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There has been an explosion in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) because of the indiscriminate use of antibiotics. In this study, we repurposed hexestrol (HXS) as an antibacterial agent to fight planktonic and biofilm-related MRSA infections. HXS is a nonsteroidal synthetic estrogen that targets estrogen receptors (ERα and ERβ) and has been used as a hormonal antineoplastic agent. In our work, the minimum inhibitory concentrations (MICs) were determined using the antimicrobial susceptibility of MSSA and MRSA strains. Anti-biofilm activity was evaluated using biofilm inhibition and eradication assays. Biofilm-related genes were analyzed with or without HXS treatment using RTqPCR analysis of S. aureus. HXS was tested using the checkerboard dilution assay to identify antibiotics that may have synergistic effects. Measurement of ATP and detection of ATPase allowed the determination of bacterial energy metabolism. As shown in the results, HXS showed effective antimicrobial activity against S. aureus, including both type strains and clinical isolations, with MICs of 16 µg/mL. Sub-HXS strongly inhibited the adhesion of S. aureus. The content of extracellular polymeric substances (EPS) and the relative transcription levels of eno, sacC, clfA, pls and fnbpB were reduced after HXS treatment. HXS showed antibacterial effects against S. aureus and synergistic activity with aminoglycosides by directly interfering with cellular energy metabolism. HXS inhibits adhesion and biofilm formation and eradicates biofilms formed by MRSA by reducing the expression of related genes. Furthermore, HXS increases the susceptibility of aminoglycosides against MRSA. In conclusion, HXS is a repurposed drug that may be a promising therapeutic option for MRSA infection., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1 , which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of ∼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The research was performed on 24 male sexually mature domestic rabbits, divided in two equal batches. The rabbits from the first batch were administered 0.5 mg/kg of body weight hexestrol diacetate intramuscularly twice a week for four consecutive weeks. Batch 2 was used for control. The testicular tissue samples obtained after orchidectomy were processed in order to obtain histological samples, stained using the Goldner's trichrome method. Examination of histological sections from the control batch showed that a natural aspect of seminal tubule epithelium, without reported injuries, even discrete. In the experimental batch were recorded a number of changes to all categories of the seminal cells. The severity and extent of damages varied greatly from one seminal tube to another and even from one part to another of the same seminal tube. These changes were the appearance of apoptotic cells and apoptotic bodies, vacuolar degenerescence of spermatocytes and spermatides, syncitialisation of spermatides, areas of necrosis accompanied by severe disruption of the seminiferous epithelium. In some areas, the lesions were so severe that the affected area of the cells forms "basal area". If lesions in the "adluminal area" affects only temporarily gametogenetic function the lesions in the "basal area" are irreversible as there are named "reserve cells" which is the starting point of spermatogenesis. Highlighted issue raised on the opportunity use of hexestrol diacetate in therapy or animal production stimulation as it gametogenetic function in males while their risk transfer to humans through consumption of foods of animal origin.

Objective: To investigate the effect of Yi Fu Ning Soft Gelatin Capsules (YFN) on reproductive endocrine-immune function of ovariectomized rats., Methods: 60 3-month old female Sprague-Dawley rats were used, 50 of them were ovariectomized and randomly divided into 5 groups: ovariectomizy (OVX) group, OVX with diethylstilbestrol tablets (DT) group, OVX with YFN (high dose, middle dose and low dose) group. The others were sham-operated group. The rats were administrated initially in the 4th week after the operation. After drugs had been given for 12 weeks the rats were sacrificed, blood serum hormone, IL-2 content and T lymphocyte subpopulation were detected with methods radioimmunoassay and flow cytometry (FCM)., Results: (1) Compared with sham group, the level of serum E2, Te and P significantly decreased (P < 0.01), FSH, LH content significantly increased; Blood T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio significantly decreased, serum IL-2 content also significantly decreased (P < 0.01). (2) Compared with model group, after treated by YFN, the level of serum E2 and P significantly increased (P < 0.01), serum FSH and LH content significantly decreased; T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio were improved significantly and serum interleukin-2 (IL-2) content increased significantly., Conclusion: YFN can increase serum sexual hormone content,reduce the level of FSH and LH, and improve imbalanced T lymphocyte subpopulation, stimulate IL-2 excretion, which means YFN can regulate inordinate reproductive endocrine-immune network in ovariectomized rats.

3(17)alpha-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17alpha-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17beta-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP(+) and the inhibitor hexoestrol refined at 2.1 A resolution is presented. Kinetic analysis and molecular-modelling studies of 17alpha- and 17beta-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (K(m)) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.

The results of in vitro experiments showed that hexestrol, a synthetic analog of estrogen hormones, at high concentrations inhibited the contractile response of isolated preparations of pregnant human uterus induced by ATP. It is suggested that estrogen hormones can interact with P2 receptors in human uterus during pregnancy.

Objective: To analyze histological aspects of ovaries as well as the ovulation of adult mice treated with the anabolic agent hexestrol., Methods: Thirty adult mice were divided into three groups of 10 animals each: (GI) the animals received a dose of 3 mg/kg of hexestrol; (GII) the animals were given a dose of 6 mg/kg of hexestrol; (GIII) the animals were injected with distilled water (vehicle). Another 10-animal group (GIV) was included, and these mice were injected with propionate testosterone (1.25 mg) after 5 days from the day of birth. Hexestrol was administered intraperitoneally once a day and the treatment lasted 30 days. The mice were then sacrificed; their ovaries and oviducts were removed, submitted to histological routine and analyzed under light microscopy., Results: In mice treated with hexestrol (6 mg/kg) (Group II), ovaries were smaller than those from the controls but highly vascularized; similar results were obtained in GIV. A great number of follicles in several stages of development were found -- however, with no corpora lutea -- in six animals in GII. No corpora lutea were found in GIV. The number of luteal bodies and oocytes in GII was lower than that in GI or GIII. No oocytes were detected in GIV. Finally, the nuclear volume of interstitial cells in GII and GIV was the largest., Conclusion: Our data suggest that the anabolic agent hexestrol in a high dose may decrease ovulation in mice.

The effects of foods and chemicals related to food hygiene on degranulation were evaluated using a method for assaying the enzyme activity of beta-hexosaminidase as an index of chemical mediator release from RBL-2H3 cells in vitro. Using a previously developed assay system, we had found a large number of inhibitors and promoters of degranulation of RBL-2H3 cells. In the present study, we examined the inhibitory effect of zinc chloride on the degranulation (beta-hexosaminidase release) from RBL-2H3 cells with or without antigen in the presence of the degranulation-promotive chemicals, namely, 4 food additives, 7 pesticides and 2 veterinary drugs. These promotive chemicals were classified into two types on the basis of inhibitory profile by zinc chloride: 1) those which showed marked degranulation-inhibitory action when the cells were stimulated with antigen, such as butylhydroxyanisole, dibutylhydroxytoluene, EPN, cis- and trans-permethrin, prothiofos, pyridaben, terbufos, 2) those which showed marked degranulation-inhibitory action whether the cells were stimulated with antigen or not, such as butyl p-hydroxybenzoate, o-phenylphenol, bitertanol, salinomycin. In conclusion, zinc had a dramatic inhibitory effect on enhanced degranulation induced by synthetic chemicals in vitro.

Little is known about the effects of residual veterinary drugs on the allergic reaction, except for the antigenicity of antibiotics and synthetic antimicrobials. Therefore, 59 kinds of veterinary drugs were investigated for their effects on the IgE receptor-mediated beta-hexosaminidase release from RBL-2H3 cells as an index of immediate allergic reaction. We found that the antibiotics chlorotetracycline, doxycycline, monensin, the synthetic antimicrobial pyrimethamine and the steroid hormone testosterone inhibited beta-hexosaminidase release. Most of the veterinary drugs showed no action, though the ionophores lasalocid, salinomycin and the steroid hormone hexestrol promoted beta-hexosaminidase release from injured cells. Based on the residual levels of these drugs and the frequencies of detection in actual food samples, it seems unlikely that these drugs have any immediate allergic effect in practice.

Activation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a property imposed on ER in cis by its ligand-binding domain (EBD). Subsequently, the derepressed ER binds specific genomic sites and regulates transcription. In addition to the natural hormone, ER binds a broad range of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduce the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F domains was activated by all the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathway. Mutant variants of the Flp-ER protein that distinguish between agonists and antagonists, and a mutant EBD that selectively lost the ability to respond to 17beta,-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distinguished in a nontranscriptional assay.

The effects of low doses of hexestrol (Hex) (2-40 micrograms/kg bw) and flutamide (FI) (10 mg/kg bw) on some endocrine mechanisms in mature intact male rats are described in the present paper. It has been shown that each preparation, administered separately for 10 days, induced a moderate decrease in the weight of the ventral prostate (VP), anterior prostate lobe or coagulating gland (CG) and seminal vesicles (SV), in the DNA content and number of cells in the VP. 5 alpha-reductase activity was also decreased; the epithelium secretory activity of the VP was suppressed. After combined FI (10 mg/kg bw) and Hex (40 micrograms/kg bw) the following castration-like effects were observed: an abrupt fall in the weight of the accessory sexual glands, a decrease of DNA level and cell number in the VP as well as a suppression of the production of 5 alpha-reductase metabolites in this structure. Histologically, a marked degenerative changes in the VP secretory epithelium was observed; on the contrary an hyperplasia of connective and smooth muscle cells was evident. When FI alone was administered to rat, the above-mentioned changes were accompanied by a pronounced elevation of plasma bio-LH and testosterone (T) levels, also an increase of testicular delta 5-3 beta-hydroxysteroid dehydrogenase activity was observed. On the contrary, when Hex was administered alone or in combination with FI, bio-LH and T levels and enzyme activity decreased. We conclude that Hex administration in low doses, in combination with FI, could be an alternative method for a complete androgen blockade of the accessory sexual glands.

Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.

The synthetic oestrogens diethylstilboestrol, hexoestrol and 17 alpha-ethynyloestradiol are known to be carcinogenic, yet they all exert antioxidant properties in vitro in that they are good inhibitors of iron ion-dependent lipid peroxidation. In rat liver microsomes incubated with Fe(III)-ascorbate or Fe(III)-ADP/NADPH and also in ox-brain phospholipid liposomes incubated with Fe(III)-ascorbate; the overall order of effectiveness of the compounds tested as inhibitors of lipid peroxidation was diethylstilboestrol > hexoestrol > 17 alpha-ethynyloestradiol > 4-hydroxytamoxifen > 17 beta-oestradiol > tamoxifen. Compounds acting as antioxidants towards lipids may also exert pro-oxidant effects towards other molecules such as DNA and thus must never be assumed to be safe for human use.

3,3'-Dihydroxy-alpha,beta-diethyldiphenylethane (I), 3,3'-dihydroxy-alpha,beta-diethylstilbene (II), hexestrol (III) and diethylstilbestrol (IV) have already been reported to show hypotensive effects on rats and exhibit phytogrowth-inhibitory activities. We have proposed that two phenolic hydroxyl groups in these compounds are necessary for the biological activities, and a structure-activity relationship for I-related compounds was accomplished using molecular-mechanics (MM) calculations. As a result, the following three findings were obtained; 1) the minimized conformational energy obtained from MM calculations, which is a parameter expressing the molecular stability, showed a relatively high correlation with the biological activities, 2) as results of quantitative structure-activity relationship (QSAR) analyses, the combination of the distance between two phenolic hydroxyl oxygens led to the regression equations with high correlation values, and 3) the idealized molecular model of the most active compound (I) showed the highest stability and had a particular conformation which differed from the other compounds (II-IV).

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.

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.

In order to study the effects of perinatal exposure to estrogens on the developing reproductive tract, outbred female mice were treated neonatally (days 1 to 5) with varying doses of diethylstilbestrol (DES) and sacrificed from 1 to 18 months of age. Uterine adenocarcinoma was observed in a time- and dose-related manner after DES treatment; at 18 months, neoplastic lesions were seen in 90% of the mice exposed neonatally to 2 micrograms/pup of DES/day, while none was observed in the corresponding control mice. These DES-induced uterine tumors were estrogen dependent; when DES-treated mice were ovariectomized before puberty, no uterine tumors developed. As a marker for neoplasia, uterine tumors were transplanted and carried as serial transplants in nude mice. The transplanted tissue retained some differentiated uterine gland structure and function and also required estrogen supplementation for maintenance. Additional groups of neonatal mice were treated with various DES analogues (hexestrol and tetrafluorodiethylstilbestrol) and steroidal estrogens. The compounds were ranked according to developmental estrogenic potency (hexestrol greater than trifluorodiethylstilbestrol greater than DES greater than 17 beta-estradiol). The combined prevalence of uterine atypical hyperplasia and adenocarcinoma follows the order of estrogenic potency. The experimental induction of these tumors will provide the basis for additional studies in mechanisms of hormonal carcinogenesis.

The significance of sex differences in the level of estrogen receptors (ER) in hepatocytes for direct effects of estrogens in male and female rat livers was investigated. 4-5-fold increase in ER level and 20-30%-elevation in plasma angiotensinogen (AG) occurred after a single injection of 0.5 microgram of hexestrol (HE) in female and gonadectomized male rats. In male liver, where the cytosol ER content is two fold lower than that in female rats, nuclear ER level was shown to be very low and unchanged after HE injection, plasma AG also did not change. Injection of 0.5 microgram of ethinylestradiol produced similar effect. Injection of a greater dose of estrogen caused an enhancement in plasma AG level in males. The existence of sex differences in hepatic ER level seems to cause in some conditions different response of metabolic processes in male and female rat liver after estrogenization.

We have reported that meso-hexestrol, a synthetic estrogen, inhibits microtubule assembly and induces microtubule proteins into twisted ribbon structures. On the other hand, Serrano et al. proved that S-tubulin, which lacks the C-terminal moiety of tubulin subunits, assembles into sheet structures in the absence of microtubule-associated proteins (MAPs). In the present investigation, we attempted to clarify whether meso-hexestrol could induce the ribbon structure from S-tubulin. meso-Hexestrol delayed the initiation of polymerization of S-tubulin into sheet structures in a dose-dependent manner below 50 microM. But the effect of meso-hexestrol on S-tubulin was reduced in the presence of either tau or microtubule-associated protein 2 (MAP2) in a MAPs-concentration-dependent manner. At concentrations higher than 100 microM, meso-hexestrol inhibited the polymerization of S-tubulin into sheet structures, without forming ribbon structures. The present results may indicate that moso-hexestrol interacts with S-tubulin, and its interaction is affected by MAPs.

Selective Birch-reduction of one of the phenolic rings of meso- or d,l-HES led to compounds 6a and 6b which exert a higher binding affinity to the androgen receptor as the respective parent HES. In vivo testing of 6a and 6b shows that 6a has the same potency in reducing accessory sex organ weights and testosterone levels in the intact mouse as has meso-HES, but strongly decreased estrogenic activity. Syntheses and testing of 7 having no ethyl side chains revealed the necessity of these groups for biological activity.

The diethylstilbestrol-related compounds 3,3'-dihydroxy-alpha, beta-diethyldiphenylethane (I), diethylstilbestrol (II) and hexestrol (III) showed hypotensive effects on spontaneously hypertensive rats (SHR) and antifungal activities against all Fusarium oxysporum sp. tested. As previously reported, I had strong hypotensive action on normotensive rats at the dose of 10 mg/kg, while II and III showed weak hypotensive effects on these rats at the same dose. In this work, all three compounds also had hypotensive actions on SHR at the same dose. I showed the strongest hypotensive effect (-80.0 +/- 5.0 mmHg, 10 mg/kg, i.v.) on both SHR and normotensive rats. The three compounds also had antifungal activities against five kinds of Fusarium oxysporum sp. tested. Especially, II strongly inhibited the growth of Fusarium oxysporum f. sp. raphani IFO-9972 (minimum inhibitory concentration (MIC): 1.0 micrograms/ml).

The synthesis of the bis-acrylate 12, the bis-beta-chloropropionate 13 and the bis-beta-bromopropionate 14 of the "partial" antiestrogen 2,3-bis(2-fluoro-4-hydroxyphenyl)-2,3-dimethylbutane (7) is described. In the case of 13 and 14 the introduction of the beta-haloester-functions moderately reduces the estrogen receptor affinity of 7. However, it was quite strongly diminished in 12. The hydrolytic stability under in vitro-receptor-assay conditions decreases in the order 12 greater than 14 greater than 13. Compared with 7 the estrogenic potency of 12-14 is increased to a great extent. The title compounds cause a strong inhibition of the hormone-dependent MXT-M3.2 mouse mammary tumor.

We previously reported on the inhibition of microtubule polymerization and the formation of ribbon structures by synthetic estrogens [Sato et al., J Biochem 101: 1247-1252, 1987]. The present investigation aimed to analyse these effects in vitro on stereochemical point of view, using hexestrol isomers ((R,R)-(+)-hexestrol, (S,S)-(-)-hexestrol and meso-hexestrol) and dl-hexestrol. Among hexestrols, dl-hexestrol showed the highest activity in ribbon formation from microtubule proteins at 100 microM. On the other hand, meso-hexestrol was distinguished from others by inhibition of microtubule assembly and formation of a large amount of aggregates from purified tubulin in the presence of MgCl2 and DMSO. These results were discussed with physico-chemical properties of hexestrols, e.g. absolute configurations as well as circular dichroism spectra and solid state carbon-13 nuclear magnetic resonance spectra.

Groups of male and female mice were gonadectomized and some implanted with a pellet of hexoestrol. Half of the animals in each group, including controls, were infected with a low-virulence strain of Toxoplasma gondii. The animals were killed 6 weeks after infection. Gonadectomy increased the relative thymic weight in both sexes, but more so in the male, and the paracortical area of the lymph node was enlarged, as was the thymus-dependent area of the spleen. Hexoestrol administration induced almost complete thymic atrophy and partial involution of the peripheral lymphoid tissues. Greater resistance to toxoplasmic infection was found in the gonadectomized mice than in controls of both sexes, possibly due to a better cell-mediated immunity in the gonadectomized animals. Overwhelming toxoplasmosis with increased mortality was found in the hexoestrol-treated mice after infection, probably resulting from a depression of cell-mediated immunity caused by hexoestrol. It is postulated that the cellular immune response is of greater importance than the formation of antibody in resistance to toxoplasmic infection. The role of the thymolmphatic--sex hormonal interrelation on the age--sex differences seen in the incidence of toxoplasmic lymphadenopathy is also discussed.

Treatment of rats, monkeys and man with diethylaminoethoxyhexestrol causes phospholipid storage in liver and other tissues. However, this drug has not been reported to alter plasma lipoprotein levels. When guinea pigs were treated with diethylaminoethoxyhexestrol, the fasting plasma triacylglycerol levels increased dramatically, from 43 to 1281 mg/dl, after only five doses of 12.5 mg/kg. Diethylaminoethoxyhexestrol-treated guinea pigs had reduced postheparin lipase activity. In addition, in vitro assays showed that this agent inhibited guinea pig postheparin lipoprotein lipase. It is hypothesized that diethylaminoethoxyhexestrol causes hypertriglyceridemia in guinea pigs because these animals are known to have low levels of serum activator for lipoprotein lipase and may be unusually susceptible to agents that inhibit lipoprotein lipase activity. The ability to produce hypertriglyceridemia in guinea pigs provides an animal model in which the metabolic consequences of hypertriglyceridemia can be studied.

The ultrastructure of the lymph node post-capillary venules (PCV) was studied in control, gonadectomised and gonadectomised-Hexoestrol treated male and female mice, infected or not with Toxoplasma gondii. The percentage of the cytoplasmic area of the endothelial cells occupied by Golgi apparatus was estimated by quantitative stereology. The endothelial cells of the PCV are higher and contain more Golgi elements in female than male controls. Oophorectomy decreases the percentage of Golgi apparatus and the height of the endothelial cells while orchidectomy does not affect the former but increases the latter. The endothelial cells react to Hexoestrol similarly in male and female mice, by increasing their height and becoming more heavily structured, with an effect particularly on the Golgi apparatus. All groups of mice infected with Toxoplasma gondii, show increased numbers of lymphocytes passing through the wall of the PCV. The endothelial cells of the latter show increased numbers of ribosomes both free and attached to rough endoplasmic reticulum. Golgi elements are decreased. In view of these findings the mechanisms for selective lymphocyte migration through the walls of the PCV are discussed.

The development of the fetus adenohypophysis analyzed under the conditions of high doses of sinestrol, administered to mothers, revealed certain morphofunctional characteristics in the group studied in comparison with the control. In normal pregnancy, a gradual increase in protein content and in nucleic acids activity are noted, as well as an increase in the enzymes in protein synthesis and transport within the cell. Under maternal hyperestrogenity, however, in the adenohypophyseal cells of 7--14 week-old fetuses, a decrease in the content of hormonal protein-precursors, nucleic acid activity, inhibition of synthesis and trnsport processes are observed.

The activity of two isoenzymes of alkaline phosphatase was studied in the uteri of ovariectomized golden hamsters. Animals belonging to different groups were daily injected with 10 micrograms of estrogen (octoestrol) once, for 4 and 16 days. The estrogen did not affect the overall activity lf alkaline phosphatase in the uterine luminal epithelium, but decreased the enzyme activity in stroma. Moreover, it was found that prolonged estrogen treatment increases significantly the proportion of alkaline phosphatase isoenzyme of the intestinal type in the uterine luminal epithelium.

After ovariectomy DMBA tumor-bearing SD rats were treated with the estrogen DES, a combination of DES plus mammary tumor-inhibiting antiestrogen (tetramethylHES, 3,3'HES and tamoxifen respectively) or with tetramethylHES alone. Small doses of DES led to a stimulation of tumor growth showing a maximum effect at 1 microgram/kg/day, whereas higher doses inhibited tumor growth. In the combination experiment only tetramethylHES reduced, i.e. antagonized, the DES effect. Tamoxifen and 3,3'HES, however, led to an increase of the tumor growth-stimulating and inhibiting effects of DES. Only the dose of 10 mg/kg/day of tetramethylHES led to a slight stimulation of the tumor growth in ovariectomized DMBA tumor-bearing rats. These results indicate that 3,3'HES and tamoxifen possibly unfold their mammary tumor-inhibiting activity by means of their estrogenic potency, whereas tetramethylHES might act as an antiestrogen, though the latter compound does not lack estrogenic activity completely.

1. Very large increases in growth rates and improvements in the efficiency of food conversion of both male and female turkeys of 70 d of age or older were effected by the subcutaneous implantation of 20 to 40 mg of trienbolone acetate (TA). 2. Implantation of TA (10 to 40 mg) into turkeys of 42 d of age or less caused growth depression, usually following an initial period of positive growth response. 3. During the period of growth depression, food conversion efficiency deteriorated and treated turkeys developed chondrodystrophic skeletal abnormalities and/or "leg weakness". 4. Implantation of oestradiol and of testosterone into 2-d-old turkeys also led to the development of skeletal abnormalities. 5. Implantation of testosterone (25 mg) into 84-d-old stag turkeys produced positive growth responses which were of smaller magnitude and lesser duration than those produced by TA. 6. Implantation of 70-d-old stag turkeys with a combination of TA (20 mg) and hexoestrol (15 mg) resulted in performances which were slightly superior in terms of the growth to those following administration of TA alone but which were markedly inferior in respect of food conversion.

The antimetastatic effect of thaliblastine alkaloid isolated from Thalictrum min. ssp. elatum was evaluated using Wexler's technique for accurate identification of experimental pulmonary metastases. Thaliblastine was found to inhibit the growth of lung metastases as compared with the control.

Steroid hormones (sex hormones and corticosteroids) were found to inhibite the activity of glutamate dehydrogenase from rat liver mitochondria in vitro. After blocking of arginine guanidine groups in the mitochondrial preparations the inhibitory effect of sex hormones was decreased and that of corticosteroids - prevented. The data obtained suggest that guanidine groups of arginine residues in glutamate dehydrogenase participate in realization of the inhibitory effect of steroid hormones.

We studied the action of E-diethylstilbestrol (E-DES), erythro-hexestrol (erythro-HES), and E,E-dienestrol (E,E-DIES) on microtubule formation. The three drugs inhibit this formation from microtubular protein; the percentages of inhibition were, respectively, 15% and 45% for 1.25 X 10-s M E-DES and E,E-DIES. With purified tubulin 6S, 7.5 X 10(-6) M E,E-DIES and erythro-HES induced a 20% inhibition. In the case of E-DES, our results are in good agreement with previous ones. These drugs partially disrupt preformed microtubules. Moreover, when E,E-DIES (5 X 10(-5) M) is added to tubulin, loosely organized aggregates composed of twisted ribbon structure are formed. In the case of erythro-HES, similar structures were observed but at higher concentrations. With E-DES, no organized structures are present.

The effect of dihydrostilbestrol diacetate and oxytocin on the Na+, K+, Ca++ and Mg++ content in uterine Wistar rats has been studied. A bilateral ovariectomy on the animals was performed. After seven days dihydrostibestrol (0.25 mg/kg a day) and oxytocine (0.025 U.I./kg a day) were administered separately and simultaneously i.p. during a period of five days. Twenty four hours after the first, third and fifth administration, five rats from each group were decapited and the above uterine cation content was determined by atomic absorption spectrometry. Dihydrostibestrol induced a marked increase and a slight decrease in K+ and Na+ contents respectively, lowering the Na/K ration. It also increased greatly Ca++ and Mg++ content. Oxytocin induced a Na+ and K+ increase, a Ca++ decrease, and slight changes in Mg++. These results partially confirmed the bioelectric and biomechanic alterations produced by the above hormones on the uterine cells.

The relative effectiveness of implanting 300 mg trenbolone acetate alone or in combination with either 15, 30 or 45 mg hexoestrol was studied in three 90-day experiments using 64 Friesian steers. In experiment 1 hexoestrol was shown to improve live-weight gain and efficiency of feed conversion in steers implanted with trenbolone acetate. In experiments 2 and 3 trenbolone acetate in combination with 30 mg hexoestrol gave a better growth response than when combined with either 15 or 45 mg. However, in experiment 3 trenbolone acetate plus 15 mg hexoestrol was shown to improve live-weight gain by about 36 per cent compared with untreated controls. In experiment 2 small differences between treated groups in mean values for plasma urea, serum albumin, plasma glucose and free fatty acids were recorded.

The effect of 4,4'-diethylamino-ethoxyhexestrol (DH) on acid esterase in rat liver was studied in vivo and in vitro. The acid esterase activity in the livers of rats treated with 0.125% DH for 1 week was found to decrease more than 60% as compared with that in untreated rats. The addition of DH to the incubation medium caused considerable inhibition of the acid esterase activity in lysosome from untreated rat liver, and the inhibition type appears to be noncompetitive. The acid lipase activity in rat liver lysosome was also inhibited by DH. Some antihistamic agents and chloroquine also inhibited the acid esterase activity in rat liver lysosome.

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.

1 Endocrine and anti-fertility studies were carried out on a fluorinated bibenzyl, bifluranol, in rats and mice. 2 A potent anti-prostatic activity of bifluranol was shown to be comparable to diethylstilboestrol (DES). In contrast, its oestrogenic potency by the oral route was about eight times less than that of DES. 3 In comparative short and long-term anti-androgenic and fertility studies in rats and in studies on sexual potency and reproductive performance in male mice, bifluranol given orally was shown to produce fully reversible suppression of accessory sexual structures without impairment of spermatogenesis and fertility. In contrast, DES administered in the same dose reduced spermatogenesis as well as accessory sexual glands. 4 Bifluranol lowered serum luteinising hormone (LH) levels without affecting follicle stimulating hormone (FSH). Under similar conditions DES reduces both LH and FSH levels. Since bifluranol does not antagonize androgen-induced stimulation of the prostate in castrated rats, its anti-prostatic effect is interpreted as a negative, hormonostatic feedback activity, mediated through a selective inhibition of LH secretion.

Two gamma-emitting estrogen analogues, (2R*,3S*)-1-[125I]iodo-2,3-bis(4-hydroxyphenyl)pentane ([125I]iodonorhexestrol) and (2R*,3S*)-1-[77Br]bromo-2,3-bis(4-hydroxyphenyl]pentane ([77Br]bromonorhexestrol), have been prepared by halide ion displacement on a labile trifluoromethanesulfonate derivative of a suitably protected precursor, followed by mild acid deprotection. Although halide displacement on a more stable tristrifluoromethanesulfonate derivative was successful, the basic conditions required for deprotection of this precursor resulted in destruction of the products by a base-induced spiroelimination reaction. In immature female rats, both of these halonorhexestrols demonstrated preferential uptake by the uterus that could be blocked selectively by coadministration of a large dose of unlabeled estradiol. In a double label comparison with 16 alpha-[125I]iodo-17 beta-estradiol the uterine uptake of [77Br]bromonorhexestrol was notably less selective. Stability studies in vitro and in vitro have indicated that both iodo- and bromonorhexestrol are quite labile, and this lability compromises the selectivity of their uptake by estrogen target tissues in vivo. p-Hydroxyphenethyl halides are known to be unusually prone to a base-catalyzed solvolysis, via cyclization of the phenolate to a spirocyclohexadienone intermediate. This unusual solvolytic mechanism may contribute to the lability of these halonorhexestrols in vivo.

We have analyzed the effect of estrogen on the kinds of unique DNA sequences which are transcriptionally expressed in chick oviduct with an "expressed" DNA probe. Steady-state nRNA in estrogen-stimulated chick oviduct represents about 25% of the complexity of total chick unique DNA. To purify this expressed DNA fraction, chick unique DNA was isolated, nick-translated, and hybridized to chemically mercurated oviduct nRNA (Hg-nRNA); the resulting hybrids were bound to sulfhydryl-Sepharose, and DNA was selectively recovered by thermal elution in formamide buffer. To compare the sequence homology between nRNAs isolated from oviduct before or up to 6 days after estrogen withdrawal, trace amounts of expressed DNA derived from estrogen-stimulated oviduct were hybridized in RNA-excess reactions. All nRNAs hybridized with equal efficiency. Furthermore, hybridization of expressed DNA to nRNA mixtures showed that nRNA from nonwithdrawn and withdrawn oviduct contained a similar set of unique sequences. The data indicate that, at most, only a small percentage (0--5%) of transcriptionally active unique DNA sequences are shut down when estrogen is removed from the circulation.