estradiol-3-glucuronide and estradiol-17-beta-glucuronide

Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response.. To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M.. The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (μmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17β-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level.. The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT.. SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Topics: Adenosine Triphosphatases; Baculoviridae; Biomarkers, Pharmacological; Drug Resistance, Multiple; Estradiol; Genetic Vectors; Glucuronides; Glutathione; Humans; Leukotriene C4; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Polymorphism, Single Nucleotide; Taurochenodeoxycholic Acid

beta-estradiol 17-(beta-D-glucuronide) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G. The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized. Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition. E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM). In summary, E217G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E23G. The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.

Topics: Allosteric Site; Animals; Biological Transport; Dose-Response Relationship, Drug; Estradiol; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Protein Binding; Rats

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATP-dependent transport of 17beta-estradiol-(17-beta-D-glucuronide) (E(2)17betaG). E(2)17betaG transport was saturable, with K(m) and V(max) values of 57.8 +/- 15 microM and 53.1 +/- 20 pmol/mg/min. By contrast, with E(2)17betaG, only modest enhancement of LTC(4) transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E(2)17betaG was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C(4) (K(i(app)), 1.5 microM), glycolithocholate 3-sulfate (K(i(app)), 34.2 microM) and MK571 (K(i(app)), 28.5 microM), and lipophilic agents such as cyclosporine A (K(i(app)), 14.4 microM). Of the inhibitors tested, LTC(4) was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E(2)17betaG indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.

Topics: Biological Transport; Cells, Cultured; Cyclosporine; Estradiol; Glycocholic Acid; Humans; Kinetics; Leukotriene Antagonists; Leukotriene C4; Multidrug Resistance-Associated Proteins; Osmotic Pressure; Propionates; Quinolines; Recombinant Proteins; Transfection

Estradiol-17beta-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow. Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis. Because glutathione and HCO3- are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis. E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively. Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time. HCO3- concentrations in bile were unchanged, so that secretion paralleled variations in bile flow. In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile. The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G. These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF.

Topics: Animals; Anions; Biliary Tract; Cholestasis; Estradiol; Female; Glutathione; Glutathione Disulfide; Rats; Rats, Sprague-Dawley

This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50nM [3H]leukotriene C(4) (LTC(4)) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5'-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D(4) antagonist, MK 571, with a k(i) of 0.67 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/min/mg protein. The properties of MRP1-mediated transport of LTC(4) are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.

Topics: Adenosine Triphosphate; Antibodies; Antibody Specificity; Binding, Competitive; Biological Transport, Active; Cell Membrane; Dose-Response Relationship, Drug; Estradiol; Glutathione; HeLa Cells; Humans; Kinetics; Leukotriene C4; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Transfection; Transport Vesicles; Tritium; Vincristine

The glucuronidation of [3H]estradiol-17 beta at the C3 vs. the C17 hydroxyl groups was determined in female Sprague-Dawley rat liver microsomes. A high-performance liquid chromatography method was developed to resolve the glucuronide conjugates which were then quantitated by liquid scintillation counting. The rates of formation of 17 beta-estradiol 3-(beta-D-glucuronide) (E(2)3G) and 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17G) were 0.49 +/- 0.03 and 0.40 +/- 0.02 nmol/min/mg of protein, respectively. The apparent Km and Vmax of estradiol glucuronidation were determined in control, pregnant (day 19 of gestation), phenobarbital-treated (80 mg/kg/day i.p. for 5 days) and ethinylestradiol-treated (5 mg/kg/day i.p. for 5 days) female rats. The least-squares estimates of Km and Vmax values as well as the confidence contours of the joint sums of squares for the parameter spaces were calculated. The Vmax (nanomoles per minute per milligram of protein) for E(2)3G was significantly decreased from 0.94 to 0.57 in pregnancy and to 0.47 as a result of ethinylestradiol treatment. The Vmax values for E(2)17G were significantly different in control (0.43), pregnant (0.31) and ethinylestradiol-treated (0.27) rats. Phenobarbital treatment slightly increased the Vmax to 0.51 for E(2)17G whereas the Vmax for E(2)3G was unchanged (0.90) compared to controls. The Km (micromolar) for E(2)3G was 144 in the controls, 112 in pregnancy and 86 and 92 as a result of treatment with ethinylestradiol and phenobarbital, respectively. The Km for E(2)17G was 60 in the controls, 40 in pregnancy, 43 and 68 as a result of ethinylestradiol and phenobarbital treatment, respectively. None of the changes in Km were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Algorithms; Animals; Estradiol; Ethinyl Estradiol; Female; Kinetics; Ovary; Phenobarbital; Pregnancy; Pregnancy, Animal; Rats; Rats, Inbred Strains

Estradiol-17 beta-(beta-D-glucuronide) (E217G) was shown to be cholestatic in the isolated perfused female rat liver. Doses of 0.85, 1.3, 1.7 and 2.1 mumol decreased bile flow maximally at 15 to 30 min by 29, 37, 68 and 76% respectively. Approximately 95% of a dose of [3H]E217G was cleared from the perfusate in 30 min and was secreted in bile, with 93, 85, 71 and 55% of the dose appearing in bile within 120 min after doses of 0.85, 1.3, 1.7 and 2.1 mumol, respectively. Neither vehicle nor 1.7 mumol of estradiol-3-(beta-D-glucuronide) (E23G) significantly altered bile flow. Infusion of taurocholate at 40 and 60 mumol/hr, respectively, partially and completely protected against the cholestasis induced by 1.7 mumol of E217G. The log dose-response curve for E217G-induced cholestasis was much steeper and was shifted to the right in the presence of a 60 mumol/hr infusion of taurocholate. Infusion of taurocholate at 80 mumol/hr in the absence of E217G or E23G decreased bile flow by 79%. However, addition of 1.7 mumol of either E217G or E23G offered marked protection against taurocholate-induced cholestasis and increased bile acid secretion. Higher doses (3.4 mumol) of E217G or E23G further increased bile flow and bile acid secretion. A model system is presented to explain the mutual protection against cholestasis.

Topics: Animals; Bile; Bile Acids and Salts; Biological Transport; Cholestasis; Dose-Response Relationship, Drug; Estradiol; Female; Perfusion; Rats; Rats, Inbred Strains; Sulfobromophthalein; Taurocholic Acid

The uptake of estradiol-17 beta(beta-D-glucuronide) (E(2)17G), estriol-16 alpha(beta-D-glucuronide (E(3)16G), estradiol-17 beta-3-(beta-D-glucuronide) (E(2)3G) and taurocholate (TC) into hepatocytes isolated from male and female rats was examined and found to be linear for at least 75 sec and to exhibit Michaelis-Menten kinetics. The Vmax (nanomoles per minute per milligram of protein) for uptake in female rat hepatocytes ranged from 0.56 for TC to 2.32 for E(3)16G and from 0.89 for TC to 1.62 for E(2)17G in males. For TC, E(2)17G and E(2)3G, the Vmax for uptake was the same or higher in males, whereas for E(3)16G the Vmax was approximately 2-fold higher in females. The Km for TC was approximately equal in males and females, whereas for E(2)17G and E(3)16G, males exhibited a 3- to 9-fold lower Km. The rate of uptake of E(2)17G (100 microM) was decreased in the presence of carbonylcyanide-m-chlorophenylhydrazone (23%), 2,4-dinitrophenol (54%), potassium cyanide (38%), iodoacetic acid (46%) and rotenone (50%) and was reduced by 30 to 40% when sodium was replaced with lithium or choline or in the presence of ouabain. The rate of uptake of all the organic anions was reduced by 80 to 85% at 0-4 degrees C and the cell/medium concentration ratios at 75 sec (37 degrees C) exceeded 1. Thus, the steroid glucuronides are taken up by the hepatocyte by a saturable process; E(2)17G uptake was found to be partially dependent upon metabolic energy and an intact sodium gradient. Substrate-dependent differences in the rate of uptake between male and female rat hepatocytes were also seen.

Topics: Animals; Estradiol; Estriol; Female; Kinetics; Liver; Male; Rats; Rats, Inbred Strains; Sex Characteristics; Taurocholic Acid

Studies were conducted to determine the absorption and metabolic fate of 3H-estradiol-17 beta-glucuronide (3H-E2-G) in swine. The conjugate, 3H-E2-G (48.7 x 10(6) DPM, 45.5 Ci/mmol), was injected into ligated 15-cm sections of duodenum, proximal jejunum, distal jejunum, ileum and spiral colon of 10 kg female pigs. Blood from the jugular and portal veins and urine were collected at .5-h intervals for 5 h. Absorption from the colon was rapid and radioactivity peaked in both portal and jugular plasma by .5 h postinjection. In contrast, the highest plasma estrogen concentration from most other sections was reached at 5 h, the last sampling time. The urinary excretion patterns were nearly identical to those seen in plasma, with the radioactivity peaking early (1.5 h) after instillation of 3H-E2-G into the colon, but still rising at the end of the experiment after instillation into the duodenum, distal jejunum and ileum. The proximal jejunum, which produced low plasma estrogen concentrations, also produced low urine concentrations. The slower absorption of 3H-E2-G compared to 14C-estradiol-17 beta is consistent with the view that the limiting factor for the absorption of the conjugate is hydrolysis to a free estrogen. The predominant metabolites in portal venous plasma from all sections of the intestine at the end of the experiments were the monoglucuronides of estrone and estradiol. Because the administered 3H-E2-G was conjugated at C-17, the presence of estrone glucuronide in portal plasma indicates that, at least in the duodenum, ileum and colon, 3H-E2-G undergoes cleavage, followed by the oxidation of estradiol to estrone, which is subsequently reconjugated by the intestinal mucosa.

Topics: Animals; Chromatography, Thin Layer; Estradiol; Estrone; Female; Intestinal Absorption; Intestinal Mucosa; Swine

In attempt to find a more reliable and earlier predictor of ovulation, radioimmunoassay methods were investigated which could measure the levels of steroid glucuronides (follicular estrogens), varying levels of which would warn of approaching ovulation well before (up to 96 hours) its occurrence so that abstinence can be practiced during the period of sperm viability before the egg is released. To this end, daily urine collections were made on 9 subjects and the steroid glucuronides were totalled. In addition, the excretion of 5 beta-pregnanediol-3 alpha-glucuronide was measured throughout the menstrual cycle. Measurement of this parameter seemed of more practical value since the ratio of estrone-3-glucuronide/pregnanediol-3 alpha-glucuronide rises during the periovulatory period and falls sharply after the formation of the corpus lutuem. This parameter, unlike measurement of total steroid glucuronides, is not dependent on the rate of excretion or on concentration. In all 9 subjects, a significant increase in this ratio (P .05) was detected 48-120 hours before the plasma lutropin maximum. For assay, this ratio value is easier to assess because the assay kit would be applicable to early morning urine samples and would not involve any measurement of urine volume or time. A short discussion follows the experimental presentation.

Topics: Estradiol; Estriol; Estrogens; Estrone; Female; Fertility; Glucuronates; Humans; Menstruation; Ovulation