci MJ, Jefcoate CR, Hannigan EV, Hokanson JA, Zhu BT. 4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis. Proc Natl Acad Sci U S A 1995;92:9220–4.[Abstract/Free Full Text]
  10. Liehr JG. Is estradiol a genotoxic mutagenic carcinogen? Endocr Rev 2000;21:40–54.[Abstract/Free Full Text]
  11. Cavalieri EL, Rogan EG. A unifying mechanism in the initiation of cancer and other diseases by catechol quinones. Ann N Y Acad Sci 2004;1028:247–57.[Abstract/Free Full Text]
  12. Liehr JG, Ricci MJ. 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc Natl Acad Sci U S A 1996;93:3294–6.[Abstract/Free Full Text]
  13. Liehr JG, Fang WF, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian hamsters. J Steroid Biochem 1986;24:353–6.[CrossRef][Medline]
  14. Newbold RR, Liehr JG. Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. Cancer Res 2000;60:235–7.[Abstract/Free Full Text]
  15. Lakhani NJ, Sarkar MA, Venitz J, Figg WD. 2-Methoxyestradiol, a promising anticancer agent. Pharmacotherapy 2003;23:165–72.[CrossRef][Medline]
  16. Lepine J, Bernard O, Plante M, et al. Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphospho-glucuronosyltransferases expressed in endometrium. J Clin Endocrinol Metab 2004;89:5222–32.[Abstract/Free Full Text]
  17. Gestl SA, Green MD, Shearer DA, Frauenhoffer E, Tephly TR, Weisz J. Expression of UGT2B7, a UDP-glucuronosyltransferase implicated in the metabolism of 4-hydroxyestrone and all-trans retinoic acid, in normal human breast parenchyma and in invasive and in situ breast cancers. Am J Pathol 2002;160:1467–79.[Abstract/Free Full Text]
  18. Duguay Y, McGrath M, Lepine J, et al. The functional UGT1A1 promoter polymorphism decreases endometrial cancer risk. Cancer Res 2004;64:1202–7.[Abstract/Free Full Text]
  19. Belanger A, Caron S, Labrie F, Naldoni C, Dogliotti L, Angeli A. Levels of eighteen non-conjugated and conjugated steroids in human breast cyst fluid: relationships with cyst type. Eur J Cancer 1990;26:277–81.[Medline]
  20. Belanger A, Labrie F, Angeli A. Unconjugated and glucuronide steroid levels in human breast cyst fluid. Ann N Y Acad Sci 1990;586:93–100.[Abstract]
  21. Adegoke OJ, Shu XO, Gao YT, et al. Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) and risk of breast cancer. Breast Cancer Res Treat 2004;85:239–45.[CrossRef][Medline]
  22. Guillemette C, De Vivo I, Hankinson SE, et al. Association of genetic polymorphisms in UGT1A1 with breast cancer and plasma hormone levels. Cancer Epidemiol Biomarkers Prev 2001;10:711–4.[Abstract/Free Full Text]
  23. Guillemette C, Millikan RC, Newman B, Housman DE. Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 and association with breast cancer among African Americans. Cancer Res 2000;60:950–6.[Abstract/Free Full Text]
  24. Cecchin E, Russo A, Corona G, et al. UGT1A1*28 polymorphism in ovarian cancer patients. Oncol Rep 2004;12:457–62.[Medline]
  25. Huang YH, Galijatovic A, Nguyen N, et al. Identification and functional characterization of UDP-glucuronosyltransferases UGT1A8*1, UGT1A8*2 and UGT1A8*3. Pharmacogenetics 2002;12:287–97.[CrossRef][Medline]
  26. Villeneuve L, Girard H, Fortier LC, Gagne JF, Guillemette C. Novel functional polymorphisms in the UGT1A7 and UGT1A9 glucuronidating enzymes in Caucasian and African-American subjects and their impact on the metabolism of 7-ethyl-10-hydroxycamptothecin and flavopiridol anticancer drugs. J Pharmacol Exp Ther 2003;307:117–28.[Abstract/Free Full Text]
  27. Bhasker CR, McKinnon W, Stone A, et al. Genetic polymorphism of UDP-glucuronosyltransferase 2B7 (UGT2B7) at amino acid 268: ethnic diversity of alleles and potential clinical significance. Pharmacogenetics 2000;10:679–85.[CrossRef][Medline]
  28. Holthe M, Rakvag TN, Klepstad P, et al. Sequence variations in the UDP-glucuronosyltransferase 2B7 (UGT2B7) gene: identification of 10 novel single nucleotide polymorphisms (SNPs) and analysis of their relevance to morphine glucuronidation in cancer patients. Pharmacogenomics J 2003;3:17–26.[CrossRef][Medline]
  29. Bernard O, Guillemette C. The main role of UGT1A9 in the hepatic metabolism of mycophenolic acid and the effects of naturally occurring variants. Drug Metab Dispos 2004;32:775–8.[Abstract/Free Full Text]
  30. Duguay Y, Baar C, Skorpen F, Guillemette C. A novel functional polymorphism in the uridine diphosphate-glucuronosyltransferase 2B7 promoter with significant impact on promoter activity. Clin Pharmacol Ther 2004;75:223–33.[CrossRef][Medline]
  31. Lanouette CM, Giacobino JP, Perusse L, et al. Association between uncoupling protein 3 gene and obesity-related phenotypes in the Quebec Family Study. Mol Med 2001;7:433–41.[Medline]
  32. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978–89.[CrossRef][Medline]
  33. Venkatakrishnan K, Von Moltke LL, Greenblatt DJ. Human drug metabolism and the cytochromes P450: application and relevance of in vitro models. J Clin Pharmacol 2001;41:1149–79.[Abstract]
  34. Houston JB, Kenworthy KE. In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos 2000;28:246–54.[Abstract/Free Full Text]
  35. Gong QH, Cho JW, Huang T, et al. Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus. Pharmacogenetics 2001;11:357–68.[CrossRef][Medline]
  36. Owens IS, Ritter JK. Gene structure at the human UGT1 locus creates diversity in isozyme structure, substrate specificity, and regulation. Prog Nucleic Acid Res Mol Biol 1995;51:305–38.[Medline]
  37. Cheng Z, Rios GR, King CD, et al. Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7. Toxicol Sci 1998;45:52–7.[Abstract/Free Full Text]
  38. Coffman BL, King CD, Rios GR, Tephly TR. The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268). Drug Metab Dispos 1998;26:73–7.[Abstract/Free Full Text]
  39. Innocenti F, Iyer L, Ramirez J, Green MD, Ratain MJ. Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7. Drug Metab Dispos 2001;29:686–92.[Abstract/Free Full Text]
  40. Barbier O, Turgeon D, Girard C, et al. 3'-azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP-glucuronosyltransferase 2B7 (UGT2B7). Drug Metab Dispos 2000;28:497–502.[Abstract/Free Full Text]
  41. Gall WE, Zawada G, Mojarrabi B, et al. Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7. J Steroid Biochem Mol Biol 1999;70:101–8.[CrossRef][Medline]
  42. Bofinger DP, Feng L, Chi LH, et al. Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium. Toxicol Sci 2001;62:299–314.[Abstract/Free Full Text]
  43. Tsuchiya Y, Nakajima M, Kyo S, Kanaya T, Inoue M, Yokoi T. Human CYP1B1 is regulated by estradiol via estrogen receptor. Cancer Res 2004;64:3119–25.[Abstract/Free Full Text]
  44. Muskhelishvili L, Thompson PA, Kusewitt DF, Wang C, Kadlubar FF. In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues. J Histochem Cytochem 2001;49:229–36.[Abstract/Free Full Text]
  45. Hanna IH, Dawling S, Roodi N, Guengerich FP, Parl FF. Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity. Cancer Res 2000;60:3440–4.[Abstract/Free Full Text]
  46. Dawling S, Roodi N, Mernaugh RL, Wang X, Parl FF. Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol estrogens: comparison of wild-type and variant COMT isoforms. Cancer Res 2001;61:6716–22.[Abstract/Free Full Text]
  47. Adjei AA, Thomae BA, Prondzinski JL, Eckloff BW, Wieben ED, Weinshilboum RM. Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics. Br J Pharmacol 2003;139:1373–82.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
C. Swanson, M. Lorentzon, L. Vandenput, F. Labrie, A. Rane, J. Jakobsson, S. Chouinard, A. Belanger, and C. Ohlsson
Sex Steroid Levels and Cortical Bone Size in Young Men Are Associated with a Uridine Diphosphate Glucuronosyltransferase 2B7 Polymorphism (H268Y)
J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3697 - 3704.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. N. Operana and R. H. Tukey
Oligomerization of the UDP-glucuronosyltransferase 1A Proteins: HOMO- AND HETERODIMERIZATION ANALYSIS BY FLUORESCENCE RESONANCE ENERGY TRANSFER AND CO-IMMUNOPRECIPITATION
J. Biol. Chem., February 16, 2007; 282(7): 4821 - 4829.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
O. Bernard, J. Tojcic, K. Journault, L. Perusse, and C. Guillemette
Influence of Nonsynonymous Polymorphisms of UGT1A8 and UGT2B7 Metabolizing Enzymes on the Formation of Phenolic and Acyl Glucuronides of Mycophenolic Acid
Drug Metab. Dispos., September 1, 2006; 34(9): 1539 - 1545.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
W. R. Harrington, S. Sengupta, and B. S. Katzenellenbogen
Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells
Endocrinology, August 1, 2006; 147(8): 3843 - 3850.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thibaudeau, J.
Right arrow Articles by Guillemette, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thibaudeau, J.
Right arrow Articles by Guillemette, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online