Characterization of UDP-Glucuronosyltransferases Involved in

Nov 5, 2012 - Characterization of UDP-Glucuronosyltransferases Involved in Glucuronidation of Diethylstilbestrol in Human Liver and Intestine. Liangli...
1 downloads 0 Views 468KB Size
Download PDF
Article pubs.acs.org/crt

Characterization of UDP-Glucuronosyltransferases Involved in Glucuronidation of Diethylstilbestrol in Human Liver and Intestine Liangliang Zhu,†,‡ Guangbo Ge,† Yong Liu,† Zhimou Guo,† Chengcheng Peng,† Feng Zhang,§ Yunfeng Cao,† Jingjing Wu,† Zhongze Fang,†,‡ Xinmiao Liang,† and Ling Yang*,† †

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China Graduate School of Chinese Academy of Sciences, Beijing, 100049, China § Chinese Academy of Inspection and Quarantine, Beijing, 100123, China ‡

ABSTRACT: Diethylstilbestrol (DES), a synthetic estrogen, is famous for its carcinogenic effects. Human exposure to this compound can occur frequently through dietary ingestion and medical treatment. Glucuronidation has been demonstrated to be a predominant metabolic pathway for DES in human. Therefore, glucuronidation metabolism may have a significant impact on its toxicities, and it is essential to clarify this metabolic pathway. Accordingly, this in vitro study is designed to characterize the UGTs involved in DES glucuronidation and, furthermore, to identify the roles of individual isoforms in the reaction in liver and intestine. Human liver microsomes (HLM) displayed much higher potential for DES glucuronidation than human intestinal microsomes (HIM). The intrinsic clearances in HLM and HIM were demonstrated to be 459 and 14 μL/min/mg protein, respectively. Assays with recombinant UGTs demonstrated that UGT1A1, -1A3, -1A8, and -2B7 could catalyze DES glucuronidation, among which UGT2B7 showed the highest affinity. Chemical inhibitors of UGT2B7 and UGT1A1/1A3 both displayed similar inhibition against the reaction in UGT2B7 and HLM. In addition, DES glucuronidation in individual HLM exhibited a large individual variability and strongly correlated to UGT2B7 activity. All evidence indicates that UGT2B7 may act as a major enzyme responsible for DES glucuronidation in human liver. For HIM, both UGT2B7 inhibitor and UGT1A1/1A3/1A8 inhibitor exerted moderate inhibition. It is suggested that although UGT2B7 contributes to DES glucuronidation in intestine, other UGTs may contribute equally. In summary, this study characterizes human UGTs involved in DES glucuronidation in human liver and intestine, which may be helpful for further study about DES-related toxicities.

■

the United States.1 It is now still accepted as an efficient therapy for the treatment of prostate and breast cancers.2,3 Aside from its wide clinical applications, DES has found widespread application as a growth promoter in feeding cattle, sheep, and poultry.4 It was estimated that in 1971 alone as much as 27600 kg of DES was used in livestock feeding in the United States.5 After widespread and extensive exposures to DES, numerous serious health problems have been encountered. DES was established to cause the rare clear cell adenocarcinoma (CCAC) of the vagina and cervix in “DES daughters” (who are exposed to DES before birth).6 In addition to CCAC, these daughters were found to suffer from malformations of their genital tracts, which can result in severe pregnancy and fertility problems.7−9 Just like DES daughters, DES sons were also found to be prone to DES damage in the genitalia.10 In company with daughters and sons, DES mothers, women who accept DES during pregnancy, can not escape the perils of DES

INTRODUCTION

Diethylstilbestrol (DES, Figure 1), a synthetic nonsteroid estrogen, is famous for its carcinogenicity. Human exposure to this compound can occur directly or indirectly through dietary ingestion and medical treatment.1−4 DES was once prescribed widely to millions of pregnant women in false hopes of preventing miscarriage and other pregnancy complications in

Received: July 6, 2012

Figure 1. Glucuronidation of DES. © XXXX American Chemical Society

A

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

■

as well. After accepting DES during pregnancy, the mothers may have increased risks of breast cancer.11 The severe transplacental toxicities make DES a model toxicant that attracts many investigations to explore any potential toxic mechanisms. It is currently accepted that most toxicities can be ascribed to its potent estrogenic activity. In addition, there are some other potential mechanisms that may also contribute to the toxicities, such as direct interaction with DNA and generation of reactive metabolites.12,13 Although a lot of investigations are conducted to explore the toxic mechanisms, little attention is paid to the detoxifying pathway that may also significantly affect the toxicities from the different perspective. Glucuronidation serves as an efficient detoxification pathway for DES, and in fact, it is also demonstrated to be the predominant metabolic pathway in human. After administration of DES in patients suffering from prostate cancer, the glucuronidated form could even account for over 96% of the circulating drug.14 It should be noted that severe toxic effects result from DES itself and its reactive metabolites, which indeed made up only a small portion in vivo in human.12,13,15 Therefore, a subtle change in individual DES glucuronidation activity may significantly affect the levels of DES and its reactive metabolites and correspondingly influence the toxicities. In retrospect, DES toxicities demonstrate two outstanding characteristics: fetuses are a susceptible population, and the reproductive tracts are easily damaged sites. Previous studies have demonstrated that reproductive tracts and liver are two major sites of DES accumulation, and the placenta can hardly limit the penetration of DES into the fetus.16 As compared with the liver, there are a very limited number of uridine 5′diphospho-glucuronosyltransferase (UGTs) expressed in the reproductive tract.17 Similarly, the fetus possesses a relatively very low glucuronidation activity in comparison with adults.18 It appears to be that the lack of UGTs with DES glucuronidation activity in the genital tract and the low glucuronidation activity in the fetus may contribute to the tissue preference of damage in offspring of DES. However, available information does not reveal which UGT isoforms are involved in DES glucuronidation, which retards our understanding of the fundamental basis of DES toxicities. There are at least 18 functional UGTs in human that can be divided into UGT1A and UGT2A and -2B subfamilies based on amino acid sequence identity.19 They are expressed in various tissues in a tissue-dependent manner.17 Each isoform displays different substrate preferences, despite usually exhibiting overlapping substrate specificities.20 Many factors, such as polymorphism, age, and coadministrated compounds, can influence the activities of UGTs, which are often in isoformdependent manners.21 Therefore, characterization of the UGTs responsible for glucuronidation of a toxicant is essential to understand the factors affecting the toxicities and to predict individual differences of potential toxicities. This study is designed to identify the UGTs involved in DES glucuronidation and their respective roles in the reaction in liver and intestine by using recombinant UGTs as well as human liver and intestinal microsomes (HLM and HIM, respectively). It is hoped that this study may provide valuable information for us to better understand the toxicities of DES, from the view of the detoxifying pathway.

Article

EXPERIMENTAL PROCEDURES

Chemicals and Reagents. DES (purity >99%) was purchased from Alfa Aesar Ltd. Phenylbutazone, androsterone, uridine 5′diphospho-glucuronic acid (UDPGA, trisodium salt), 17-β-estradiol, 17-β-estradiol-3-O-glucuronide, 3′-azido-3′-deoxy-thymidine (AZT), D-saccharicacid 1,4-lactone, and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO). AZT glucuronide was obtained from Toronto Research Chemicals Inc. (North York, ON, Canada). All of the other reagents were of HPLC grade. Enzyme Sources. Pooled HLMs (n = 25) and HIMs (n = 10) were purchased from Research Institute for Liver Diseases (RILD, Shanghai, China). Individual human liver samples were obtained from Dalian Medical University (Dalian, China) with the approval of the local ethics committee at the university. Any information on the medication history of the samples was not gained. HLM was prepared according to the methods described by our previous study.22 Sprague− Dawley rats (n = 10, male; weight, 180−220 g) were purchased from Dalian Medical University with the approval of the local Institutional Animal Care & Use Committee. The animals had free access to tap water and pellet diet. The rats were euthanized by decapitation, and the livers were rapidly excised and pooled for preparation of microsomes. A panel of recombinant human UGT isoforms (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10, -2B4, -2B7, -2B15, and -2B17) expressed in baculovirus-infected insect cells were purchased from BD Gentest Corp. (Woburn, MA). DES Glucuronidation Assays and Analysis Method. DES was incubated with either HLM or recombinant UGT isoforms in a reaction mixture of 200 μL of 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl2, 10 mM D-saccharicacid 1,4-lactone, 4 mM UDPGA, and alamethicin (5% microsomal protein concentrations) when HLM and HIM were used. Prior to reaction initiation by the addition of UDPGA, all of the reaction mixtures were preincubated at 37 °C for 5 min. The reaction was terminated by adding 100 μL of methanol. The samples were then centrifuged at 20000g for 10 min to remove protein, and supernatants were analyzed for glucuronides formation. The ultrafast liquid chromatography (UFLC)-diode array detector (DAD) and mass spectrum (MS) system, used to analyze glucuronidation samples, has been described previously.20 A Shimpack XR-ODS (50.0 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) analytical column with an ODS guard column (5 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) was used to separate DES and its glucuronide. The mobile phase consisted of CH3CN (A) and Millipore water containing 0.2% formic acid (B). Used were the following gradient conditions: 0−9 min, 90% B to 30% B; 9−12.5 min, 5% B; 12.5−16 min, balance to 90% B. The flow rate was 0.3 mL/min, and the detector wavelength was set at 250 nm. DES glucuronidation was quantified by the standard curve of the glucuronide, which was linear from 0.01 to 10 μM (the correlation coefficient was >0.999) and had intra- and interday variance less than 5%. Mass detection was performed in both negative and positive ion modes from m/z 100 to 800. The detector voltage was set at +1.55 and −1.55 kV, for positive and negative ion detection, respectively. The curved desolvation line (CDL) temperature and the block heater temperature were both set at 250 °C. Other MS detection conditions were as follows: interface voltage, 4 kV; CDL voltage, 40 V; nebulizing gas (N2) flow, 1.5 L/min; and drying gas (N2) pressure, 0.06 MPa. Data processing was performed using the software LCMS Solution version 3.41. Estradiol Glucuronidation Assays and Analysis Method. Estradiol (10 μM) was incubated with individual HLM for 20 min, with a final protein concentration of 0.25 mg/mL. Estradiol glucuronidation samples were analyzed on the UFLC system (as described above). A Shim-pack XR-ODS (50.0 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) analytical column with an ODS guard column (5 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) was used and kept at 40 °C. The mobile phase consisted of acetonitrile (A) and 0.2% formic acid (B) at a flow rate of 0.3 mL/min, with the following gradient: 0−9 min, 90% B to 30% B; 9−12.5 min, 5% B; 12.5−16 min, balance to 90% B. The B

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

performed in triplicate determinations, and the kinetic parameters were expressed as the mean ± computer-calculated SE. DES Glucuronidation in the Absence or Presence of Chemical Inhibitors. Androsterone and phenylbutazone have been reported as inhibitors of UGT2B7 and UGT1As, respectively.23 They were employed in the chemical inhibition study to decipher the roles of individual UGTs in hepatic glucuronidation of DES. In view of the possibility of substrate-dependent inhibition occurring, their inhibitory effects on DES glucuronidation in individual UGTs were tested first in the present study. DES (5 μM) was incubated with UGT1A1, -1A3, -1A8, -2B7, HIM, or HLM in the absence and presence of androsterone (100 μM) or phenylbutazone (500 μM). Furthermore, the IC50 values for the inhibition of androsterone on HLM and UGT2B7, representing the inhibitor concentrations that inhibit 50% of the control activity, were determined by nonlinear curve fitting as described previously.24 DES Glucuronidation in Individual HLM and Correlation Analysis. To reveal potential interindividual differences of hepatic DES glucuronidation, DES (5 μM) was incubated with individual HLM from 11 different donors, respectively. Meanwhile, estradiol (10 μM) and AZT (500 μM) glucuronidation in individual HLM (0.25 mg protein/mL) were used as probe reactions of respective UGT1A1 and 2B7, respectively. DES glucuronidation activity of 11 individual HLM was correlated to estradiol and AZT glucuronidation activity, respectively. All incubations were performed in triplicate determinations. The correlation parameter was expressed by the linear regression coefficient (r), and p < 0.05 was considered as being statistically significant.

detector wavelength was set at 250 nm. The standard curve of the estradiol-3-O-glucuronide was used to quantify glucuronides formation. AZT Glucuronidation Assays and Analysis Method. AZT (0.5 mM) was incubated with individual HLM for 20 min, with a final protein concentration of 0.5 mg/mL. AZT glucuronidation samples were analyzed on the HPLC system (Shimadzu, Kyoto, Japan), equipped with a SCL-10A system controller, two LC-10AT pumps, a SIL-10A autosampler, and a SPD-10AVP UV detector. A C-18 column (250 mm × 4.6 mm i.d., 5 μm, Kromasil) was used to separate AZT and its glucuronide. The mobile phase was acetonitrile (A) and 0.2% formic acid (B) at a flow rate of 1.0 mL/min, with an isocratic 0−25 min, 90% B. The detector wavelength was set at 260 nm. The standard curve of AZT glucuronide was used to quantify the glucuronide formation. Biosynthesis of DES Glucuronide. The glucuronides were biosynthesized using pooled liver microsomes from rats and human (90% RLM and 10% HLM) and purified for structure elucidation and quantitative analysis. In brief, 0.15 mg/mL DES (stocked in DMSO) was incubated with pooled liver microsomes (0.4 mg protein/mL) in 50 mM Tris-HCl (pH 7.4), containing 5 mM MgCl2, Brij 58 (0.1 mg/ mg protein), and 2 mM UDPGA in 200 mL of final incubations for 4 h at 37 °C. The reaction was terminated by adding 100 mL of methanol, and the vessel was then put in an ice bath for 20 min. Proteins were removed by centrifugation at 20000g for 10 min at 4 °C, and the combined supernatants were loaded on a SPE cartridge (C18 and anion exchange resin, 1000 mg, Dalian Sipore, CN). The SPE cartridge was preconditioned by sequential washing with 6 mL of methanol and 6 mL of Millipore water. After sample loading, the SPE cartridge was sequentially eluted with 6 mL of Millipore water, 12 mL of methanol, and 12 mL of methanol containing 5% formic acid. The entire process was monitored by UFLC-DAD, and the glucuronides were assembled in methanol containing 5% formic acid. After vacuum evaporation, 8.9 mg of metabolite was obtained, and the purity was greater than 95% by UFLC-DAD analysis. The structure of the metabolite was determined by NMR spectra including 1H NMR and 13C NMR. All experiments were carried on a Bruker Avance-500 NMR spectrometer (Bruker, Switzerland). The purified metabolites were stored at −20 °C before dissolving in methanol-d4 (Euriso-Top, Saint-Aubin, France) for NMR analysis. Chemical shifts were given on δ scale and referenced to tetramethylsilane (TMS) at 0 ppm for 1H NMR (500 MHz) and 13 C NMR (125 MHz). Screening of UGTs with DES Glucuronidation Activity. DES (10 μM) was incubated with 0.1 mg/mL 12 commercial recombinant UGT isoforms (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10, -2B4, -2B7, -2B15, and -2B17) for 60 min. UFLC-DAD was used to detect the formation of glucuronide. Kinetic Analyses for DES Glucuronidation by Recombinant UGTs, HLM and HIM. Kinetic analyses were performed in HLM, HIM, and involved recombinant UGT enzymes. Preliminary experiments were performed to ensure that glucuronide was formed in the linear range of both reaction time (0−60 min) and microsomal concentration (0.01−0.1 mg/mL). For HLM, DES (0−100 μM) was incubated with pooled HLM (0.0125 mg/mL) at 37 °C for 30 min. For HIM, DES (0−100 μM) was incubated with pooled HIM (0.1 mg/mL) at 37 °C for 30 min. For UGT1A1, DES (0−200 μM) was incubated with the recombinant enzyme (0.05 mg/mL) at 37 °C for 30 min. For UGT1A3, DES (0−64 μM) was incubated with the recombinant enzyme (0.1 mg/mL) at 37 °C for 30 min. For UGT1A8, DES (0−100 μM) was incubated with the recombinant enzyme (0.05 mg/mL) at 37 °C for 30 min. For UGT2B7, DES (0−64 μM) was incubated with the recombinant enzyme (0.02 mg/mL) at 37 °C for 30 min. After removal of protein through centrifugation at 20000g for 10 min, 30 μL of supernatants was analyzed on UFLC-DAD. To estimate the kinetic parameters, a substrate inhibition model was employed: v = Vmax[S]/(Ks + [S] + [S]2/Ksi), where v is the rate of glucuronidation reaction, [S] is the substrate concentration, Vmax is the apparent maximum reaction rate, Ks is the substrate affinity constant, and Ksi is the substrate inhibition constant. All incubations were

■

RESULTS Identification of DES Glucuronidation. Incubation of DES with HLM, in the presence of UDPGA, yielded a single metabolite peak. This metabolite peak was absent in the control samples without either microsomes, UDPGA or DES (Figure 2). Mass spectrometry in the negative ion mode showed m/z

Figure 2. Representative LC profiles of DES and its glucuronide. The representative UPLC chromatograms of control samples without either microsomes, UDPGA or DES, were also included. The glucuronide and DES were eluted at 5.8 and 8.0 min, respectively. The m/z of the glucuronide in negative ion mode is displayed as the inset.

443.2 for the deprotonated metabolite (Figure 2), a value that corresponds well to DES (268 minus 1) with the m/z 176 of the glucuronosyl substitution. The DES glucuronide was purified for structure elucidation by 1H NMR and 13C NMR analyses (Table 1). As compared to the parent compound, DES, the 13C NMR spectrum of the metabolite showed that C-A2-1 shifted upfield to δ 157.44 (Δδ −0.6) due to the glycosidation shift of phenolic compound. In addition, the G1 proton and carbon exhibited characteristic chemical shifts near 5 and 100 ppm, and the G6 carbon (−COOH) showed chemical shifts about 170 ppm. All of the evidence pointed that the location of glucuronosyl substitution was at the A2 phenolic group. C

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

Table 1. Proton and Carbon NMR Chemical Shift Assignments for DES and the Glucuronide DES δ H (no. of protons)

glucuronide

1

position A1-1, A2-1 A1-4, A2-4 A1-2, A2-2 A1-3, A2-3 A1-5, A2-5 A1-6, A2-6 1, 6 2, 5 3, 4 G1 G2 G3 G4 G5 G6

6.77−7.00 (8H)

δ H (no. of protons) 1

δ13C

δ13C

156.82

156.92, 157.44

140.14

139.73, 140.55

115.82

6.78−7.13 (8H)

115.85, 117.61

130.77

130.74, 130.74

130.77

130.74, 130.74

115.82

115.85, 117.61

0.74−0.77 (6H)

13.69

2.10−2.14 (4H)

29.51

0.74−0.77 (6H) 2.08−2.16 (4H)

135.15 4.95 3.64 3.32 3.52 4.00

(1H) (1H) (1H) (1H) (1H)

13.63, 13.63 29.45, 29.52 130.82, 130.82 102.7 74.65 76.56 73.03 77.38 172.28

Figure 4. Kinetics of DES glucuronidation by HLM, HIM, and the commercial recombinant UGT1A1, -1A3, -1A8, and -2B7. Data points represent the mean of triplicate independent determinations, and error bars represent the calculated SD.

UGTs with DES Glucuronidation Activity. To identify the isoform involved in DES glucuronidation, a panel of 12 recombinant human UGT isoforms was investigated. Results were displayed in Figure 3. It was found that UGT1A1, -1A3, -1A8, and -2B7 could convert DES to its glucuronide, whereas other recombinant UGTs could not catalyze the formation of DES glucuronide.

nmol/min/mg protein, respectively. For pooled HIM, the higher Ks value and the lower Vmax value were observed. The Ks, Ksi, and Vmax values were 24.2 μM, 20.4 μM, and 0.36 nmol/ min/mg protein, respectively. The intrinsic clearance (CLint, Vmax/Ks) of HLM and HIM were calculated to be 459 and 15 μL/min/mg protein, respectively. Among recombinant UGTs with DES glucuronidation activity, UGT2B7 displayed the lowest Ks value and the highest CLint value. The CLint values for UGT1A1, -1A3, -1A8, and -2B7 were calculated to be 17, 24, 9, and 144 μL/min/mg protein, respectively. It should be noted that Ks and Ksi values of UGT2B7 (2.16 and 12.9 μM, respectively) were very similar with those of HLM, suggesting that UGT2B7 played an important role in DES glucuronidation in HLM. Kinetic parameters for all enzyme sources, expressed in mean ± calculated SE, are displayed in Table 2. Chemical Inhibition Studies. To reveal the roles of UGTs in hepatic and intestinal glucuronidation of DES, chemical inhibition studies were conducted for the involved UGTs, HIM, and HLM. The inhibitory effects of androsterone and phenylbutazone on DES glucuronidation in HLM, HIM, UGT1A1, -1A3, and -2B7 are displayed in Figure 5. Inhibition studies on individual UGTs demonstrated that androsterone acted as a potent inhibitor of UGT2B7, while phenylbutazone acted as an inhibitor of UGT1A enzymes, which were in agreement with the previous study.23 Androsterone (100 μM) exhibited potent inhibition against UGT2B7, whereas it showed none or muted inhibition against UGT1As. In the presence of androsterone (100 μM), the remaining activities of UGT2B7, UGT1A1, -1A3, and -1A8 were 12, 58, 84, and 58%, respectively. Contrary to androsterone, phenylbutazone (500 μM) strongly inhibited the activities of UGT1A1, -1A3, and -1A8, reducing DES glucuronidation activities of to be 31, 19, and 20% of the control, while lacking significant inhibition against UGT2B7.

Figure 3. Formation of DES glucuronide by recombinant UGT isoforms. DES (10 μM) was incubated with various recombinant human UGTs (0.1 mg/mL) at 37 °C for 60 min. Data columns and error bars represent the mean and SD of triplicate determinations, respectively.

Kinetic Analyses of DES Glucuronidation. To better understand the catalytic properties of these UGTs involved in DES glucuronidation, the kinetic experiments were performed in pooled HLM, HIM, and recombinant UGT1A1, -1A3, -1A8, and -2B7. All of the UGTs with DES glucuronidation activities exhibited substrate inhibition kinetic behavior with a large activity variability (Figure 4). HLM showed much higher activity toward DES glucuronidation than HIM. For pooled HLM, Ks, Ksi, and Vmax values were 2.44 μM, 19.4 μM, and 1.12 D

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

Table 2. Kinetic Parameters of DES Glucuronidation by HLM, HIM, and Recombinant UGTsa enzyme sources HLM HIM UGT1A1 UGT1A3 UGT1A8 UGT2B7 a

Ks (μM) 2.44 24.2 156 10.3 34.0 2.16

± ± ± ± ± ±

0.07 7.6 71 2.5 16.0 0.35

Ksi (μM) 19.4 20.4 16.1 7.34 6.79 12.9

± ± ± ± ± ±

Vmax (nmol/min/mg protein)

CLint (μL/min/mg protein)

± ± ± ± ± ±

459 15 17 24 9 144

1.6 5.1 7.4 1.78 3.14 2.3

1.12 0.36 2.72 0.25 0.30 0.31

0.04 0.19 1.09 0.05 0.12 0.03

Data represent the mean ± SE of computer calculations.

DES Glucuronidation in Individual HLM and Correlation Study. Glucuronidation rates of DES, estradiol, and AZT were determined in individual HLM from 11 different donors. Human liver microsomal DES glucuronidation activities exhibited significant individual variability, ranging from 0.01 to 0.3 nmol/min/mg protein with a CV value of 60% (Figure 7). Glucuronide formation rates of DES in individual HLM

Figure 5. Inhibitory effects of phenylbutazone (500 μM) and androsterone (100 μM) on glucuronidation of DES (5 μM) by HLM, HIM, and recombinant UGT1A1, -1A3, -1A8, and -2B7. Data columns represent the mean of duplicate determinations. Figure 7. DES glucuronidation in a panel of 11 individual HLM. Data columns represent the mean of triplicate determinations, and error bars represent the calculated SD.

Inhibition of androsterone and phenylbutazone on HIM and HLM was further conducted. As was shown in Figure 5, androsterone (100 μM) displayed potent inhibition in HLM, with the remaining activity of 28% of the control, while phenylbutazone (500 μM) lacked significant inhibition, decreasing the activity to be 80% of the control. Even more, androsterone exhibited similar inhibition potential in HLM and UGT2B7 with similar IC50 values of 20.5 and 18.1 μM, respectively (Figure 6). For HIM, both androsterone and phenylbutazone exerted moderate inhibition (Figure 5). In the presence of phenylbutazone (500 μM), DES glucuronidation activity of HIM was decreased to be 65% of the control. Similarly, androsterone (100 μM) decreased HIM activity to be 61% of the control.

highly correlated with those of AZT, a probe substrate for UGT2B7 (Figure 8a, r = 0.90, p < 0.001),25 while weakly

Figure 8. Correlation analyses between DES (5 μM) glucuronidation activity and (A) UGT2B7-specific AZT glucuronidation and (B) UGT1A1-specific esradiol-3-O-glucuronidation in a panel of 11 HLMs.

correlating to eatradiol-3-O-glucuronidation, a probe reaction for UGT1A1 (Figure 8b).26 The correlation study indicated that UGT2B7 played an important role in DES glucuronidation in HLM, while UGT1A1 contributed limitedly to this pathway.

■

DISCUSSION

UGTs-mediated glucuronidation is the dominant metabolic pathway of DES in human, which may have significant impacts on DES toxicities. Our study characterizes the DES glucuronidation pathway for the first time, expanding the basis for us to better understand DES-related toxicities, from the view of detoxification by UGTs.

Figure 6. Inhibition of androsterone on DES glucuronidation by UGT2B7 and HLM. DES (5 μM) was incubated with various concentrations of androsterone (0−100 μM). Each data point represents the mean of duplicate determinations. E

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

no UGT2B7 transcripts were detected in fetal liver at 20 weeks' gestation.29 Therefore, DES may undergo glucuronidation in a very low level in fetus and has plenty of opportunities to manifest hazardous effects. Furthermore, because the fetus displays low DES glucuronidation activity, the level and time of fetus exposure to DES may be dependent on UGT2B7 activities of their mothers. Accordingly, carcinogenic effects of DES on the sons and daughters may also be significantly influenced by mothers' UGT2B7 activities. Therefore, information pertaining to UGT2B7, such as polymorphisms, is warranted to develop therapeutic strategies. A recent study demonstrated that by comparison with the wild-type (UGT2B7*1), mutant UGT2B7 (UGT2B7*2, UGT2B7*5, and UGT2B7*71S), all exhibited relatively low glucuronidation activity.30 This may give a helpful hint in nursing DES daughters and sons: if their mothers carry mutant UGT2B7 genes, they may be at higher risk of cancers. Because many factors can affect UGT2B7 activity, the activity of this enzyme displays large individual variability,21 which further results in that human liver microsomal DES glucuronidation activity displays significant individual differences with DES glucuronidation activity ranging from 0.01 to 3.0 nmol/min/mg (Figure 7). This suggests that the toxicity may correspondingly exhibit large individual variability in UGT2B7 activity-dependent manner. In fact, there is a controversy of whether DES can result in increasing risks of cervix, ovary, and other cancers in DES mothers.31 On the basis of this study, carcinogenic effects of DES may be more prominent in those people with lower UGT2B7 activities, which may help throw light on the study about increasing cancer risks of DES exposure. In summary, the current study characterizes the DES glucuronidation pathway in human, and UGT2B7 is demonstrated to play an important role. From the view of the detoxifying function, DES toxicities might be UGT2B7 activity dependent. It is suggested that those people with low UGT2B7 activities may be susceptible to the injury of this synthetic estrogen.

Assays with a panel of recombinant UGTs demonstrated that UGT1A1, -1A3, -2B7, and extrahepatic 1A8 could catalyze DES glucuronidation (Figure 3). All UGTs with DES glucuronidation activity were not detected in the reproductive system including ovary, cervix, and testes.17 In fact, in these tissues, only UGT1A7, -2B15, and -2B17 are expressed;17 nevertheless, these UGTs can not catalyze DES glucuronidation. The lack of the UGTs results in that DES can not be timely detoxicated via glucuronidation and therefore freely exerts the severe damage in the reproductive system. Unlike the reproductive tract, UGT1A1, -1A3, and -2B7 are abundant in the liver,17 in which DES can be effectively glucuronidated, and thereby displays fewer damages. This can explain, at least partly, why injury of DES prefers the reproductive systems other than liver, despite the fact that both of these two tissues are DES accumulation sites. Although HLM and HIM can both catalyze DES glucuronidation, their catalytic activities are dramatically different. The DES glucuronidation activity of HLM is as high as 30-fold of that of HIM (Table 2). This is in agreement with the relative expression levels of UGTs with DES glucuronidation activity in these two tissues.17 In comparison with liver, UGT1A1, -1A3, and -2B7 are expressed in very low levels in the intestine. Although UGT1A8 is mainly expressed in the intestine rather than the liver, its expression level in the intestine is also very low. As a result, it seems that the glucuronidation in the intestine may contribute limitedly to the systemic elimination. Accordingly, the liver becomes a major detoxifying tissue for DES. Kinetic assays with UGTs demonstrated that UGT2B7 showed the highest activity (Table 2). On the basis of our previous study, the activity of UGT2B7 in commercial recombinant system is lower than that in liver, while the case of UGT1A1 is contrary.20 It can be anticipated that UGT2B7 may play an important role in DES glucuronidation in HLM. This assumption was further conformed in subsequent chemical inhibition studies and correlation analyses. It should be noted here that this study only investigated the respective roles of individual UGTs in DES glucuronidation at 5 μM substrate concentration. At higher concentrations, the relative contributions of UGTs may differ from the results obtained in this study. In human liver, the contribution of UGT1A1 and -1A3 may increase at the high DES concentration (>5 μM), while in human intestine, the roles of UGT1A1 and -1A8 may become more important. However, as an oral chemical, in most cases, the concentrations of DES in plasma are far lower than 5 μM (22 nM).27 As is displayed in Table 2, among the individual UGTs with DES glucuronidation activity, UGT2B7 displays the highest affinity (the lowest Ks value, 2.16 μM). Because the other UGTs exhibit much lower affinity to DES, it is conceivable that UGT2B7 may play a more important role at the physiological DES concentration. Therefore, given that glucuronidation can protect humans from DES damage, the activity of UGT2B7 may significantly affect the toxicities. Among many factors that can affect the activity of UGT2B7, the age deserves particular attention for the interpretation of toxicities related with DES. The most intriguing trait of the DES toxicities is that fetuses are vulnerable groups. From the view of detoxification mechanisms, this trait can result from the detoxifying UGTs with low activities in fetuses. Interestingly, the rate of morphine glucuronidation, a probe reaction for UGT2B7, in human fetal liver was much lower than that in adult liver. 28 Particularly, Strassburg and co-workers stated that

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by the National Science & Technology Pillar Program of China (2009BADB9B02), International Science & Technology Cooperation Program of China (2012DFG32090), and the National Natural Science Foundation of China (81273590 and 81001473). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS DES, diethylstilbestrol; UDPGA, uridine 5′-diphospho-glucuronic acid; UGT, uridine 5′-diphospho-glucuronosyltransferase; AZT, 3′-azido-3′-deoxy-thymidine; HLM, human liver microsomes; HIM, human intestinal microsomes; RLM, rat liver microsomes; Clint, intrinsic clearance

■

REFERENCES

(1) Noller, K. L., and Fish, C. R. (1974) Diethylstilbestrol usage: Its interesting past, important present, and questionable future. Med. Clin. N. Am. 58, 793−810.

F

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

(2) Smith, D. C., Redman, B. G., Flaherty, L. E., Li, L., Strawderman, M., and Pienta, K. J. (1998) A phase II trial of oral diethylstilbesterol as a second-line hormonal agent in advanced prostate cancer. Urology 52, 257−260. (3) Peethambaram, P. P., Ingle, J. N., Suman, V. J., Hartmann, L. C., and Loprinzi, C. L. (1999) Randomized trial of diethylstilbestrol vs. tamoxifen in postmenopausal women with metastatic breast cancer. An updated analysis. Breast Cancer Res. Treat. 54, 117−122. (4) McMartin, K. E., Kennedy, K. A., Greenspan, P., Alam, S. N., Greiner, P., and Yam, J. (1978) DiethylstilbestrolReview of its toxicity and use as a growth promotant in food-producing animals. J. Environ. Pathol. Toxicol. 1, 279−313. (5) Li, S., Hursting, S. D., Davis, B. J., McLachlan, J. A., and Barrett, J. C. (2003) Environmental exposure, DNA methylation, and gene regulation: lessons from diethylstilbesterol-induced cancers. Ann. N.Y. Acad. Sci. 983, 161−169. (6) Herbst, A. L., Scully, R. E., and Kurman, R. J. (1972) Vaginal and cervical abnormalities after exposure to stilbestrol in utero. Obstet. Gynecol. 40, 287−298. (7) Goldberg, J. M., and Falcone, T. (1999) Effect of diethylstilbestrol on reproductive function. Fertil. Steril. 72, 1−7. (8) Kaufman, R. H., Adam, E., Hatch, E. E., Noller, K., Herbst, A. L., and Palmer, J. R. (2000) Continued follow-up of pregnancy outcomes in diethylstilbestrol-exposed offspring. Obstet. Gynecol. 96, 483−489. (9) Palmer, J. R., Hatch, E. E., Rao, S. R., Kaufman, R. H., Herbst, A. L., and Noller, K. L. (2001) Infertility among women exposed prenatally to diethylstilbestrol. Am. J. Epidemiol. 154, 316−321. (10) Wilcox, A. J., Baird, D. D., Weinberg, C. R., Hornsby, P. P., and Herbst, A. L. (1995) Fertility in men exposed prenatally to diethylstilbestrol. N. Engl. J. Med. 332, 1411−1416. (11) Titus-Ernstoff, L., Hatch, E. E., Hoover, R. N., Palmer, J., Greenberg, E. R., Ricker, W., Kaufman, R., Noller, K., Herbst, A. L., Colton, T., and Hartge, P. (2001) Long-term cancer risk in women given diethylstilbestrol (DES) during pregnancy. Br. J. Cancer 84, 126−133. (12) Hinrichs, B., Zahid, M., Saeed, M., Ali, M. F., Cavalieri, E. L., and Rogan, E. G. (2011) Formation of diethylstilbestrol-DNA adducts in human breast epithelial cells and inhibition by resveratrol. J. Steroid Biochem. Mol. Biol. 127, 276−281. (13) Saeed, M., Rogan, E., and Cavalieri, E. (2009) Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: The defining link to natural estrogens. Int. J. Cancer 124, 1276−1284. (14) Fergusson, J. D. (1961) Tracer experiments showing the distribution and fate of injected phosphorylated oestrogens in cancer of the prostate. Br. J. Urol. 33, 442−447. (15) Cunningham, A., Klopman, G., and Rosenkranz, H. S. (1996) The carcinogenicity of diethylstilbestrol: structural evidence for a nongenotoxic mechanism. Arch. Toxicol. 70, 356−361. (16) Shah, H. C., and McLachlan, J. A. (1976) The fate of diethylstilbestrol in the pregnant mouse. J. Pharmacol. Exp. Ther. 197, 687−696. (17) Ohno, S., and Nakajin, S. (2009) Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab. Dispos. 37, 32− 40. (18) Court, M. H., Zhang, X., Ding, X., Yee, K. K., Hesse, L. M., and Finel, M. (2012) Quantitative distribution of mRNAs encoding the 19 human UDP-glucuronosyltransferase enzymes in 26 adult and 3 fetal tissues. Xenobiotica 42, 266−277. (19) Mackenzie, P. I., Bock, K. W., Burchell, B., Guillemette, C., Ikushiro, S., Iyanagi, T., Miners, J. O., Owens, I. S., and Nebert, D. W. (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet. Genomics 15, 677−685. (20) Zhu., L., Ge, G., Zhang, H., Liu, H., He, G., Liang, S., Zhang, Y., Fang, Z., Dong, P., Finel, M., and Yang, L. (2012) Characterization of hepatic and intestinal glucuronidation of magnolol: application of the

relative activity factor approach to decipher the contributions of multiple UDP-glucuronosyltransferase isoforms. Drug Metab. Dispos. 40, 529−538. (21) Court, M. H. (2010) Interindividual variability in hepatic drug glucuronidation: studies into the role of age, sex, enzyme inducers, and genetic polymorphism using the human liver bank as a model system. Drug Metab. Rev. 42, 209−224. (22) Zhang, J. W., Liu, Y., Zhao, J. Y., Wang, L. M., Ge, G. B., Gao, Y., Li, W., Liu, H. T., Liu, H. X., Zhang, Y. Y., Sun, J., and Yang, L. (2008) Metabolic profiling and cytochrome P450 reaction phenotyping of medroxyprogesterone acetate. Drug Metab. Dispos. 36, 2292−2298. (23) Uchaipichat, V., Mackenzie, P. I., Elliot, D. J., and Miners, J. O. (2006) Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human udp-glucuronosyltransferases. Drug Metab. Dispos. 34, 449− 456. (24) Zhang, Y. Y., Liu, Y., Zhang, J. W., Ge, G. B., Liu, H. X., Wang, L. M., Sun, J., and Yang, L. (2009) C-7 configuration as one of determinants in taxanes metabolism by human cytochrome P450 enzymes. Xenobiotica 39, 903−914. (25) Barbier, O., Turgeon, D., Girard, C., Green, M. D., Tephly, T. R., Hum, D. W., and Bélanger, A. (2000) 3′-Azido-3′-deoxythimidine (AZT) is glucuronidated by human UDP-glucuronosyltransferase 2B7 (UGT2B7). Drug. Metab. Dispos. 28, 497−502. (26) Zhou, J., Tracy, T. S., and Remmel, R. P. (2011) Correlation between bilirubin glucuronidation and estradiol-3-gluronidation in the presence of model UDP-glucuronosyltransferase 1A1 substrates/ inhibitors. Drug Metab. Dispos. 39, 322−329. (27) Kemp, H. A., Read, G. F., Riad-Fahmy, D., Pike, A. W., Gaskell, S. J., Queen, K., Harper, M. E., and Griffiths, K. (1981) Measurement of diethylstilbestrol in plasma from patients with cancer of the prostate. Cancer Res. 41, 4693−4697. (28) Pacifici, G. M., Säwe, J., Kager, L., and Rane, A. (1982) Morphine glucuronidation in human fetal and adult liver. Eur. J. Clin. Pharmacol. 22, 553−558. (29) Strassburg, C. P., Strassburg, A., Kneip, S., Barut, A., Tukey, R. H., Rodeck, B., and Manns, M. P. (2002) Developmental aspects of human hepatic drug glucuronidation in young children and adults. Gut 50, 259−265. (30) Wang, H., Yuan, L., and Zeng, S. (2011) Characterizing the effect of UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A9 genetic polymorphisms on enantioselective glucuronidation of flurbiprofen. Biochem. Pharmacol. 82, 1757−1763. (31) Veurink, M., Koster, M., and Berg, L. T. (2005) The history of DES, lessons to be learned. Pharm. World Sci. 27, 139−143.

G

dx.doi.org/10.1021/tx300310k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX