Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.     

Diethylstilbestrol treatment increases the amount of choline kinase in rooster liver

Studies have been performed on the mechanism by which diethylstilbestrol stimulates the activity of choline kinase in livers from cockerels. The enzyme was purified 700–900-fold by affinity chromatography. The increased enzyme activity could not be accounted for by diethylstilbestrol alteration of the kinetic constants of the enyzme. Rabbit antibody was raised to the purified enzyme. Titration studies with antiserum demonstrated a 2-fold increase in the amount of choline kinase in diethylstilbestrol-treated cytosol, which correlated with a 2-fold elevation in the activity of the enzyme. We conclude that diethylstilbestrol stimulates the activities of choline kinase in cockerel liver by promoting a corresponding increase in the amount of enzyme.     

Competitive affinity chromatography of human alpha fetoprotein on immobilized diethylstilbestrol]

Human alpha-fetoprotein (AFP) was isolated by affinity chromatography method on immobilized diethylstilbestrol from butanol extract of abortive material. Elution from the column was performed with 10% aqueous buffered butanol solution, pH 8.6. During one procedure human AFP-preparation containing about 10% of AFP and about 90% of albumin was obtained, with the yield about 60%. The preliminary incubation of extract of the abortive material with estrone raised AFP yield up to 85% with the increase of AFP content in the preparation up to 35%, and preincubation with estriol and estradiol caused the increase of the yield up to 88-92%, and AFP content in the preparation was 50% and 65%, respectively. The preincubation of human AFP with diethylstilbestrol lowers the yield of this protein, which testified to the possible binding of human AFP with free diethylstilbestrol; testosterone, hydrocortisone and desoxycorticosterone caused the increase of the yield of AFP. So the competitive variant of the affinity chromatography on immobilized diethylstilbestrol makes it possible to raise human AFP preparation purity and yield by decreasing the competition between AFP, and not binding free steroid hormones, ad albumin for immobilized diethylstilbestrol.     

Metabolism of diethylstilbestrol by horseradish peroxidase and prostaglandin-H synthase. Generation of a free radical intermediate and its interaction with glutathione

Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide-glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound.     

Affinity chromatography of mouse and rat alpha-fetoproteins on immobilized diethylstilbestrol]

Alpha-fetoproteins (AFP) from amniotic fluid of mouse and rat demonstrate high affinity and specificity during their binding with immobilized diethylstilbestrol, which allows to isolate these two proteins by one step using the method of affinity chromatography on Sepharose with immobilized diethylstilbestrol. Meanwhile the yield of mouse AFP was 42%, and rat AFP--75%. The preliminary incubation of the amniotic fluid of rat and mouse with free estradiol results in abrupt fall of AFP outcome, which may testify to the binding of estradiol and diethylstilbestrol by the same receptor sites on AFP molecule.     

Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

14C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.     

乙烯雌酚对间情期比格犬发情的诱导

目的实验分析口服乙烯雌酚诱导间情期比格犬发情的效果,研究适合可行的比格犬诱导发情的方案。方法根据比格犬由间情期到发情期血清内雌二醇浓度逐步升高的变化规律,设计逐渐给实验犬增加口服乙烯雌酚的剂量,诱导比格犬发情;同时对照组给以恒定剂量的乙烯雌酚诱导发情;空白对照组给以淀粉片。结果实验组比格犬的诱导发情率为50%,怀孕率为25%;对照组的发情率为31.25%,怀孕率为18.75%;空白对照组的发情数为零。结论隔天给药,逐渐加量乙烯雌酚组诱导发情的效果明显好于隔天给药,恒定剂量组乙烯雌酚组诱导发情的效果。     

Action of diethylstilbestrol on the NADH-dehydrogenase region of the respiratory chain

Diethylstilbestrol reversibly inhibits electron transfer in particle-bound mitochondrial NADH-dehydrogenase (Keilin-Hartree preparation) at a site located in the vicinity of, or superimposed on, the piericidin A- and rotenone-specific sites. At this site, diethylstilbestrol half-maximal inhibitory concentration varies from 0.2 μm (at 37 μm NADH) to 2.6 μm (at 180 μm or higher NADH concentrations). The postulated localization of the diethylstilbestrol site is supported by (a) the similar effects of the diethylstilbestrol, piericidin A, and rotenone on the kinetics of the NADH-induced changes of the 470–500-nm chromophore; (b) the lack of action of diethylstilbestrol on the NADH-ferricyanide reductase and the NADH-acetylpyridine-adenine dinucleotide transhydrogenase reactions; (c) the lack of action of diethylstilbestrol on the NADH-2,3-dimethoxy-2′-methyl-1,4-benzoquinone reaction at infinite concentration of UQ₀; (d) the similar inhibitions of the NADH-UQ₂ reaction and NADH-cytochrome b_k reaction; (e) the noncompetitive inhibition of the NADH-UQ₂ reaction; (f) the similar effects of diethylstilbestrol and rotenone on the kinetics of the NADH-UQ₀ reaction; and (g) the inhibition of the NADH-cytochrome b_k reaction, in contrast with the insensitivity of the succinate-cytochrome b_k reaction. The interaction of diethylstilbestrol with the NADH-dehydrogenase protein is supported by the summation of diethylstilbestrol, piericidin A, rotenone, and p-chloromercuribenzoate inhibitions. Diethylstilbestrol inhibition involves specific structural requirements as shown by the lesser (if any) inhibitory potency of hexestrol, diethylstilbestrol monomethyl ether, diethylstilbestrol dimethyl ether, and hexestrol dimethyl ether.     

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.     

Estrogen-induced luteolysis in the rhesus monkey: Reversal with indomethacin

Normally cycling Rhesus monkeys were treated with diethylstilbestrol (25 mg/day) alone or in combination with indomethacin (25 mg/day) for five consecutive days beginning in the early luteal and mid-luteal phase of the menstrual cycle. Blood specimens were obtained daily to monitor corpus luteum function (progesterone), and the length of each menstrual cycle was recorded. Diethylstilbestrol alone cause premature luteolysis as indicated by decreasing plasma progesterone and shortened menstrual cycle, and indomethacin effectively blocked the luteolytic action of diethylstilbestrol. These results suggest that the probable mechanism of diethylstilbestrol action in causing luteolysis is mediated via the prostaglandins.     

Relationship of ovarian and thyroid function

In an investigation of relationships between ovarian function and thyroid function, three groups of ten women each were studied by means of radioactive iodine uptake. In the first group no significant changes were noted during normal menstrual cycles. The second group, women who had dysfunctional uterine bleeding and were under treatment with diethylstilbestrol, most of the patients had no significant change in the uptake of radioactive iodine. Three patients did show a small increase in the uptake. (A fourth patient had a very bizarre result with a very great increase in the uptake, but it is felt that some undetected error was involved.)The third group was made up of women without evident ovarian function. Under treatment with diethylstilbestrol one patient showed a small increase in iodine uptake. The other nine had no significant change. No convincing evidence was found of any change in thyroid function as measured by the uptake of radioactive iodine-either during normal menstrual cycles or following the administration of diethylstilbestrol in dosages of 3 mg. daily for two to three weeks.     

NEWS AND NOTES

In an investigation of relationships between ovarian function and thyroid function, three groups of ten women each were studied by means of radioactive iodine uptake. In the first group no significant changes were noted during normal menstrual cycles.The second group, women who had dysfunctional uterine bleeding and were under treatment with diethylstilbestrol, most of the patients had no significant change in the uptake of radioactive iodine. Three patients did show a small increase in the uptake. (A fourth patient had a very bizarre result with a very great increase in the uptake, but it is felt that some undetected error was involved.)The third group was made up of women without evident ovarian function. Under treatment with diethylstilbestrol one patient showed a small increase in iodine uptake. The other nine had no significant change.No convincing evidence was found of any change in thyroid function as measured by the uptake of radioactive iodine—either during normal menstrual cycles or following the administration of diethylstilbestrol in dosages of 3 mg. daily for two to three weeks.     

Chronic estrogen treatment causes an alteration in uterine estrogen receptor dynamics of rats

Estrogen receptor content and dynamics in the uteri obtained from chronically estrogenized rats were analyzed. 12 day treatment with a subcutaneous implantation of a diethylstilbestrol pellet resulted in maximal stimulation of uteri with regard to wet tissue weight, DNA content, as well as progesterone receptor content without significant alteration of the estrogen receptor level. Estrogen receptor dynamics in just ovariectomized or ovariectomized and diethylstilbestrol-stimulated rats elicited by a single injection of estradiol were next examined using the exchange methods. The cytosol receptor content rapidly declined, with a small and temporary accumulation of the nuclear receptor in the uterus from rats continuously exposed to diethylstilbestrol during the preceding 12 days. A relatively rapid cytosol receptor replenishment was also observed in rats pretreated with diethylstilbestrol. This was accompanied by a rapid decrease in the nuclear receptor level to 70% of the preinjection value at 5 h after estradiol administration. These data are in contrast to findings on uteri of ovariectomized and nonestrogen-treated rats, in which a single injection of estradiol resulted in a prolonged nuclear receptor retention and a delayed cytosol receptor replenishment. Adrenalectomy did not result in a significant change of receptor dynamic patterns, suggesting that adrenal steroids do not play a role in the alteration of receptor dynamics elicited by continuous stimulation with diethylstilbestrol. These observations suggest that a continuous exposure of rat uteri to the estrogen causes an altered regulation of estrogen receptor dynamics by the homologous steroid compared to those in chronically estrogen-deprived rats.     

Effect of progesterone, estrogen withdrawal and secondary estrogen treatment of mannosylphosphoryldolichol synthesis in chick oviduct membranes

Oviduct membranes from chicks treated with diethylstilbestrol have a fully induced level of an enzyme that transfers mannose from GDP-Man to form mannosylphosphoryldolichol (Lucas, J.J. and Levin, E. (1977) J. Biol. Chem.252, 4330--4336). Withdrawal of diethylstilbestrol for 5 days causes a decrease in oviduct weight, lysozyme, and 60% of the mannosyltransferase activity. Chicks withdrawn from treatment for 10 days followed by secondary stimulation with diethylstilbestrol exhibit a more rapid increase in the mannosyltransferase activity than chicks that have not been previously treated with diethylstilbestrol. Further experiments indicate that the decrease in mannosylphosphoryldolichol synthesis after hormonal withdrawal may be the result of decreased levels of endogenous dolichyphosphate in the membrane preparations.     

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.     

米曲霉M-4降解己烯雌酚的条件优化

【目的】以米曲霉(Aspergillus oryzae)M-4对己烯雌酚(Diethylstilbestrol,DES)的降解率为响应值,对其降解条件进行优化。【方法】采用Plackett-Burman法对培养基组分和降解条件筛选显著性影响因素,并通过Box-Bohnken设计试验优化降解条件。【结果】最优培养基配方为:蛋白胨1.3%,CaCl_2 0.045%,葡萄糖0.5%,K_2HPO_4 0.15%,KH_2PO_4 0.05%,NaCl 0.05%,Tween 80 0.2%,DES质量浓度44 mg/L;最优培养条件为:初始p H 7.5,种龄72 h,转速140 r/min,培养温度28°C,培养时间72 h。【结论】在最优条件下菌株M-4对DES降解率为83.89%,比优化前(60.98%)提高1.38倍,差异极显著(P0.01)。     

Effect of hormone treatments on chick oviduct β-N-acetylglucosaminidase isozymes and other acid hydrolases

The effects of several hormone treatments on chick oviduct acid hydrolases were studied; including the effect of those treatments on the isozymes of β-N-acetylglucosaminidase. Chicks were treated for 10 days with diethylstilbestrol after which they were treated with progesterone alone, diethylstilbestrol alone, progesterone and diethylstilbestrol, or withdrawn from all hormone treatment. Protease and acid phosphatase were increased four- to fivefold upon hormone withdrawal, but they were not increased by any of the other treatments. β-N-Acetylglucosaminidase, however, increased fourfold upon hormone withdrawal and progesterone alone or progesterone and diethylstilbestrol treatment. In addition to the increase in β-N-acetylglucosaminidase activity, isozyme II increased from 20 to 35% of the total activity upon withdrawal (but not the other treatments). Isozyme I is the only form of the enzyme found in egg white.     

Sensitivity of expression of perivitelline membrane glycoprotein ZP1 mRNA in the liver of Japanese quail (Coturnix japonica) to estrogenic compounds

Avian perivitelline membrane protein, ZP1, is synthesized and secreted by the liver with the stimulation of estrogens. In the present study, we measured the expression of ZP1 gene in the liver of immature male quail treated with various estrogenic compounds and in the liver of male quail embryos that were developed in the fertilized eggs laid by mother quail injected with various estrogenic compounds during vitellogenesis. Total RNA extracted from the liver was reverse-transcribed and cDNA was subjected to real-time PCR. Both diethylstilbestrol and ethinyl estradiol caused significant effect on the increase in mRNA in immature male quail. In contrast, diethylstilbestrol administered via the route of maternal injection was not effective for induction of embryonic mRNA, although the effect of ethinyl estradiol administered via the same route was prominent. These results showed that direct administration of estrogenic compounds, diethylstilbestrol and ethinyl estradiol, stimulates the induction of ZP1 gene, but the rate of accumulation of these compounds in the yolk is different during vitellogenesis. The present studies suggest that although ZP1 gene is a sensitive biomarker to evaluate the effects of endocrine disruptors, the route of administration is an important factor to compare the effectiveness.     

Affinity labelling of the estrogen binding site of glutamate dehydrogenase with iodoacetyldiethylstilbestrol. Selective alkylation of cysteine-89.

Iodoacetyldiethylstilbestrol was used as an affinity label to alkylate the estrogen binding site of bovine liver glutamate dehydrogenase. This reagent induced inactivation and alkylation of the enzyme. The non-alkylating analogues diethylstilbestrol and estradiol protected the enzyme towards alkylation. The apparent constant of alkylation was of the order of magnitude of I50 for the allosteric inhibition by diethylstilbestrol. These two results suggest that alkylation occurred at the estrogen binding site. The stoichiometry of alkylation was between one and two, depending on the experimental conditions. When the stoichiometry was found to be less than or equal to 1, 90% of the label was bound on cystein residues, 70% of which was carried by cysteine-89, a cysteine residue which is known to be inacessible to iodoacetamide in phosphate buffer in the same conditions of temperature and pH.