Dry olive leaf extract attenuates DNA damage induced by estradiol and diethylstilbestrol in human peripheral blood cells in vitro.

Phenolic groups of steroidal or nonsteroidal estrogens can redox cycle, leading to oxidative stress, where creation of reactive oxygen species are recognized as the main mechanism of their DNA damage properties. Dry olive (Olea europaea L.) leaf extract is known to contain bioactive and antioxidative components and to have an ability to modulate the effects of various oxidants in cells. The main goal of this study was to investigate antigenotoxic potential of a standardized dry olive leaf extract on DNA damage induced by 17ß-estradiol and diethylstilbestrol in human whole blood cells in vitro, using comet assay. Our results indicated that both hormones showed a genotoxic effect at a concentration of 100 µM (P < 0.05, n = 6). Dry olive leaf extract was efficient in reducing number of cells with estrogen-induced DNA damage at tested concentrations (0.125, 0.5 and 1 mg/mL) (P < 0.05, n = 6) and under two experimental protocols, pre-treatment and post-treatment, exhibiting antigenotoxic properties. Analysis of antioxidant properties of the extract revealed moderate ABTS radical scavenging properties and reducing power. Overall, our results suggested that the protective potential of dry olive leaf extract could arise from the synergistic effect of its scavenging activity and enhancement of the cells' antioxidant capacity.

[Estrogen Receptor Subtype-mediated Luciferase Reporter Gene Assays for Determining (anti) Estrogen Effect of Chemicals].

OBJECTIVE: To establish gene assays for determining (anti) estrogen effect of environmental chemicals; and to compare the reactivity and sensitivity of two assays with different estrogen subtype. METHODS: Human estrogen receptor a (hERalpha) and hERbeta mediated reporter gene assays employing firefly luciferase (Luc) were developed. The expression plasmid hERalpha or hERbeta was constructed and transiently co-transfected into LLC-MK2 cells with pERE-minP-Luc2P reporter plasmid and the control plasmid pGL4.74. Estradiol (E2) and diethylstilbestrol (DES) served as positive test substances to verify the performance of the assays. The effectiveness of the assays for detecting anti-estrogenic activity was tested using 10(-5) mol/L ICI 182, 780 under different concentrations of E2. The performance of the two subtype-mediated assays was verified and compared using bisphenol A (BPA) and genistein (GS). RESULTS: The hERalpha mediated assay found expression of reported gene at 1.9 x 10(-11) mol/L E2; and the largest luciferase activity was shown at 10(-8) mol/L E2, resulting in 30.7-fold of vehicle control. The hERbeta mediated assay found expression of reporter gene at 2.2 x 10(11) mol/L E2, and the largest luciferase activity was shown at 10(-8) mol/L E2, resulting in 14.4-fold of vehicle control. ICI 182, 780 inhibited estrogenic activity of E2 significantly. In both assays, E2 failed to induce luciferase activity without hER-pcDNA3.1. BPA and GS induced luciferase activity. CONCLUSION: Both assays have high sensitivity and reproducibility for detecting (anti) estrogen effect. The pGL4-based hERbeta has lower sensitivity than the hERalpha- mediated reporter gene assay. BPA shows stronger estrogenic activity than GS in hERalpha mediated reporter gene assay; whereas, GS shows stronger estrogenic activity than BPA in hERbeta mediated reporter gene assay.

Diallyl sulfide inhibits diethylstilbesterol-induced DNA adducts in the breast of female ACI rats.

Diethylstilbestrol (DES) is metabolized to reactive intermediates that produce DNA adducts and ultimately cancer. Diallyl sulfide (DAS) has been shown to inhibit the metabolism of several procarcinogens. The ability of DES to produce DNA adducts in microsomal, mitochondrial, and nuclear in vitro metabolic systems and in the breast of female ACI rats, as well as ability of DAS to inhibit DNA adducts were investigated. Microsomes, mitochondria, and nuclei isolated from breast tissue of female ACI rats were used to catalyze oxidation reactions. Female ACI rats were treated i.p. as follows: (1) corn oil, (2) 200mg/kg DES, (3) 200mg/kg DES/200mg/kg of DAS, (4) 200mg/kg DES/400mg/kg DAS. DES produced DNA adducts in each metabolic system. The relative adduct levels were 2.1 x 10(-4), 6.2 x 10(-6), and 2.9 x 10(-7) in microsomal, mitochondrial, and nuclear reactions, respectively. DAS inhibited DNA adducts in each metabolic system. The percent inhibition ranged from 86% in microsomes to 93% in nuclei. DES produced DNA adducts in mtDNA and nDNA. DAS completely inhibited the DES-induced mtDNA adducts and caused a dose dependent decrease in nDNA adduct formation. These findings suggest that DAS could inhibit DES-induced breast cancer by inhibiting its metabolism.

Diallyl sulfide inhibits diethylstilbestrol-induced lipid peroxidation in breast tissue of female ACI rats: implications in breast cancer prevention.

Diallyl sulfide (DAS) is a component of garlic and prevents cancer in several animal models in various organs. The chemopreventive effects of DAS are attributed to modulation of enzymes to alter the bioactivation of xenobiotics. Diethylstilbestrol (DES) is a synthetic estrogen that causes breast cancer in female ACI rats subsequent to metabolism with concurrent free radical production. This study assessed the effect of DAS on DES-induced reactive oxygen species (ROS) using lipid peroxidation as an empirical endpoint. We have demonstrated that acute exposure to DES results in a significant increase in lipid hydroperoxides (LPH) in breast tissue and DAS attenuated DES-induced LPH concentrations. Two-week exposure to DES caused significant increases in LPH concentrations in breast and liver tissues. DES-induced LPH concentrations were decreased by coadministration of DAS at this time point. There were no statistical differences in the concentrations of LPH in breast and liver tissues of rats treated for 4/6 weeks with DAS/DES. These results demonstrate that DAS inhibits the production of ROS which suggests that DAS effectively inhibits DES bioactivation in female ACI rats which may have implications for chemopreventive intervention strategies. Our results suggest that garlic consumption might be useful for the prevention of human breast cancers.

Mechanisms of diethylstilbestrol-induced relaxation in rat aorta smooth muscle.

The mechanisms of diethylstilbestrol (1 to 30 microM)-induced relaxation on noradrenaline (30 nM)-raised tone in the rat aorta smooth muscle were studied. Neither the increase of calcium content in the medium (3, 6 and 9 mM) nor Bay K 8644 (3, 10 and 100 nM) reversed diethylstilbestrol relaxation. Tamoxifen (3 microM), the quaternary derivate (tamoxifen ethyl bromide, 3 microM), actinomycin D (30 microM), cycloheximide (100 microM), Rp-cAMPS (30 microM), TPCK (1 microM) and difluoromethylornithine (1 mM) inhibited diethylstilbestrol-induced relaxation. Incubation with 2 microg/ml pertussis toxin, propranolol (1 microM), H-7 (10 microM), 2',3'- and 2',5'-dideoxiadenosine (10 and 30 microM, respectively) and methylene blue (10 microM) did not modify diethylstilbestrol-induced relaxation. Our results showed that presumably an activation of membrane mechanisms, protein kinase A activation, genomic mechanisms and polyamine synthesis might participate in diethylstilbestrol-elicited relaxation in addition to the increase in K(ATP) permeability, as previously described. Actinomycin D produces a synergistic effect, with tamoxifen, difluoromethylornithine and glibenclamide antagonizing the effect of diethylstilbestrol. In the case of the association of actinomycin D and glibenclamide, the antagonism of relaxation is complete. The fact that tamoxifen- and difluoromethylornithine-dependent mechanisms participate in diethylstilbestrol relaxation inhibited by glibenclamide suggests that two transduction pathways are involved in the relaxation. Therefore, K(ATP) channels and genomic mechanisms, both modulated by cyclic AMP (cAMP)-dependent mechanisms, are associated with diethylstilbestrol relaxation.

Effects of postpubertal treatment with progesterone on protein expression in the vagina and uterus of mice exposed neonatally to diethylstilbestrol.

Postpubertal progesterone injections slightly inhibited the proliferation and cornification of the vaginal epithelium induced by neonatal injections of diethylstilbestrol (DES). In vaginae of neonatally DES-exposed mice (DES mice), 9 protein expressions (PEX) appeared newly; 13 PEX decreased; 5 PEX increased compared to those in the controls. In vaginae of DES mice, PEX were altered by postpubertal injections of progesterone (DES-P); 3 PEX disappeared; 11 PEX were reversed to those in the controls. Progesterone injections impaired the proliferation of the vaginal epithelium, reversing 11 PEX compared to those in DES mice. In uteri of DES-P mice, 3 PEX increased, 2 PEX decreased, 2 PEX appeared and 1 PEX disappeared compared to those in DES mice. In conclusion, it was shown that postpubertal injections of progesterone alter the protein expression in the vagina and uterus of DES mice.

Subacute toxicity of diethylstilboestrol and hexoestrol in the female rat, and the effects of clomiphene pretreatment.

Two groups, each of ten female rats, were orally dosed with the synthetic oestrogens diethylstilboestrol (DES; 6 mg/kg day) and hexoestrol (60 mg/kg/day) for 6 wk. A further group of ten animals received clomiphene citrate (2 mg/kg/day) and DES (6 mg/kg/day), while two groups, each of ten animals, acted as controls. One animal treated with DES died within the 42-day study period. Its death was associated with a bleeding disorder related to severe liver damage. The remaining treated animals showed little overt toxicity. The major haematological finding in the treated animals that survived was a moderate, stable, normocytic, normochromic anaemia. The terminal bone marrows of these animals showed a modest degree of erythroid hypoplasia. There were stimulatory and other changes in the reproductive tract and there were gains in the liver, adrenal and pituitary weights. These effects were more marked in the rats treated with hexoestrol, the gains in the relative organ weights being particularly striking: liver, 51%, adrenals, 210%, uterus, 79% and pituitary, 500%. The oestrogens caused reductions in body weight (18% in both cases) and appetite, a modest degree of fatty change in the liver, and alterations in the serum proteins. The Thrombotest clotting times of the hexoestrol-treated rats were prolonged. Clomiphene citrate decreased the adverse effects of DES on appetite and body weight, and sharply decreased the gains in the pituitary and uterine weights from 243 to 80% and 59 to 14%, respectively. The toxic effects of the oestrogens in the rat were more closely related to the dose administered than to their hormonal potency. Oestrogens are less toxic to the rat than they are to the cat, dog and ferret, and the toxic effects are in many respects qualitatively different.

Inhibition of follicle-stimulating hormone/diethylstilbestrol-stimulated ovarian growth by extracts of pregnancy urine.

We devised an in vivo biological assay for ovarian growth inhibiting activity to examine extracts of human pregnancy urine for the presence of ovarian growth inhibiting factor. Diethylstilbestrol (DES) capsules were implanted sc in immature hypophysectomized female rats; FSH was injected sc with or without test substance for 5 days. Rats with unstimulated ovaries were implanted with blank capsules and given the vehicle without FSH. Twenty four hours after the last injection, the ovaries were removed and weighed. The ovarian growth inhibition of the ovarian weight gain achieved in rats treated with DES and FSH. Crude commercial human CG (hCG) preparations, extracted from pregnancy urine, were chromatographed on Sephadex G-100, and the fractions were tested for ovarian growth inhibiting activity. The peak of ovarian growth inhibiting activity was found in fractions eluting from the column in the mol wt range of 12,000-20,000. Ovarian growth inhibiting activity was heat sensitive, not extracted by ether, and precipitated by acetone. Ovarian growth inhibiting activity was stable in acid at pH 2, but was inactivated by digestion with trypsin. The ovarian growth inhibiting activity was purified by chromatography on Concanavalin A-Sepharose and diethylaminoethyl-Sephacel. The active material contained hCG alpha, hCG beta, and beta-carboxyterminal peptide-immunoreactivity and its inhibiting activity could be removed from solution by immunoadsorption with antisera specific for hCG beta. The ovarian growth inhibiting activity was further purified on an anti-hCG alpha-immunoglobulin G affinity column. The activity was eluted from the affinity column at low pH, and eluted material contained all of the immunodeterminants of hCG. Virtually identical dose-response curves of ovarian inhibition were obtained using equivalent doses of beta-carboxyterminal peptide immunoreactivity of purified inhibitor and purified hCG (CR123). The inhibiting activity reached plateau of 80-90% at doses of 50-100 ng hCG/rat. Upon rechromatography on Sephadex G-100, the ovarian growth activity that was pooled from fractions corresponding to the 12,000-20,000 mol wt range was recovered in fractions corresponding to the elution position of hCG. We conclude that the low mol wt inhibiting activity observed in the crude pregnancy extracts is due to hCG and that hCG is a very potent inhibitor of FSH/DES stimulation of ovarian growth.

Effects of pergolide on diethylstilbestrol-induced rat pituitary hyperplasia.

Hyperplastic anterior pituitary glands were produced in female rats by treatment with 10 mg of diethylstilbestrol in Silastic tubing. This led to increased numbers of immunoreactive prolactin cells and increased serum prolactin levels. After 6 weeks of diethylstilbestrol treatment, one group of rats was treated with daily injections of pergolide for 3 weeks. Pergolide produced a significant decrease in pituitary gland weight and in serum prolactin levels but did not change the percentage of prolactin cells significantly, compared with that of control rats. Ultrastructural studies showed a significant increase in the numbers of prolactin secretory granules and numerous large intracellular bodies with associated secretory granules in pituitaries from rats treated with pergolide. In one group of rats in which the diethylstilbestrol was discontinued for 3 weeks after 6 weeks of treatment there was a significant decrease in pituitary gland weight and serum prolactin and a significant decrease in the percentage of prolactin cells, compared with values in the rats treated with diethylstilbestrol for 9 weeks. These results indicate that pergolide causes decreased release of prolactin from secretory granules in anterior pituitary prolactin cells and an increase in the numbers of PRL secretory granules per cell but does not change the percentage of prolactin-producing pituitary cells after 3 weeks of treatment.

The inhibitory effect of caffeine on hormone-induced rat breast cancer.

Studies have associated coffee and/or caffeine with human fibrocystic breast disease. Two animal studies have implicated caffeine as a promoter in rat mammary cancer. The current investigation examines the effect of two caffeine doses in ACI rats with and without diethylstilbestrol (DES). Without DES, cancer did not develop in any of the rats receiving either of the two caffeine dosages. With DES, increasing caffeine dosage lengthened the time to first cancer, decreased the number of rats that developed cancers, and decreased the number of cancers overall. The presence or amount of caffeine did not cause detectable histologic differences in the breast cancers. The presence or amount of caffeine did not influence animal weight or mortality, although the rats without DES weighed more and survived better into old age. The presence or amount of caffeine did not influence pituitary weights and prolactin levels, although values of the DES groups were three times higher than the values for the group without DES (P less than 0.05). In conclusion, chronic caffeine ingestion inhibits rat breast cancer, neither by interfering with the high prolactin levels--a necessary step in murine tumor development--nor by causing hypocaloric intake.

Fibrosis y cirrosis en el conejo por acción del dietilestilbestrol y su inhibición por la progesterona / Fibrosis and cirrhosis in the rabbit induced by diethylestilbestrol and its inhibition with progesterone

El dietilestilbestrol administrado por boca en forma prolongada (más de tres meses) produce en conejos una intensa fibrosis o cirrosis hepática. Se investigó el efecto de la progesterona inyectada en forma intramuscular día por medio (33 mg) respecto a esta fibrosis hormonal. Se estudiaron cuatro lotes de conejos. En el primero (lote A) compuesto por once conejos, se administró el estrógeno, se realizaron biopsias quirúrgicas seriadas y se comprobó la existencia de fibrosis después de los tres meses de iniciado el estrógeno. Al suspender el dietilestilbestrol la fibrosis desapareció paulatinamente hasta que no era histológicamente comprobable a los ocho meses de la suspensión. En el segundo (lote B) se administró conjuntamente el dietilestilbestrol y la progesterona en nueve conejos. El estudio histológico demostró que la progesterona impedía totalmente o disminuía la formación del tejido conectivo. El tercero (lote C) estaba compuesto por trece conejos, ocho recibieron dietilestilbestrol durante un período de cuatro a doce meses y sirvieron de testigos a cinco conejos que recibieron dietilestilbestrol durante seis meses, y a éstos sin suspender el estrógeno, se les añadió progesterona. Los conejos se sacrificaron a los cuatro meses y a los nueve meses de recibir la progesterona. Se comprobó que la fibrosis era mucho menor que en los conejos que no habían recibido progesterona. Se hicieron tambíen mediciones de enzimas, bilirrubina, etc., a todos los lotes sin que se comprobaran alteraciones con respecto a los valores normales, y se realizó también el estudio histológico de conejos que habían recibido solamente progesterona en forma prolongada sin encontrar anormalidades. Se concluye que en la fibrosis y cirrosis producida en conejos por la administración prolongada de dietilestilbestrol, la progesterona administrada simultáneamente impide totalmente o disminuye al mínimo el aumento del tejido conectivo. Administrada una vez producida la fibrosis y sin eliminar el estrógeno, la progesterona disminuye el aumento del tejido conectivo provocado por el dietilestilbestrol

Fibrosis y cirrosis en el conejo por acción del dietilestilbestrol y su inhibición por la progesterona / Fibrosis and cirrhosis in the rabbit induced by diethylestilbestrol and its inhibition with progesterone

El dietilestilbestrol administrado por boca en forma prolongada (más de tres meses) produce en conejos una intensa fibrosis o cirrosis hepática. Se investigó el efecto de la progesterona inyectada en forma intramuscular día por medio (33 mg) respecto a esta fibrosis hormonal. Se estudiaron cuatro lotes de conejos. En el primero (lote A) compuesto por once conejos, se administró el estrógeno, se realizaron biopsias quirúrgicas seriadas y se comprobó la existencia de fibrosis después de los tres meses de iniciado el estrógeno. Al suspender el dietilestilbestrol la fibrosis desapareció paulatinamente hasta que no era histológicamente comprobable a los ocho meses de la suspensión. En el segundo (lote B) se administró conjuntamente el dietilestilbestrol y la progesterona en nueve conejos. El estudio histológico demostró que la progesterona impedía totalmente o disminuía la formación del tejido conectivo. El tercero (lote C) estaba compuesto por trece conejos, ocho recibieron dietilestilbestrol durante un período de cuatro a doce meses y sirvieron de testigos a cinco conejos que recibieron dietilestilbestrol durante seis meses, y a éstos sin suspender el estrógeno, se les añadió progesterona. Los conejos se sacrificaron a los cuatro meses y a los nueve meses de recibir la progesterona. Se comprobó que la fibrosis era mucho menor que en los conejos que no habían recibido progesterona. Se hicieron tambíen mediciones de enzimas, bilirrubina, etc., a todos los lotes sin que se comprobaran alteraciones con respecto a los valores normales, y se realizó también el estudio histológico de conejos que habían recibido solamente progesterona en forma prolongada sin encontrar anormalidades. Se concluye que en la fibrosis y cirrosis producida en conejos por la administración prolongada de dietilestilbestrol, la progesterona administrada simultáneamente impide totalmente o disminuye al mínimo el aumento del tejido conectivo. Administrada una vez producida la fibrosis y sin eliminar el estrógeno, la progesterona disminuye el aumento del tejido conectivo provocado por el dietilestilbestrol (AU)

[Effect of tamoxifen on the feminization of chick embryo testis]. / Action du tamoxifène sur la féminisation du testicule embryonnaire de poulet.

The problem was approached whether the feminizing action of oestrogens or androgens on the chick embryo testes could be antagonized by anti-oestrogens or anti-androgens. 100 micrograms of either tamoxifen, cyproterone acetate or compound RU 23908 were injected into the chick embryo after 4 days of incubation and 100 micrograms of either oestradiol benzoate or diethylstilboestrol or 1 mg of either androsterone, dehydroepiandrosterone, 5 alpha-dihydrotestosterone or 5 alpha-androstanedione were given one day later. The surviving embryos were sacrificed when 14-17 days old and their gonads were examined morphologically and in some cases also histologically. Control embryos were only given oestrogens or androgens. Out of 20 surviving embryos treated with oestradiol only, 10 were genetic females and 10 strongly feminized males. After tamoxifen treatment, out of 20 embryos, 11 were females, 7 normal males and 2 slightly feminized males. Thus, tamoxifen suppressed the feminizing action of oestradiol. It antagonized also the feminizing action of diethylstilboestrol. Neither cyproterone acetate nor compound RU 23908 suppressed the feminizing action of androsterone or diethylstilboestrol. But tamoxifen interfered with the androgens, preventing the feminization of the testes. It is suggested that androgens bind to the oestrogen receptor.

[Feminization of embryonic chicken testicle by diethylstilbestrol and antagonistic action of tamoxifen]. / Féminisation du testicule embryonnaire de poulet par le diéthylstilboestrol et action antagoniste du tamoxifène.

The question was asked whether the feminization of the chick embryo testis by diethylstilboestrol involves oestrogen secretion. The left gonads of male embryos treated with diethylstilboestrol at the stage of 5 days of incubation were removed at the stage of 17 days and cultured in vitro in the presence of radiolabelled androstenedione, dehydroepiandrosterone, progesterone or sodium acetate. Radiochromatography and repeated crystallizations to constant specific activity were used to demonstrate the formation of oestrone and oestradiol. The results show that oestrogen synthesis takes place from any of the 4 precursors. The normal testis does not produce oestrogens or produces them only in small amounts. Thus, it can be concluded that diethylstilboestrol induces oestrogen synthesis in the chick embryo testis. In a second part of the work, it is shown that tamoxifen antagonizes the feminizing action of diethylstilboestrol on the testes, both at the morphological and biochemical levels.

Diethylstilbestrol potentiation of the dopamine-elicited formation of adenosine 3',5'-monophosphate in incubated male rat hypothalamus.

Dopamine (DA) stimulates the cAMP-generating system in the male rat hypothalamus only to a very low extent (25% above control). Diethylstilbestrol (DES), a synthetic estrogen, was found to be extremely potent (a 4- and 16-fold stimulation at 20 micron and 100 micron, respectively). Addition of either one to an incubation medium containing varying concentrations of the other resulted in a synergistic response. The potentiation by 20 micron DES of the effect elicited by 100 micron DA was the most remarkable, namely, a 3-fold stimulation of the combined response. A 4- and 7.5-fold stimulation of cAMP accumulation was observed when adenosine (100 micron) or adenosine (100 micron) + DA (100 micron) were present in the incubation medium. Theophylline (0.5 mM), an adenosine antagonist, could effectively reduce this effect, as did adenosine deaminase (10 microgram/ml). Clomiphene (50 micron), an estrogen antagonist, exhibited a marked decrease in DES + DA-elicited cAMP formation. Pimozide (40 micron) had the ability to significantly block the stimulatory effects of DES and DA.