A glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes for voltammetric determination of the illicit growth promoter dienestrol in animal urine.

An electroanalytical method for determining dienestrol (DNL) in bovine urine samples is described. A glassy carbon electrode (GCE) modified with silver nanoparticles and functionalized multi-walled carbon nanotubes was used as working sensor. The modified GCE displays substantial analytical improvements including an amplified signal, fast electron transfer kinetics, and resistance to fouling. The irreversible oxidation signal of DNL is pH-dependent. Best reactivity is found at pH 3.0, where a typical anodic peak is recorded at 0.8 V (vs. Ag/AgCl). Square-wave voltammetry revealed a 8.4 nM detection limit (1.9 µg L-1), good repeatability and reproducibility (RSDs <5.0%), and good accuracy (93.2-99.4% recovery from spiked samples). The modified electrode is highly stable even in the presence of ions (Na+ and K+), urea and uric acid. The electrochemical sensor fulfills all requisites to be used as forensic device in surveillance of illegal livestock practices. Graphical abstract Schematic presentation of the construction of a glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes. This sensor exhibited a remarkable performance for voltammetric detection of the illicit growth promoter dienestrol in animal urine.

Multiplug filtration cleanup method with multi-walled carbon nanotubes for the analysis of malachite green, diethylstilbestrol residues, and their metabolites in aquatic products by liquid chromatography-tandem mass spectrometry.

The food safety supervision in aquatic products has raised public concern in recent years. In this study, a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantification and identification of four residues of the ever widely used analytes (including malachite green, leucomalachite green, diethylstilbestrol, and dienestrol) in aquaculture samples was developed. For sample preparation, a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used, which was initially developed for pesticide residue analysis. For cleanup procedure, low-temperature cleanup method was combined with multiplug filtration cleanup (m-PFC) method based on multi-walled carbon nanotubes (MWCNTs). The volume of water, extraction solvent, cleanup sorbents, and m-PFC procedure were optimized for carp, striped bass, and giant salamander matrices. It was validated by analyzing four residues in each matrix spiked at three concentration levels of 0.5, 5, and 50 µg/kg (n = 5). The method was successfully validated according to the 2002/657/EC guidelines. After optimization, spike recoveries were within 73-106 % and <15 % relative standard deviations (RSDs) for all analytes in the tested matrices. Limits of quantification (LOQs) for the proposed method ranged from 0.10 to 0.50 µg/kg. Matrix-matched calibrations were performed with the coefficients of determination >0.998 between concentration levels of 0.5 and 200 µg/kg. The developed method was successfully applied to the determination of residues in market samples. Graphical abstract Flow chart of multi-plug filtration cleanup combined with low-temperature cleanup method.

Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test.

BACKGROUND: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). OBJECTIVE: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. METHODS: Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. RESULTS: Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.

[Simultaneous determination of nine estrogens in eel by ultrafast liquid chromatography-tandem mass spectrometry with isotope dilution technique and solid-phase extraction].

OBJECTIVE: Developing a rapid and sensitive analytical method based on ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) with solid-phase extraction (SPE) for the simultaneous determination of nine estrogens (dienestrol, diethylstilbestrol, estrone, hexestrol, 17-alpha-estradiol, 17-beta-estradiol, estriol, 17alpha-ethinylestradiol and estradiol valerate) in eel. METHODS: After the sample was extracted by acetonitrile and cleaned by Waters Oasis HLB solid-phase extraction cartridge, the UFLC separation was performed on a Shim-pack XR-ODS II column (100 mm x 2.0 mm, 2.2 microm) with a linear gradient elution program of methanol solution containing 0.04% ammonia (v/v) and 0.04% ammonia aqueous solution (v/v) as the mobile phase. Electrospray ionization was applied and operated in the negative multiple reaction monitoring (MRM) mode. The quantitation was used by isotope internal standard dilution technique. RESULTS: The results showed that the limits of quantitation (LOQs, S/N(10) were in the range of 0.07-0.4 microg/kg, the calibration curves were in good linearities for the nine analytes in the range of 0.5-50.0 microg/L with the correlative coefficients (r2) more than 0.998, the recoveries were between 81.0% and 110.0% with the relative standard deviations (RSDs) of 1.92%-8.24%. Additional, the mass spectra characterization of the nine estrogens was discussed and the fragmentation pathways were speculated. CONCLUSION: The developed method is rapid, sensitive, specific and reproducible, and adapts not only to the simultaneous determination of the nine trace estrogens including the epimer of 17-alpha-estradiol and 17-beta-estradiol but also to the identified detection in other fish tissues.

Trace analysis of diethylstilbestrol, dienestrol and hexestrol in environmental water by Nylon 6 nanofibers mat-based solid-phase extraction coupled with liquid chromatography-mass spectrometry.

A simple, rapid and sensitive method for the determination of diethylstilbestrol (DES), dienestrol (DE) and hexestrol (HEX) was developed by using the Nylon 6 nanofibers mat-based solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS). These estrogens were separated within 8 min by LC using an ODS column and methanol/water (80/20, v/v) at a flow rate of 1.0 mL min(-1). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of the estrogens. Under the optimum SPE conditions, all target analytes in 50 mL environmental water samples can be completely extracted by 1.5 mg Nylon 6 nanofibers mat at flow rate of 3.0 mL min(-1) and easily eluted by passage of 500 µL mobile phase. By using the novel SPE-LC/MS method, good linearity of the calibration curve (r(2) ≥ 0.9992) was obtained in the concentration range from 0.10 ng L(-1) to 1.0 mg L(-1) (except for DE which was 0.20 ng L(-1) to 1.0 mg L(-1)) for all analytes examined. The limits of detection (S/N = 3) of the three estrogens ranged from 0.05 ng L(-1) to 0.10 ng L(-1). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several water samples were collected from Jinchuan River and Xuanwu Lake, and in Jinchuan River water DES was detected at 0.13 ng L(-1). The recoveries of estrogens spiked into tap water were above 98.2%, and the relative standard deviations were below 4.78%.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37-6.25 µg/kg bw/day], linuron (LIN) [1.5-50 mg/kg bw/day], flutamide (FLU) [3.5-50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 µg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+µg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (µg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.

Ability of Group IVB metallocene polyethers containing dienestrol to arrest the growth of selected cancer cell lines.

BACKGROUND: Monomeric Group IVB (Ti, Zr and Hf) metallocenes represent a new class of antitumor compounds. There is literature on the general biological activities of some organotin compounds. Unfortunately, there is little information with respect to the molecular level activity of these organotin compounds. We recently started focusing on the anti-cancer activity of organotin polymers that we had made for other purposes and as part of our platinum anti-cancer effort. METHODS: For this study, we synthesized a new series of metallocene-containing compounds coupling the metallocene unit with dienestrol, a synthetic, nonsteroidal estrogen. This is part of our effort to couple known moieties that offer antitumor activity with biologically active units hoping to increase the biological activity of the combination. The materials were confirmed to be polymeric using light scattering photometry and the structural repeat unit was verified employing matrix assisted laser desorption ionization mass spectrometry and infrared spectroscopy results. RESULTS: The polymers demonstrated the ability to suppress the growth of a series of tumor cell lines originating from breast, colon, prostrate, and lung cancers at concentrations generally lower than those required for inhibition of cell growth by the commonly used antitumor drug cisplatin. CONCLUSION: These drugs show great promise in vitro against a number of cancer cell lines and due to their polymeric nature will most likely be less toxic than currently used metal-containing drugs such as cisplatin. These drugs also offer several addition positive aspects. First, the reactants are commercially available so that additional synthetic steps are not needed. Second, synthesis of the polymer is rapid, occurring within about 15 seconds. Third, the interfacial synthetic system is already industrially employed in the synthesis of aromatic nylons and polycarbonates. Thus, the ability to synthesize large amounts of the drugs is straight forward.

Oral exposure of rats to dienestrol during gestation and lactation: Effects on the reproductive system of male offspring.

This study was aimed at determining whether dienestrol (DIES) affects reproduction in male offspring of rats following oral maternal exposure during gestation and lactation. Pregnant rats were treated from GD 6 to PND 21. Animals received 0 (control-vehicle), 0.75, 1.5, 3.12, 6.25, 12.5, 50, 75 µg/kg bw/d of DIES. A control group -without vehicle-was also included. High DIES concentrations caused abortions at 75 and 50 µg/kg bw/d, while at 12.5 µg/kg bw/d had still miscarriages. Ten male rats per group were kept alive until PND 90 to ensure sexual maturity. Body and organ weights, anogenital distance (AGD) at PNDs 21 and 90, biochemical and sperm parameters like motility, viability, morphology, spermatozoa and resistant spermatid counts, and histopathology for sexual organs and liver were determined. An increase in organ weight (liver and sexual organs) and a decrease in AGD due to vehicle were found. A reduction of sperm motility and viability, and an increase of abnormal sperm morphology were caused by DIES, which provoked a dose-dependent prostatitis. Maternal exposure to DIES induced toxicity on the reproductive system of the male offspring, which could affect the capacity of fertilization.

[Determination of four phenolic estrogens in water samples by pressure-assisted electrokinetic injection coupled with capillary electrophoresis].

A capillary electrophoresis (CE) method was developed for the simultaneous separation and determination of four phenolic estrogens (PEs), in which pressure-assisted electrokinetic injection (PAEKI) was adopted as the on-line concentration method to enrich the PEs. Several parameters affecting PAEKI conditions such as injection voltage and injection time, were systematically investigated and compared with the usual parameters. Under the optimized PAEKI conditions (-9 kV, 0.3 psi (ca. 2.1 kPa), 0.4 min), the four PEs separated completely within 7 min. Good linearities were attained in the range of 0.05-5 mg/L for hexestrol and dienestrol, and 0.1-10 mg/L for bisphenol A and diethylstilbestrol, with correlation coefficients (r) over 0.9936. The limits of detection (S/N=3) were 0.0071-0.017 mg/L, and enrichment factors ranged from 11 to 15 compared to normal hydrodynamic injection. The combined micellar electrokinetic chromatographic-PAEKI method developed was used to determine the PEs in tap water and lake water samples; limits of quantification (S/N=10) of 0.029-0.064 mg/L and 0.033-0.079 mg/L were attained, respectively, by the two sample types. Furthermore, recoveries ranged from 75.6% to 110.1% with relative standard deviations (n=5) within 4.6%-11.8%. To use this PAEKI method, researchers would only need to adjust the parameters of the CE apparatus to perform on-line enrichment of analytes, without using other reagents; this demonstrates the simplicity, rapidity, and highly automated nature of this method.

[Role of insufficient hormone production in development of osteopenia in consequence of physical loads defficiency].

Experiments with rats deprived of support loading on hind limbs by tail-suspension showed that injection of hormones participating in bone metabolism impeded the development of tibial spongy osteopenia; also, normalization of longitudinal bone growth was observed in several cases. Investigations were aimed to evaluate the effectiveness of growth hormone, sex hormones, thyreoid hormones, calcitonin, CNS stimulating ephedrine and strychnine, and graded support loads. The best results were obtained after injection of calcitonin combined with sinestrol (synthetic analog of estradiol) and graded support loads which acted as initiators and amplifiers of hormonal effects. The combination of calcitonin, sinestrol and support loads prevented osteopenia and provided the normal bone growth in length during rat's suspension.

Oxidative metabolism of diethylstilbestrol by prostaglandin synthetase.

The cooxidative metabolism of the transplacental carcinogen, diethylstilbestrol (DES), was examined using ram seminal vesicle microsomes. The major extractable metabolite was beta-dienestrol (Z,Z-DIES) and represented about 35% of the added DES in 3-min incubations supplemented with arachidonic acid. Its formation was dependent upon the presence of arachidonic acid, whereas reduced nicotinamide adenine dinucleotide phosphate failed to elicit Z,Z-DIES above background. Indomethacin and 1-phenyl-3-pyrazolidone, known inhibitors of prostaglandin synthetase, blocked Z,Z-DIES formation, probably by inhibiting the cyclooxygenase and the hydroperoxidase activities, respectively. Hydrogen peroxide and 15-hydroperoxyarachidonic acid (cosubstrates of the prostaglandin synthetase-hydroperoxidase), when replacing arachidonic acid in incubations, also supported oxidative metabolism of DES catalyzed by ram seminal vesicle microsomes. 1-Phenyl-3-pyrazolidone, but not indomethacin, inhibited the 15-hydroxyperoxyarachidonic acid-dependent formation of Z,Z-DIES. Incubation conditions which supported efficient Z,Z-DIES formation also resulted in the formation of 3,3-di(p-hydroxyphenyl)hexan-4-one and the cis-isomer of DES as well as nonextractable, protein-associated radioactivity indicating the presence of reactive intermediates. The implications of the peroxidative metabolism of DES for its toxic activity are obvious.

Involvement of prostaglandin synthetase in the peroxidative metabolism of diethylstilbestrol in Syrian hamster embryo fibroblast cell cultures.

The mechanism for induction of tumors by estrogens is still unresolved. Neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol (DES) suggests that established principles of chemical carcinogenesis may be involved. The Syrian hamster embryo fibroblast cells provide a system in which now the question can be asked whether the metabolic activation of DES is a prerequisite for its biological activity in this system. In this study, Syrian hamster embryo fibroblast cell cultures were shown to oxidatively metabolize DES to cis,cisdienestrol (Z,Z-DIES) which is a DES metabolite commonly found in vivo. The only other metabolic conversion of DES detectable in these cell cultures was the formation of the glucuronides of DES and Z,Z-DIES. Z,Z-DIES is formed more efficiently in incubations with rapidly growing cells than in cultures approaching confluence. When arachidonic acid was added to the medium, Z,Z-DIES formation was enhanced, whereas indomethacin added to the cell cultures inhibited the formation of this metabolite. These data suggest the involvement of prostaglandin synthetase in the oxidative metabolism of DES by Syrian hamster embryo fibroblast cells in culture and suggest that cooxidation may play a role for its biotransformation in whole cells. Moreover, since many competing metabolic pathways are available to DES in vivo, this present study adds important additional support to the hypothesis that metabolism of DES via a peroxidative route plays a role in its carcinogenicity.

Preparation and characterization of magnetic molecularly imprinted polymers for selective trace extraction of dienestrol in seawater.

Highly selective and efficient magnetic molecularly imprinted polymers (MMIPs) were prepared using Fe3O4@SiO2 as a magnetic supporter, 3-methacryloxypropyltrimethoxy-silane (MPS) as a silane coupling agent, DIS as a template, methacrylic acid (MAA) as a functional monomer and ethyleneglycol dimethacrylate (EGDMA) as a cross-linker for the extraction of trace residuals of the synthetic estrogen dienestrol (DIS) in seawater, which is a concern worldwide for its endocrine disruption and carcinogenic danger to human health. The obtained MMIPs were demonstrated to have spherical morphologies, core-shell structures, large binding capacities, high efficiency and selectivity. These were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and adsorption experiments. Owing to the specific binding sites, the MMIPs exhibited an almost three times higher adsorption capacity towards DIS (Qmax=4.68mgg-1) than magnetic molecularly non-imprinted polymers (MNIPs) (Qmax=1.72mgg-1). DIS in spiked seawater samples from the Weihai Bay of China was extracted and enriched by MMIPs, and satisfactory recoveries (87.3%-96.4%) with low relative standard deviation (RSD) values (2.03%-5.18%, n=5) were obtained. The limit of detection (LOD) of the method obtained was 0.16µgL-1, and the limit of quantitation was 0.52µgL-1 after MMIPs. No significant deterioration of the adsorption capacity of the MMIPs was observed after six rounds of regeneration. The results further demonstrated the applicability of the MMIPs method, a simple and straightforward method for the extraction and enrichment of DIS in seawater without any time-consuming procedures.

Synthesis of the catechols of natural and synthetic estrogens by using 2-iodoxybenzoic acid (IBX) as the oxidizing agent.

A method for the synthesis of 2-hydroxyestrone/estradiol, 4-hydroxyestrone/estradiol, 3'-hydroxydiethylstilbestrol, 3'-hydroxyhexestrol, and 3'-hydroxydienestrol is reported, in which 2-iodoxybenzoic acid (IBX) and the corresponding phenolic estrogen are reacted. Treatment of the natural estrogens, estrone/estradiol, with stoichiometric amounts of IBX in dimethylformamide initially yielded a mixture of estrone/estradiol-2,3- and -3,4-quinones, which were reduced in situ to the corresponding catechols by treatment with a 1 M aqueous solution of ascorbic acid. Chromatographic separation of the reaction products afforded 2- and 4-hydroxyestrone/estradiol in good overall yields (79%). In the case of the synthetic estrogens containing two identical phenolic rings, protection of one ring is a prerequisite for the synthesis of the monocatechol. Thus, diethylstilbestrol and dienestrol were protected at one phenol ring as their methyl ethers. The resulting monophenols were treated with stoichiometric amounts of IBX for 1 h, followed by treatment with 1 M aqueous ascorbic acid to obtain the corresponding catechols in more than 70% yield. Furthermore, the catechol of diethylstilbestrol, protected at one ring, was reduced by catalytic hydrogenation at the C3-C4 double bond to obtain 3'-hydroxyhexestrol in 90% yield. Removal of the protected methoxy groups of the synthetic estrogen catechols was carried out by treatment with a 1 M solution of boron tribromide in dichloromethane. This method is highly efficient for the preparative scale synthesis of catechols of both natural and synthetic estrogens.

[Functional state of calcitonin-producing cells of the female rats' thyroid as a result of experimental osteopenia therapy by graduated support loading combined with oestrogens and calcitonin injection].

Effects of graduated (2-hr.) support loading, synestrol and myocalcic on the thyroid C-cells was evaluated with the immunocytochemistry and cytomorphometry techniques. Graduated support loading was shown to partially recondition degraded functioning of C-cells. Synestrol injection suppressed the stimulating effect of support loading; myocalcic had a little effect on the C-cell activity. Simultaneous injection of two substances cancelled out the stimulating effect of support loading.

Estrogen stimulation of phosphatidylinositol metabolism in mouse uterine tissue.

The effect of estrogens on incorporation of labeled precursor into inositol-containing phospholipids (PI) from ovariectomized mouse uterus was investigated. The results indicate that diethylstilbestrol (DES) a potent mitogen, stimulated incorporation of myo-[3H]inositol into uterine PI in 1-3 h, with maximal incorporation occurring at 6 h. This activity followed a dose-dependent increase, with a maximal effect at 5 micrograms/kg. Incorporation of radiolabeled phosphorous into the polyphosphoinositides was also increased in estrogen-stimulated uterine tissue. Studies using a weak uterotropic DES derivative, Z,Z-dienestrol, produced an early stimulation of the PI response comparable to DES. This activity was increased by Z,Z-dienestrol, with minimal estrogen receptor occupancy, and did not result in stimulation of DNA synthesis. These findings would suggest that uterine PI stimulation may not occur via an estrogen receptor-mediated mechanism related to tissue proliferation induced by estrogens.

Diethylstilbestrol stimulates persistent phosphatidylinositol lipid turnover by an estrogen receptor-mediated mechanism in immature mouse uterus.

The effect of estrogen on phosphoinositide (PI) metabolism was evaluated in the immature mouse uterus, a tissue which undergoes estrogen-induced proliferation. Uteri isolated from untreated mice or from mice injected ip with diethylstilbestrol (DES) were incubated with [3H]myo-inositol and assessed for incorporation of label into PI lipids or inositol phosphate generation. DES administration elicited a rapid increase in [3H]myo-inositol incorporation, which persisted until at least 18 h post treatment. This effect could not be duplicated by incubation of uteri with DES in vitro, although [3H]myo-inositol incorporation in uteri removed from DES-treated mice remained elevated for 3 h of in vitro incubation. Stimulation of PI lipid metabolism by DES was blocked by ICI 164,384, a specific estrogen receptor antagonist. The effect of DES on PI metabolism consisted of a time-dependent increase in the specific activity of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and a significant increase of inositol (1,4,5)-trisphosphate mass by 12 h post treatment. These changes occur before the onset of estrogen-induced DNA synthesis. The results indicate that estrogens rapidly modulate PI lipid turnover through an estrogen receptor-mediated mechanism. Since the metabolic products of PI lipids are important for signal transduction and cellular proliferation, altered metabolism of these lipids may play an integral role in estrogen-induced mitogenesis.

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation.

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.

Qualitative study of the interaction mechanism of estrogenic drugs with tubulin.

Synthetic estrogenic drugs (E-diethylstilbestrol, erythro-hexestrol and E,E-dienestrol) inhibit tubulin assembly and erythro-hexestrol and E,E-dienestrol lead to the formation of twisted ribbon structures. For the inhibitory effect on tubulin assembly, estrogenic drugs seem to interact directly with tubulin 6S on site(s) analogous to the colchicine-site, but independent of the GTP- and vinblastine-sites. This binding does not involve tubulin tryptophanyl residues or sulfhydryl groups. The influence of temperature, calcium and magnesium on the formation of twisted ribbon structures induced by the binding of estrogenic drugs to microtubular protein and tubulin has also been studied. This formation is strongly magnesium-dependent whereas preformed twisted ribbon structures are calcium- and chilling-insensitive.