Hepatic steroid sulfatase critically determines estrogenic activities of conjugated equine estrogens in human cells in vitro and in mice.

Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women. Extracted from pregnant mare urine, CEEs are composed of nearly a dozen estrogens existing in an inactive sulfated form. To determine whether the hepatic steroid sulfatase (STS) is a key contributor to the efficacy of CEEs in HRT, we performed estrogen-responsive element (ERE) reporter gene assay, real-time PCR, and UPLC-MS/MS to assess the STS-dependent and inflammation-responsive estrogenic activity of CEEs in HepG2 cells and human primary hepatocytes. Using liver-specific STS-expressing transgenic mice, we also evaluated the effect of STS on the estrogenic activity of CEEs in vivo We observed that CEEs induce activity of the ERE reporter gene in an STS-dependent manner and that genetic or pharmacological inhibition of STS attenuates CEE estrogenic activity. In hepatocytes, inflammation enhanced CEE estrogenic activity by inducing STS gene expression. The inflammation-responsive estrogenic activity of CEEs, in turn, attenuated inflammation through the anti-inflammatory activity of the active estrogens. In vivo, transgenic mice with liver-specific STS expression exhibited markedly increased sensitivity to CEE-induced estrogenic activity in the uterus resulting from increased levels of liver-derived and circulating estrogens. Our results reveal a critical role of hepatic STS in mediating the hormone-replacing activity of CEEs. We propose that caution needs to be applied when Premarin is used in patients with chronic inflammatory liver diseases because such patients may have heightened sensitivity to CEEs due to the inflammatory induction of STS activity.

Biodegradation of estrogen conjugates by bacteria isolated from river sediments.

The objective of this study was to investigate the ability of E. coli in river sediments to degrade estrogen conjugates. Biodegradation experiments on glucuronide estrogens (E1-GLU, E2-GLU and E3-GLU) using E. coli, non-E. coli bacteria as well as sediment crude extracts were carried out in batch mode. A pure identified E. coli strain (KCTC 2571) was used for comparison of enzyme activity. The results showed that the degradation rate of estrogen conjugates by KCTC 2571 and E. coli isolated from sediments followed a similar trend. Fecal bacteria showed a high ability to deconjugate glucuronided estrogens. Approximately 50% of glucuronide moieties were cleaved within 4 h of contact time in experiments using pure E. coli. The degradation rate was slower in experiments using crude extracts of sediments, and conjugated estrogens were not completely degraded even after 12 h of reaction. These results provide a clear understanding of the fate and behavior of estrogen by bacteria in the environment.

The molecular etiology of breast cancer: evidence from biomarkers of risk.

Estrogens can become endogenous carcinogens via formation of catechol estrogen quinones, which react with DNA to form specific depurinating estrogen-DNA adducts. The mutations resulting from these adducts can lead to cell transformation and the initiation of breast cancer. Estrogen metabolites, conjugates and depurinating DNA adducts in urine samples from 46 healthy control women, 12 high-risk women and 17 women with breast cancer were analyzed. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of the ratios of depurinating DNA adducts to their respective estrogen metabolites and conjugates were significantly higher in high-risk women (p < 0.001) and women with breast cancer (p < 0.001) than in control subjects. The high-risk and breast cancer groups were not significantly different (p = 0.62). After adjusting for patient characteristics, these ratios were still significantly associated with health status. Thus, the depurinating estrogen-DNA adducts are possible biomarkers for early detection of breast cancer risk and response to preventive treatment.

Molecular characterization of a B-ring unsaturated estrogen: implications for conjugated equine estrogen components of premarin.

Conjugated equine estrogens (CEEs) are routinely used for hormone replacement therapy (HRT), making it important to understand the activities of individual estrogenic components. Although 17beta-estradiol (17beta-E2), the most potent estrogen in CEE, has been extensively characterized, the actions of nine additional less potent estrogens are not well understood. Structural differences between CEEs and 17beta-E2 result in altered interactions with the two estrogen receptors (ERalpha and ERbeta) and different biological activities. To better understand these interactions, we have determined the crystal structure of the CEE analog, 17beta-methyl-17alpha-dihydroequilenin (NCI 122), in complex with the ERalpha ligand-binding domain and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator. NCI 122 has chemical properties, including an unsaturated B-ring and 17alpha-hydroxyl group, which are shared with some of the estrogens found in CEEs. Structural analysis of the NCI 122-ERalpha LBD-GRIP1 complex, combined with biochemical and cell-based comparisons of CEE components, suggests that factors such as decreased ligand flexibility, decreased ligand hydrophobicity and loss of a hydrogen bond between the 17-hydroxyl group and His524, contribute significantly to the reduced potency of CEEs on ERalpha.

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP.

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E1-G), estradiol-3- and 17-glucuronides (E2-3G and E2-17G), as well as estriol-3- and 16-glucuronides (E3-3G and E3-16G) are found in human plasma and urine. Unlike E2-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E1-G, E2-3G, E3-3G, E3-16G and estrone-3-sulfate (E1-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E1-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E1-G and E2-3G, were still transported by BCRP at 10-fold higher rates than E1-S. BCRP also transported E3-16G at higher rates than the studied MRPs, while it transported E3-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E1-G, E2-3G, E3-3G and E3-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high Km values, between 180 and 790 µM. MRP3 transported all the tested glucuronides at rather similar rates, at Km values below 20 µM, but lower Vmax values than other transporters. In the case of E3-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E3-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.

Identification of an estrogenic hormone receptor in Caenorhabditis elegans.

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.

The differential association of conjugated equine estrogen and esterified estrogen with activated protein C resistance in postmenopausal women.

OBJECTIVES: Clinical trials have demonstrated that oral conjugated equine estrogen (CEE) therapy with or without medroxyprogesterone (MPA) increases venous thrombotic risk but this safety issue has not been investigated for other oral estrogens. Based on observational study findings that esterified estrogen (EE) was not associated with venous thrombotic risk whereas CEE was, we hypothesized that CEE users would be more resistant to activated protein C (APC), a prothrombotic phenotype, than EE users. METHODS: We conducted an observational, cross-sectional study of postmenopausal women 30-89 years old who were controls in a case-control study of venous thrombosis. Use of CEE, EE, and MPA at the time of phlebotomy was determined using computerized pharmacy records. APC resistance was measured in plasma by the endogenous thrombin potential normalized APC sensitivity ratio. Adjusted mean APC resistance values were compared across estrogen type and CEE:EE ratios are presented. RESULTS: There were 119 CEE and 92 EE users at the time of phlebotomy. Compared with EE users, CEE users had APC resistance measures that were 52% higher (1.52; 95% confidence intervals: 1.07-2.17) in adjusted analyses. Restricting to modal dose users (0.625 mg) and stratifying by MPA use did not materially change associations. CONCLUSIONS: CEE use was associated with higher levels of APC resistance when compared with EE use in postmenopausal women. These findings might provide an explanation for the higher risk of venous thromboembolism previously observed with CEE compared with EE use and, if replicated, may have safety implications for women when choosing an estrogen for symptom relief.

Select estrogens within the complex formulation of conjugated equine estrogens (Premarin) are protective against neurodegenerative insults: implications for a composition of estrogen therapy to promote neuronal function and prevent Alzheimer's disease.

BACKGROUND: Results of the Women's Health Initiative Memory Study (WHIMS) raised concerns regarding the timing and formulation of hormone interventions. Conjugated equine estrogens (CEE), used as the estrogen therapy in the WHIMS trial, is a complex formulation containing multiple estrogens, including several not secreted by human ovaries, as well as other biologically active steroids. Although the full spectrum of estrogenic components present in CEE has not yet been resolved, 10 estrogens have been identified. In the present study, we sought to determine which estrogenic components, at concentrations commensurate with their plasma levels achieved following a single oral dose of 0.625 mg CEE (the dose used in the WHIMS trial) in women, are neuroprotective and whether combinations of those neuroprotective estrogens provide added benefit. Further, we sought, through computer-aided modeling analyses, to investigate the potential correlation of the molecular mechanisms that conferred estrogen neuroprotection with estrogen interactions with the estrogen receptor (ER). RESULTS: Cultured basal forebrain neurons were exposed to either beta-amyloid(25-35) or excitotoxic glutamate with or without pretreatment with estrogens followed by neuroprotection analyses. Three indicators of neuroprotection that rely on different aspects of neuronal damage and viability, LDH release, intracellular ATP level and MTT formazan formation, were used to assess neuroprotective efficacy. Results of these analyses indicate that the estrogens, 17alpha-estradiol, 17beta-estradiol, equilin, 17alpha-dihydroequilin, equilinen, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, and Delta8,9-dehydroestrone were each significantly neuroprotective in reducing neuronal plasma membrane damage induced by glutamate excitotoxicity. Of these estrogens, 17beta-estradiol and Delta8,9-dehydroestrone were effective in protecting neurons against beta-amyloid25-35-induced intracellular ATP decline. Coadministration of two out of three neuroprotective estrogens, 17beta-estradiol, equilin and Delta8,9-dehydroestrone, exerted greater neuroprotective efficacy than individual estrogens. Computer-aided analyses to determine structure/function relationships between the estrogenic structures and their neuroprotective activity revealed that the predicted intermolecular interactions of estrogen analogues with ER correlate to their overall neuroprotective efficacy. CONCLUSION: The present study provides the first documentation of the neuroprotective profile of individual estrogens contained within the complex formulation of CEE at concentrations commensurate with their plasma levels achieved after an oral administration of 0.625 mg CEE in women. Our analyses demonstrate that select estrogens within the complex formulation of CEE contribute to its neuroprotective efficacy. Moreover, our data predict that the magnitude of neuroprotection induced by individual estrogens at relatively low concentrations may be clinically undetectable and ineffective, whereas, a combination of select neuroprotective estrogens could provide an increased and clinically meaningful efficacy. More importantly, these data suggest a strategy for determining neurological efficacy and rational design and development of a composition of estrogen therapy to alleviate climacteric symptoms, promote neurological health, and prevent age-related neurodegeneration, such as AD, in postmenopausal women.

Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump.

Previous studies have identified the ATP-dependent export of glutathione conjugates as a physiological function of the multidrug resistance protein (MRP). The involvement of MRP in the transport of endogenous and xenobiotic conjugates was investigated further using membrane vesicles from MRP-transfected HeLa cells. The ATP-dependent transport of the glutathione conjugates [(3)H]leukotriene C(4), S-(2,4-dinitrophenyl)-[(3)H]glutathione, and (3)H- labeled oxidized glutathione was characterized by determination of the transport efficiency V(max):K(m) amounting to 1031, 114, and 7.1 ml multiplied by min(-1), respectively. Additional endogenous substrates for MRP-mediated transport included the steroid conjugate 17 beta- glucuronosyl [(3)H]estradiol and the bile salt conjugates [6 alpha-(14)C]glucuronosylhyodeoxycholate and 3 alpha-sulfatolithocholyl [(3)H]taurine. The K(m) value of MRP for 17-beta-glucuronosyl [(3)H]estradiol was 1.5 +/- 0.3 microM, with a V(max):K(m) ratio of 42 ml multiplied by mg protein(-1) multiplied by min(-1), and a K(i) value of 0.7 microM for the leukotriene receptor antagonist MK 571. MRP-mediated ATP-dependent transport was observed for the anticancer drug conjugates glucuronosyl [(3)H]etoposide and monocholoro-mono[(3)H]glutathionyl melphalan, but not for unmodified [(14)C]doxorubicin, [(3)H]daunorubicin, or [(3)H]vinblastine. Our results establish that MRP functions as an ATP-dependent export pump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds.

16-Hydroxylation of estrone-3-sulfate and estrone in the guinea pig in vivo.

[6,7-3H]Estrone-3-sulfate or [6,7-3H]-estrone of high specific activity was injected into adult female English Shorthair guinea pigs. Blood, liver, kidney, gall bladder bile, and urine were obtained and investigated for metabolites. Chromatographic procedures followed by enzymatic or solvolytic cleavage of conjugates and subsequent crystallization with appropriate carrier steroids revealed the pattern of metabolites formed. Injected estrone sulfate was partially hydrolyzed and reconjugated, resulting in the production of estrone and estradiol glucuronides. The main metabolites, however, were monosulfates of 16alpha-hydroxyestrone, 16-keto-17beta-estradiol, and estriol as well as disulfates of 16alpha-hydroxyestrone, estriol, and 16beta-hydroxyestrone. Particularly high amounts of these were found in urine. By far the main metabolites of injected estrone were glucuronides of estrone and estradiol, although the pattern of mono- and disulfated steroids was qualitatively similar to that found after estrone sulfate injection. It is concluded that the guinea pigs employed in the study hydroxylated estrogen in the 16alpha- and 16beta-configurations and that this activity was much more pronounced after injection of estrone sulfate than after estrone.

Hydrolysis of estrogen conjugates by the lungs of the dog in vivo.

Although hydrolysis of certain estrogen conjugates released into the systemic circulation is now known to occur in the human and in the dog, the site or sites of such hydrolysis remained unidentified. In an attempt to identify such sites, radioactive estrone was infused intravenously into a series of male dogs and serial paired samples of blood were obtained from the right ventricle (RV) and a peripheral artery (A). The samples were analysed for total radioactivity, unconjugated radioactive estrogens, radioactive estrone, estradiol-17beta, estrone sulfate, estrone glucosiduronate and estradiol-17beta glucosiduronate(s). The mean percent extraction across the lungs (see article) was calculated for each radioactive moiety analyzed. There was no significant percent extration of total radioactivity or of estrone sulfate. The mean percent extractions for unconjugated estrogens and for estrone and estradiol-17beta were -24.7, -21 and -34.3 respectively (P less than 0.01), indicating net production by the lungs. The mean percent extractions for estrone glucosiduronate and estradiol-17beta glucosiduronate(s) were +19.8 and +10.0 respectively (P less then 0.01), indicating net uptake of these conjugates by the lungs. Statistical analysis showed that the two processes were related. Our findings support the hypothesis that the lungs are one site, possibly the main site, at which hydrolysis of estrogen glucosiduronates occurs in the dog.

Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice.

We have developed a novel osteotropic prodrug of estradiol (E(2)) conjugated with L-Asp-hexapeptide (E(2).3D(6)), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E(2) x 3D(6) to mice, the half-time for elimination from plasma was about 100 min; however, E(2) was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E(2), the half-time in plasma was about 70 min, whereas E(2) was highly distributed to the uterus, and the bone concentration of E(2) was only slightly increased at 6 h. When E(2) (0.37 micromol/kg, sc, every third day) or E(2) x 3D(6) (0.11 to 1.1 micromol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E(2) increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E(2) x 3D(6) increased only the BMD, in a dose-dependent manner. E(2) x 3D(6) enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen alpha) of OVX mice at 4 h after administration, but E(2) did very slightly. These results indicate that the E(2) prodrug was delivered to the bone, where it gradually released E(2), thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.

Sulfohydrolase activity for estrone sulfate and dehydroepiandrosterone sulfate in human fetal membranes and decidua around the time of parturition.

We examined the distribution and kinetic parameters of sulfohydrolase activity in human amnion, chorion, and decidua using estrone sulfate (E1S) and dehydroepiandrosterone sulfate as substrates. Amnion contained low levels of sulfatase activity. Chorion had active sulfohydrolase activity for both substrates, but a significantly greater maximum velocity (Vmax) for E1S. The Km was not different between the two substrates. However, there was a slight but statistically significant decrease in Km and increase in Vmax for sulfohydrolase activity using E1S in chorion from patients delivering vaginally after the spontaneous onset of labor compared to those delivering by elective cesarean section before the onset of labor but at a similar gestational age. Decidua possessed sulfohydrolase for E1S with similar Km and Vmax as chorion. There were no changes occurring around the onset of labor. Using dehydroepiandrosterone sulfate as substrate, the decidua had a similar Km as the chorion, but its Vmax was significantly less. In both tissues for both substrates, the enzyme had highest specific activity in the 105,000 X g pellet, with almost no activity in the soluble fraction. The greatest total sulfohydrolase activity was contained in the 800 X g pellet despite several methods of homogenization and washing of the 800 X g pellet. We conclude that the sulfohydrolase activity of human chorion and decidua may be an important factor in regulating free steroid levels within the pregnant uterus. The significant change in the kinetic parameters of E1S sulfatase may partially explain the increased ability of chorion to hydrolyze E1S which occurs in association with the spontaneous onset of labor.

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions.

The constant infusion of [3H]equilin sulfate ([3H]EqS) was used to estimate the MCR of equilin sulfate (EqS) and to measure the conversion of this estrogen to equilin (Eq), equilenin (Eqn), equilenin sulfate (EqnS), 17 beta-dihydroequilin (17 beta-Eq), 17 beta-dihydroequilin sulfate (17 beta-EqS), 17 beta-dihydroequilenin (17 beta-Eqn), and 17 beta-dihydroequilenin sulfate (17 beta-EqnS) in normal postmenopausal women and men. Infusion of [3H]EqS was started in five postmenopausal women and two men 30 min after a priming dose and continued at a constant rate of 12-15 microCi/h for 3 h. Blood samples were taken 15 min before the end of infusion, at the end of the infusion, and 15 min after the end of infusion. Unconjugated and sulfate-conjugated Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated from plasma. The mean MCR of EqS was calculated to be 280 +/- 24 L/day or 170 +/- 18 L/day.m2. The mean conversion ratios for precursor EqS to product 17 beta-EqS, EqnS, 17 beta-EqnS, 17 beta-Eq, Eq, Eqn, and 17 beta-Eqn were 0.300, 0.190, 0.100, 0.020, 0.016, 0.008, and 0.004 respectively. In both the sulfate-conjugated and unconjugated forms, 17 beta-Eq was the most abundant metabolite formed. 17 beta-Eq estrogen is a potent uterotropic agent and has a much higher affinity for estrogen receptors than Eq. Its formation may be of importance in the overall biological activity of EqS present in conjugated equine estrogen preparations.

Estriol conjugates in human breast cyst fluid and in serum of premenopausal women.

A sensitive method was developed for the assay of the four major estriol (E3) conjugates in human breast cyst fluid and in serum during the menstrual cycle. In the cyst fluid, estriol-3-sulfate (E3-3S) was found in each of ten samples, the concentrations ranging from 240-4310 pg/ml. Estriol-16-glucosiduronate (E3-16G) was detected in six samples at levels of 19-153 pg/ml; estriol-3-glucosiduronate (E3-3G) in five samples, 13-79 pg/ml and estriol-3-sulfate-16-glucosiduronate (E3-3S-16G) in four samples, 28-152 pg/ml. Unconjugated estriol was found in three of the ten cases (12-30 pg/ml). Serum samples obtained in the follicular and luteal phases of the cycle from eight different subjects were assayed in the same way. There was considerable variation between subjects and many values were indistinguishable from zero. But preliminary data suggest that E3-3G is the predominant E3 conjugate in the serum and E3-3S-16G is quantitatively least important. It appears that E3 conjugates in the cyst fluid are not derived from the blood directly, but are produced locally from precursors which have not been identified.

Specific metabolic pathways of steroid sulfates in human liver microsomes.

The hydroxylation of steroid sulfates has been studied in liver microsomal preparations from adult humans. Ten different C18, C19, and C21 steroid sulfates and the corresponding unconjugated steroids were used as substrates. In several cases it was found that steroid sulfates were efficiently hydroxylated in a way that differed both qualitatively and quantitatively from the hydroxylation of the corresponding unconjugated substrates. Only the hydrophobic (nonsulfurylated) end of the steroid sulfate molecule was hydroxylated. The steroids sulfurylated in position 3 were generally better substrates for the liver microsomal hydroxylase system than those sulfurylated in position 17. The findings that unconjugated and sulfoconjugated steroids are metabolized along different pathways in the liver may be of general significance. Sulfoconjugation and subsequent hydroxylation may also be an important pathway in the metabolism of xenobiotics in man.

Differential permeability of uterine and liver vascular beds to estrogens and estrogen conjugates.

The role of capillary membrane permeability and the effect of plasma protein binding on the influx of unconjugated and conjugated estrogens into a target organ, the uterus, and a metabolic organ, the liver, were studied in anesthetized rats. In the absence of plasma proteins, estrone (E1) and estradiol (E2) were freely diffusible through the uterine capillaries, but influx was significantly reduced for estriol (E3) and estetrol. In the uterus, the influx of the conjugated estrogens was markedly restricted and approximated the influx of dextran, a vascular space marker. The polarity of the compound (based on the number of hydrogen bond-forming functional groups and the presence of charged groups) appeared to predict uterine endothelial membrane permeability better than the octanol/Ringer's partition coefficient. In contrast to the selective permeability properties of the uterine endothelial barrier, the limiting membrane lining the hepatic microcirculation, the hepatocyte cell membrane, was highly permeable to all unconjugated and conjugated estrogens. The addition of 4% albumin to the injection solution led to a significant inhibition of uterine influx of E2, but not E1 or E3. In the liver, only the influx of E1 sulfate was slightly diminished by 4% albumin. In all cases, the influx of estrogens greatly exceeded the rate that would be expected if only the fraction that was free (dialyzable) in vitro was diffusible in vivo. Human sera containing sex hormone-binding globulin and albumin caused inhibition of influx of E1 and E2 through the uterine capillary barriers, whereas in the liver, the influx of E2 sulfate, and E3 glucuronide were diminished. The results are compatible with a difference in permeability of the microvasculature of the two organs and a differential availability of protein-bound estrogen for influx into liver and uterus. With the exception of E1, which is nearly completely diffusible into both organs, the influx of estrogens and estrogen conjugates into liver is greatly amplified compared to that into a peripheral organ such as the uterus.

Studies of phenolic steroids in human subjects. XIX. Renal conjugation of 15alpha-hydroxyestradiol-3-sulfate.

In order to elucidate further the role of the kidney in the conjugation and excretion of 15alpha-hydroxy-estrogens, we synthesized doubly and singly labeled 15alpha-hydroxyestradiol-3-sulfate (15alpha-OHE-S) and determined its urinary excretory pattern after administering differently labeled 15alpha-OHE2-S into the renal artery and concomitantly into a peripheral vein and by the infusion of doubly labeled 3H-15alpha-OHE2-S-35S into the renal artery. The excretion of radioactivity in the early urinary samples and the conjugate pattern in these urines were determined. About twice as much radioactivity was excreted in the early urine samples originating from the renal artery injection than from samples obtained following peripheral administration, indicating renal conjugation and excretion of the 15alpha-OHE2-S. The latter was preponderantly excreted as the S-NAG with only a small part (about 10%) of the administered sulfate being hydrolyzed to the aglycone. The latter was then either conjugated with N-acetylglucosamine (NAG) or glucuronide, probably also in the kidney. Most of the 15-alpha-OHE2-S could not be accounted for, possibly because of biliary excretion and enterohepatic circulation. The results obtained and those already published indicate that the human kidney is capable of conjugating 15alpha-OHE2, its NAG, and its sulfate conjugates.

A naturally occurring mutation in MRP1 results in a selective decrease in organic anion transport and in increased doxorubicin resistance.

The human 190 kDa multidrug resistance protein, MRP1, is a polytopic membrane glycoprotein that confers resistance to a wide range of chemotherapeutic agents. It also transports structurally diverse conjugated organic anions, as well as certain unconjugated and conjugated compounds, in a reduced glutathione-stimulated manner. In this study, we characterized a low-frequency (<1%) naturally occurring mutation in MRP1 expected to cause the substitution of a conserved arginine with serine at position 433 in a predicted cytoplasmic loop of the protein. Transport experiments with membrane vesicles prepared from transfected human embryonic kidney cells and HeLa cells revealed a two-fold reduction in the ATP-dependent transport of the MRP1 substrates, leukotriene C4 (LTC4) and oestrone sulphate. Kinetic analysis showed that this reduction was due to a decrease in Vmax for both substrates but Km was unchanged. In contrast, 17beta-oestradiol-17beta-(D-glucuronide) transport by the Arg433Ser mutant MRP1 was similar to that by wild-type MRP1. Fluorescence confocal microscopy showed that the mutant MRP1 was routed correctly to the plasma membrane. In contrast to the reduced LTC4 and oestrone sulphate transport, stably transfected HeLa cells expressing Arg433Ser mutant MRP1 were 2.1-fold more resistant to doxorubicin than cells expressing wild-type MRP1, while resistance to VP-16 and vincristine was unchanged. These results provide the first example of a naturally occurring mutation predicted to result in an amino acid substitution in a cytoplasmic region of MRP1 that shows an altered phenotype with respect to both conjugated organic anion transport and drug resistance.