Estriol Production Research Articles

HCV Antiviral Drugs Have the Potential to Adversely Perturb the Fetal-Maternal Communication Axis through Inhibition of CYP3A7 DHEA-S Oxidation.

The hepatitis C virus (HCV) poses a great risk to pregnant people and their developing fetus, yet no HCV antiviral treatment guidelines have been established. While there has been a substantial increase in the development of HCV antivirals, the effect they have on the developing fetus remains poorly defined. Many of these drugs are metabolized through the cytochrome P450 CYP3A pathway, which is mediated by cytochrome P450 3A7 (CYP3A7) in the fetus and developing infant. In this study, we sought to investigate the effect HCV antivirals have on CYP3A7 metabolism, as this CYP enzyme plays a vital role in proper fetal and neonatal development. Of the 13 HCV antivirals we investigated, 8 (∼62%) inhibited CYP3A7 metabolic activity by 50% or more at a concentration of 20 µM. Furthermore, paritaprevir, asunaprevir, simeprevir, danoprevir, and glecaprevir all had observed half-maximal inhibitory concentrations between the range of 10 and 20 µM, which is physiologically relevant in comparison with the Km of dehydroepiandrosterone-sulfate (DHEA-S) oxidation (reported to be between 5 and 20 µM). We also discovered that paritaprevir is a time-dependent inhibitor of CYP3A7, which shifts the IC50 ∼twofold from 11 µM to 5 µM. Upon further characterization, paritaprevir inactivates DHEA-S metabolism by CYP3A7, with KI and Kinact values of 4.66 µM and 0.00954 minute-1, respectively. Depending on treatment plan and off-label drug use, HCV treatment could adversely affect the fetal-maternal communication axis by blocking fetal CYP3A7 metabolism of important endogenous hormones. SIGNIFICANCE STATEMENT: The prevalence of HCV in pregnant people is estimated at between 1% and 8% of the global population, yet little to no information exists about the risk antiviral treatment poses to the developing fetus. There is a potential risk of drugs adversely affecting mother-fetal communication by inhibiting fetal hepatic CYP3A7, an integral enzyme for estriol production. We discovered that five HCV antivirals inhibited DHEA-S metabolism by CYP3A7, and paritaprevir inactivated the enzyme. Our studies demonstrate the potential threat these drugs pose to proper fetal development.

A comprehensive analysis of metabolomics and transcriptomics to reveal major metabolic pathways and potential biomarkers of human preeclampsia placenta.

Background: The etiology of preeclampsia (PE) remains unclear. With the utilization of metabolomics, dysregulated production of several metabolic components in human plasma, such as lipids, amino acids, androgens and estrogens, was found to be important in the pathogenesis of PE. Transcriptomics adds more in-depth information, and the integration of transcriptomics and metabolomics may yield further insight into PE pathogenesis than either one alone. Objectives: We investigated the placental metabolomics and transcriptomics of PE patients to identify affected metabolic pathways and potential biological targets for exploring the disease pathogenesis. Methods: Integrated transcriptomics and metabolomics were used to analyze five paired human placentas from patients with severe PE and normal pregnancies. This was followed by further validation of our findings in a publicly available dataset of 173 PE vs. 157 control placentas. In addition, weighted gene coexpression network construction was performed to assess the correlation between genetic alterations and diseases. Results: We identified 66 and 41 differentially altered metabolites in negative and positive ion modes, respectively, in the PE group compared to the control group, and found 2,560 differentially expressed genes. Several pathways were aberrantly altered in the PE placenta at both the metabolic and transcriptional levels, including steroid hormone biosynthesis, the cAMP signaling pathway, neuroactive ligand–receptor interactions, taste transduction and prion diseases. Additionally, we found 11 differential metabolites and 11 differentially expressed genes involved in the steroid hormone biosynthesis pathway, indicating impaired metabolism of steroid hormones in the PE placenta. Furthermore, we found that CYP11A1, HSD3B2, and HSD17B6 are highly correlated with diseases. Conclusion: Our findings provide a profile of the dysregulated steroid hormone biosynthesis in PE placenta, we observed a dysregulated cortisol-to-cortisone ratio, testosterone accumulation, decreased testosterone downstream metabolites, impaired production of estrone and estriol, and aberrant hydroxylation and methylation of estradiol. Disorders of placental steroid hormone metabolism might be a consequence or a compensatory change in pathological placentation in PE, which underscores the need to investigate the physiology of steroid hormone metabolites in the etiology of PE.

CYP3A7 drug‐hormone Interactions in the Neonate: Adrenal Insufficiency and Low Birthweight in the HIV infected Neonate caused by Inhibition of CYP3A7 DHEA‐S Oxidation by Ritonavir

In the neonate and developing infant, CYP3A7 is the predominant CYP3A isoform and is responsible for the majority of metabolism of CYP3A substrates. However, its primary function is the 16α-hydroxylation of the endogenous hormone DHEA-S, which is important for maintenance of hormone homeostasis in the adrenal gland and production of estriol during the third trimester of pregnancy. Low maternal estriol levels have been reported to lead to low birth weight and premature birth. It has been previously reported that cohorts of HIV infected mothers and their newborns on a combined therapeutic regimen of Lopinavir and Ritonavir have increased adrenal insufficiency and low birth weight, as well as substantially elevated levels of DHEA-S, leading to adverse outcomes in this fragile patient group. Since Ritonavir is a known potent inhibitor of CYP3A4, we hypothesized that it might also be an inhibitor of CYP3A7-mediated DHEA-S oxidation and that this inhibition might be responsible for the adrenal insufficiency and low birth weight associated with Lopinavir-Ritonavir treatment. In order to test this hypothesis, we employed both recombinant CYP enzyme and neonatal human liver microsomes (nHLMs). We observed significant inhibition of CYP3A7 DHEA-S hydroxylation activity in the presence of Ritonavir with an IC50 of 0.05 µM. Furthermore, Ritonavir displayed time-dependent inhibition of DHEA-S oxidation, with a Ki of 0.336 µM and a Kinact of 0.1077 min-1, suggesting that it may be a mechanism based inactivator of CYP3A7. Lopinavir was also capable of inhibiting CYP3A7, albeit to a much lesser extent than Ritonavir with an IC50 of 5.88 µM for DHEA-S 16α-hydroxylation, and without the associated time-dependency. Interestingly, we observed that Lopinavir alone had a lower intrinsic clearance (Clint) rate in nHLMs compared to adult HLMs, suggesting that a pharmacokinetic enhancer adjuvant may not be necessary in this patient population in order to slow drug clearance and maintain efficacy. At a minimum, our results suggest that closer monitoring of the effective dose of the pharmacokinetic enhancer in neonatal HIV therapy may be required in order to prevent significant inhibition of CYP3A7 DHEA-S oxidation and the associated adverse effects.

CYP3A7 drug‐hormone Interactions in the Neonate: Adrenal Insufficiency and Low Birthweight in the HIV infected Neonate caused by Inhibition of CYP3A7 DHEA‐S Oxidation by Ritonavir

In the neonate and developing infant, CYP3A7 is the predominant CYP3A isoform and is responsible for the majority of metabolism of CYP3A substrates. However, its primary function is the 16α‐hydroxylation of the endogenous hormone DHEA‐S, which is important for maintenance of hormone homeostasis in the adrenal gland and production of estriol during the third trimester of pregnancy. Low maternal estriol levels have been reported to lead to low birth weight and premature birth. It has been previously reported that cohorts of HIV infected mothers and their newborns on a combined therapeutic regimen of Lopinavir and Ritonavir have increased adrenal insufficiency and low birth weight, as well as substantially elevated levels of DHEA‐S, leading to adverse outcomes in this fragile patient group. Since Ritonavir is a known potent inhibitor of CYP3A4, we hypothesized that it might also be an inhibitor of CYP3A7‐mediated DHEA‐S oxidation and that this inhibition might be responsible for the adrenal insufficiency and low birth weight associated with Lopinavir‐Ritonavir treatment. In order to test this hypothesis, we employed both recombinant CYP enzyme and neonatal human liver microsomes (nHLMs). We observed significant inhibition of CYP3A7 DHEA‐S hydroxylation activity in the presence of Ritonavir with an IC50 of 0.05 μM. Furthermore, Ritonavir displayed time‐dependent inhibition of DHEA‐S oxidation, with a Ki of 0.336 μM and a Kinact of 0.1077 min−1, suggesting that it may be a mechanism based inactivator of CYP3A7. Lopinavir was also capable of inhibiting CYP3A7, albeit to a much lesser extent than Ritonavir with an IC50 of 5.88 μM for DHEA‐S 16α‐hydroxylation, and without the associated time‐dependency. Interestingly, we observed that Lopinavir alone had a lower intrinsic clearance (Clint) rate in nHLMs compared to adult HLMs, suggesting that a pharmacokinetic enhancer adjuvant may not be necessary in this patient population in order to slow drug clearance and maintain efficacy. At a minimum, our results suggest that closer monitoring of the effective dose of the pharmacokinetic enhancer in neonatal HIV therapy may be required in order to prevent significant inhibition of CYP3A7 DHEA‐S oxidation and the associated adverse effects.Support or Funding InformationThis work was supported by the National Institutes of Health, Institute of General Medical Sciences [Grant R01 GM128508‐03] to JNL.

On the trail of steroid aromatase: The work of Kenneth J. Ryan

On the trail of steroid aromatase: The work of Kenneth J. Ryan

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3β,20S-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.

The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production

Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy.

Duration of hexobarbital sleep as a marker for resilience to postraumatic stress disoder (PTSD) in rats

tion about the concentrations before the 12th week of pregnancy is missing. Methods: The aim of this pilot study was to verify whether the sensitivity of a newly developed enzyme immunoassay of IBL International is sufficient for the determination of salivary estriol during very early pregnancy (N=25, 6th–12th week). Saliva samples were collected weekly between 10:00 am and 14.00 pm. Results: Salivary estriol was detectable from the beginning of the 6th week of pregnancy (M=3.17pg/ml, SD=2.13) and the concentrations ranged from .10 to 10.94 pg/ml. Salivary estriol concentrations showed a steep significant increase between the 10th and the 11th gestational week (t(11) =−3.847, p< .01). Conclusion: With the new enzyme immunoassay low concentrations of salivary estriol can be measured during very early pregnancy. Concordant with blood concentrations, salivary estriol starts to increase by the 10th week, when under stimulation of ACTH the placenta takes over the production of estriol from the corpus luteum. Salivary estriol may therefore serve as a promising marker for future research on the mechanisms and consequences of prenatal stress during very early pregnancy.

Higher HPA axis activity in healthy participants compared to depressed patients after MR blockade: Evidence for attenuated MR function in depression

tion about the concentrations before the 12th week of pregnancy is missing. Methods: The aim of this pilot study was to verify whether the sensitivity of a newly developed enzyme immunoassay of IBL International is sufficient for the determination of salivary estriol during very early pregnancy (N=25, 6th–12th week). Saliva samples were collected weekly between 10:00 am and 14.00 pm. Results: Salivary estriol was detectable from the beginning of the 6th week of pregnancy (M=3.17pg/ml, SD=2.13) and the concentrations ranged from .10 to 10.94 pg/ml. Salivary estriol concentrations showed a steep significant increase between the 10th and the 11th gestational week (t(11) =−3.847, p< .01). Conclusion: With the new enzyme immunoassay low concentrations of salivary estriol can be measured during very early pregnancy. Concordant with blood concentrations, salivary estriol starts to increase by the 10th week, when under stimulation of ACTH the placenta takes over the production of estriol from the corpus luteum. Salivary estriol may therefore serve as a promising marker for future research on the mechanisms and consequences of prenatal stress during very early pregnancy.

A Unique Co-culture Model for Fundamental and Applied Studies of Human Fetoplacental Steroidogenesis and Interference by Environmental Chemicals

Background: Experimental tools for studying the complex steroidogenic interactions that occur between placenta and fetus during human pregnancy are extremely limited.Objectives: We aimed to develop a co-culture model to study steroidogenesis by the human fetoplacental unit and its disruption by exposure to environmental contaminants.Methods: We cultured BeWo human choriocarcinoma cells, representing the villous cytotrophoblast, and H295R human adrenocortical carcinoma cells, representing the fetal unit, in a carefully adapted co-culture medium. We placed H295R cells in 24-well plates and BeWo cells on transwell inserts with or without pesticide treatment (atrazine or prochloraz) and assessed CYP19 activity and hormonal production after 24 hr of co-culture.Results: The co-culture exhibited the steroidogenic profile of the fetoplacental unit, allowing a synergistic production of estradiol and estriol (but not of estrone) of 133.3 ± 11.3 pg/mL and 440.8 ± 44.0 pg/mL, respectively. Atrazine and prochloraz had cell-type specific effects on CYP19 activity and estrogen production in co-culture. Atrazine induced CYP19 activity and estrogen production in H295R cells only, but did not affect overall estrogen production in co-culture, whereas prochloraz inhibited CYP19 activity exclusively in BeWo cells and reduced estrogen production in co-culture by almost 90%. In contrast, prochloraz did not affect estradiol or estrone production in BeWo cells in monoculture. These differential effects underline the relevance of our co-culture approach to model fetoplacental steroidogenesis.Conclusions: The co-culture of H295R and BeWo cells creates a unique in vitro model to reproduce the steroidogenic cooperation between fetus and placenta during pregnancy and can be used to study the endocrine-disrupting effects of environmental chemicals.Citation: Hudon Thibeault AA, Deroy K, Vaillancourt C, Sanderson JT. 2014. A unique co-culture model for fundamental and applied studies of human fetoplacental steroidogenesis and interference by environmental chemicals. Environ Health Perspect 122:371–377; http://dx.doi.org/10.1289/ehp.1307518

Aromatase inhibition by 4 beta,5 beta-epoxides of 16 alpha-hydroxyandrostenedione and its 19-oxygenated analogs, potential precursors of estriol production in the feto-placental unit.

To gain insight into the nature of the substrate binding site and the catalytic function of aromatase, we studied the inhibition of androstenedione aromatization by 4beta,5beta-epoxy-16alpha-hydroxyandrostenedione (4) and its 19-hydroxy and 19-oxo derivatives, 5 and 6, as well as the biochemical aromatization of these steroids in human placental microsomes. The 19-methyl and 19-oxo compounds, 4 and 6, were weak competitive inhibitors of aromatase, with apparent K(i) values of 246 microM and 270 microM, respectively, whereas the 19-hydroxy compound 5 inhibited aromatase in a non-competitive manner with the K(i) of 135 microM. The 19-methyl compound 4 inactivated aromatase in a time-dependent manner with k(inact) of 0.213 min(-1) in the presence of NADPH in air, but the other two did not cause it. The conversion of the three epoxides into estrogen, as well as 19-oxygenation of 19-methyl steroid 4 with human placental microsomes in the presence of NADPH in air, were not detected by gas chromatography-mass spectrometry. The present results are consistent with the two binding sites theory in the active site of aromatase.

Changes in rates of salivary estriol increases before parturition at term

Objective: The aim of this study was to characterize the increases of salivary estriol concentrations before the onset of labor at term. Study Design: Salivary estriol concentrations were measured in weekly patient-collected samples by means of a sensitive (mean ± SD threshold, 0.025 ± 0.001 ng/mL; coefficient of variation, 3.8%) direct enzyme immunoassay in a microtiter plate format. The salivary estriol concentrations in 16 healthy pregnant women were characterized from 30 weeks' gestation until the time of parturition and delivery. Samples were stored frozen at collection and analyzed in batches after delivery. Results: The median salivary estriol concentration profile revealed a nonlinear rise beginning from 30 weeks' gestation (0.89 ng/mL) until term (2.70 ng/mL, an increase of 201%). At 35 weeks' gestation the salivary estriol concentration median value increased sharply (positive inflection point, 50%-93% increase) at a demarcation between a slower increase during early pregnancy and a more rapid increase during late pregnancy. This positive inflection point associated with a late pregnancy increase characterized subgroups of pregnancies according to the lengths of gestation as follows: early term (delivered at <38 weeks 1 day's gestation), middle term (delivered at 38 weeks 1 day–40 weeks' gestation), and late term (delivered at >40 weeks' gestation). Five weeks before delivery the mean (±SEM) rate of rise in salivary estriol concentration was 0.50 ± 0.13 ng/mL per week to 0.84 ± 0.26 ng/mL per week in the early term group. The increase in rate for the middle term group was 0.32 ± 0.06 ng/mL per week to 0.37 ± 0.26 ng/mL per week, whereas in the late term group the rate of salivary estriol concentration rise was 0.37 ± 0.03 ng/mL per week to –0.03 ± 0.25 ng/mL per week. Conclusion: These data demonstrate in normal pregnancies (1) that a direct, nonradiometric measure of salivary estriol concentration can be used to monitor the late pregnancy increase in estriol production, (2) that 35 weeks' gestation marks a positive inflection point of the onset of increased estriol production, and (3) that the late pregnancy rise in salivary estriol concentration shows distinct patterns that tend to be characteristic of the length of pregnancy. These data support the concept that the rate of increase of estriol production is related to the timing of the onset of labor. (Am J Obstet Gynecol 2001;184:123-30.)

Gas chromatography-mass spectrometric determination of activity of human placental aromatase using 16alpha-hydroxyandrostenedione as a substrate.

Aromatization of 16alpha-hydroxyandrostenedione (16alpha-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using 12,4,6,6,9alpha,16beta,17alpha-2H7]estriol as an internal standard. 16alpha-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH4. The reduced product, estriol, was isolated by Sep-Pak C18 cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent Km and Vmax values of the microsomal aromatase for 16alpha-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competitive manner with 25 and 30 nM of apparent Ki values, respectively.

Comparison between oral and intramuscular dexamethasone in suppressing unconjugated estriol levels during the third trimester

Objectives : Unconjugated estriol production depends on fetal adrenal androgen precursors. Fetal exposure to exogenous glucocorticoids results in adrenal suppression with a subsequent decrease in maternal serum unconjugated estriol levels. We compared the efficacy between oral and intramuscular dexamethasone in maternal serum unconjugated estriol suppression at 48 hours after the initial dose among women at risk for preterm delivery. Study Design: Twenty-four gravidas at risk for preterm delivery were randomized to receive either 6 mg intramuscular or 8 mg oral dexamethasone every 12 hours for a total of 4 doses. Blood samples (9 mL) were obtained before the initial dexamethasone administration and again after the fourth dose. Serum was separated and frozen at –70°C and subsequently underwent batch analysis. Unconjugated estriol levels were determined by radioimmunoassay with intra-assay and interassay coefficients of variation of 7.9% and 5.5%, respectively. All values are reported as mean ± SD. The primary statistical analysis was a t test, with P < .05 considered significant. Results: At the time of dexamethasone administration, gestational ages in both groups were similar. Predexamethasone and postdexamethasone unconjugated estriol levels were also similar between the intramuscular and oral groups (5.39 ± 3.99 vs 1.80 ± 2.49 ng/mL and 6.05 ± 3.00 vs 1.61 ± 1.03 ng/mL, respectively, P > .05). No difference in percent decrease in unconjugated estriol levels was found between the intramuscular (0.67 ± 0.24) and oral (0.65 ± 0.39) groups. Conclusion: Oral dexamethasone (8 mg) produces similar maternal serum unconjugated estriol suppression compared with intramuscular dexamethasone (6 mg) when evaluated 48 hours after administration.

Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol.

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although [3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the "Estriol Hypothesis" on the protective role for estriol in opposing carcinogenic effects of estradiol.

Molecular and Kinetic Basis for the Mixed Agonist/Antagonist Activity of Estriol

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although[ 3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the “Estriol Hypothesis” on the protective role for estriol in opposing carcinogenic effects of estradiol.

The effects of the phosphorothioate insecticide fenitrothion on mammalian cytochrome P450-dependent metabolism of estradiol.

Phosphorothioate insecticides, such as fenitrothion, are suicide substrates of cytochromes P450 (P450). These compounds undergo oxidative desulfuration by P450 resulting in the release and subsequent binding of atomic sulfur to the enzyme. Consequently, the P450-dependent metabolism of certain endogenous substrates could be inhibited by exposure to these insecticides. Formation of 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 16 alpha-hydroxyestrone (16 alpha-OHE1), and estriol in mammals occurs by P450-dependent hydroxylation of estradiol at various positions on the steroid nucleus. In the present study, pretreatment of male Swiss Webster mice with increasing doses of fenitrothion resulted in dose-dependent biphasic decreases in 2-OHE2 and 4-OHE2 production in mouse hepatic microsomes compared to control, with substantial decreases even at a dosage as low as 7 mg/kg. Fenitrothion pretreatment also resulted in dose-dependent biphasic increases in 16 alpha-OHE1 and estriol production, along with substantial increases in estrone formation, probably as a result of shunting from the inhibition of 2- and 4-hydroxylation. These data suggest that exposure to fenitrothion might alter estradiol metabolism by inhibition of certain P450 isozymes.

The Effects of the Phosphorothioate Insecticide Fenitrothion on Mammalian Cytochrome P450-Dependent Metabolism of Estradiol

Phosphorothioate insecticides, such as fenitrothion, are suicide substrates of cytochromes P450 (P450). These compounds undergo oxidative desulfuration by P450 resulting in the release and subsequent binding of atomic sulfur to the enzyme. Consequently, the P450-dependent metabolism of certain endogenous substrates could be inhibited by exposure to these insecticides. Formation of 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 16α-hydroxyestrone (16α-OHE1), and estriol in mammals occurs by P450-dependent hydroxylation of estradiol at various positions on the steroid nucleus. In the present study, pretreatment of male Swiss Webster mice with increasing doses of fenitrothion resulted in dose-dependent biphasic decreases in 2-OHE2 and 4-OHE2 production in mouse hepatic microsomes compared to control, with substantial decreases even at a dosage as low as 7 mg/kg. Fenitrothion pretreatment also resulted in dose-dependent biphasic increases in 16/-OHE1 and estriol production, along with substantial increases in estrone formation, probably as a result of shunting from the inhibition of 2- and 4-hydroxylation. These data suggest that exposure to fenitrothion might alter estradiol metabolism by inhibition of certain P450 isozymes.

Corticotropin-releasing hormone and pituitary-adrenal hormones in pregnancies complicated by chronic hypertension

OBJECTIVE: We hypothesized that maternal plasma corticotropin-releasing hormone levels are elevated in chronic hypertension and that elevations modulate maternal and fetal pituitary-adrenal function. STUDY DESIGN: Venous blood samples and 24-hour urine speciemsn were obtained in normal and hypertensive pregnancies at 21 to 40 weeks of gestatin. Corticotropin-releasing hormone, corticotropin, cortisol, dehydroepiandrosterone sulfate, and total estriol levels were measured by radioimmunoassay. Mean hormone levels were compared by unpaired t test or two-way analysis of variance. RESULTS: Plasma corticotropin-releasing hormone levels were elevated early in hypertensive pregnancies but did not increase after 36 weeks. Levels of pituitary and adrenal hormones were not different in normal and hypertensive women. However, maternal plasma estriol levels were lower in hypertensive pregnancies compared with normal pregnancies. CONCLUSIONS: Fetal 16-hydroxy dehydroeplandrosterone sulfate, the major precursors to placental estriol production, has been reported to be lower than normal in hypertensive pregnancies, possibly explaining the decreased plasma estriol levels reported here. Early stimulation of placental corticotropin-releasing hormone production or section may be related to accelerated maturation of placental endocrine function in pregnancies complicated by chronic hypertension.

Estriol production and metabolism in normal women

Studies on estriol metabolism and production were carried out in normal reproductive-aged and post-menopausal women using pulse injections and constant infusions of radiolabeled estriol, estrone and estradiol. The circulating levels were measured by radioimmunoassay. Following an intravenous pulse of [ 3H]estriol, the disappearance of radioactivity as estriol could be described as a function which was sum of two exponentials with t 1 2 's of 3.6 and 64 min. The initial volume of distribution was 201. Using the constant infusion technique the metabolic clearance rate (MCR) of estriol was 2100 1/day in the follicular phase of the cycle and similar in the luteal phase. In postmenopausal women the MCR was 1890 1/day. The circulating levels of estriol were 7 and 11 pg/ml in the follicular and luteal phases respectively and 6 pg/ml in post-menopausal women. The production rates of estriol were 14 and 23 μg/day in the follicular and luteal phases of the cycle and 11 μg/day in post-menopausal women. In many of the women infused with radiolabeled estrone or estradiol, no radioactivity could be identified in the blood as estriol The maximal conversion of estrone and estradiol was < 0.4%. Estriol circulates at low but relatively steady levels in the blood. In some women estriol appears to be secreted by the ovary, especially in the luteal phase.