The effect of a single dose of oestradiol on tamoxifen-induced uterine hyperaemia and growth in the rat.

1. The effect of oestradiol on tamoxifen - (single doses of each agent) induced responses in the uterus of the mature ovariectomized rat was investigated. The parameters examined were uterine blood flow (measured by the microsphere technique), weight and oestrogen receptor concentrations. 2. Initially the addition of oestradiol to the tamoxifen regimen produced a reduction in uterine blood flow and dry weight which was reflected by a reduction in nuclear oestrogen receptor. However, as the tamoxifen response became more established from 48 h onwards the addition of oestradiol produced an additive effect. These changes were not mirrored by increases in nuclear oestrogen receptor. 3. The results suggest that tamoxifen and oestradiol may, in part, act via different mechanisms; the role of possible mediatory systems such as histamine release and eosinophil migration is discussed. Heterogeneous control systems may also be involved in the early and late uterine response.     

Modification of the rat uterine response to oestrogen and tamoxifen by thromboxane antagonists.

1. The thromboxane receptor antagonists EP092, AH23848 and BM 13.505 were used to investigate the role of thromboxane in the uterotrophic response to oestradiol and tamoxifen. 2. The parameters examined were uterine blood flow (measured by the microsphere technique), uterine wet and dry weights and the concentrations of cytosolic and nuclear oestrogen receptors. 3. Only EP092 potentiated the hyperaemic response to oestrogen but all three thromboxane antagonists inhibited oestradiol-stimulated uterine growth. This inhibition was accompanied by a decrease in nuclear oestrogen receptor concentration. 4. The uterotrophic response to tamoxifen was unaffected by the thromboxane antagonists. 5. The mechanism by which the thromboxane antagonists may be exerting their growth inhibitory effect is discussed, although, whether this effect can be attributed to blockade of thromboxane receptors or to some other mechanism is not clear from this study.     

A study on the effect of a single dose of tamoxifen on uterine hyperaemia and growth in the rat.

1 The effect of a single subcutaneous dose of tamoxifen on the rat uterotrophic response was investigated. 2 The parameters examined were uterine blood flow (measured by the microsphere technique), uterine wet and dry weights and the concentrations of cytosolic and nuclear oestrogen receptors. 3 Tamoxifen or its metabolites proved to be capable of eliciting a uterotrophic response of 35-42 days duration. The changes seen in uterine blood flow and weight are discussed in relation to oestrogen receptor distribution.     

Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery.

1. The effect of oestradiol 17 beta on vascular smooth muscle proliferation was examined in segments of the pig left anterior descending coronary artery (LAD). It was established by cytochemical techniques that out-growth from the segments was composed of vascular smooth muscle cells. 2. [3H]-thymidine uptake by pig LAD segments was used as an index of vascular smooth muscle cell proliferation. Nitroprusside and forskolin significantly inhibited [3H]-thymidine uptake and were used as positive controls. 3. Oestradiol 17 beta (180-360 nM) inhibited thymidine uptake by pig LAD segments (P < 0.05). The inhibition was observed only in the absence of phenol red, which is a weak oestrogen receptor agonist. The anti-oestrogens tamoxifen and its more potent metabolite 4-hydroxytamoxifen, both of which are partial oestrogen receptor agonists, also significantly inhibited thymidine uptake. However, pretreatment with either tamoxifen or 4-hydroxytamoxifen did not significantly block oestradiol 17 beta-induced inhibition of thymidine uptake. 4. The LAD segments bound [3H]-oestradiol 17 beta in a time-dependent manner and about 20 to 30% was displaced by an excess of unlabelled oestradiol 17 beta. Autoradiography showed [3H]-oestradiol 17 beta was evenly distributed in the cytosol and nuclei of cells in the three layers of the vessel wall. 5. The data suggest that oestradiol 17 beta inhibits smooth muscle cell proliferation in porcine LAD segments, possibly through an oestrogen receptor mechanism. This in vitro effect suggests an in vivo role for oestradiol 17 beta in directly protecting coronary arteries against myointimal proliferation in premenopausal women.     

The pure anti-oestrogen ICI 182,780 (Faslodex) activates large conductance Ca(2+)-activated K(+) channels in smooth muscle

Oestrogen and tamoxifen activate large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle through a non-genomic mechanism that depends on the regulatory beta1 subunit and an extracellular binding site. It is unknown whether a "pure" anti-oestrogen such as ICI 182,780 (Faslodex), that has no known oestrogenic properties, would have any effect on BK(Ca) channels. Using single channel patch clamp techniques on canine colonic myocytes, the hypothesis that ICI 182,780 would activate BK(Ca) channels was tested. ICI 182,780 increased the open probability of BK(Ca) channels in inside-out patches with an EC(50) of 1 microM. These data suggest that molecules with the ability to bind nuclear oestrogen receptors, regardless of oestrogenic or anti-oestrogenic nature, activate BK(Ca) channels through this nongenomic, membrane-delimited mechanism. The identity and characteristics of this putative binding site remain unclear; however, it has pharmacological similarity to oestrogen receptors alpha and beta, as ICI 182,780 interacts with it.     

Effects of the Environmental Oestrogens Bisphenol A,Tetrachlorobisphenol A,Tetrabromobisphenol A, 4‐Hydroxybiphenyl and 4,4′‐Dihydroxybiphenyl on Oestrogen Receptor Binding,Cell Proliferation and Regulation of Oestrogen Sensitive Proteins in the Human Breast Cancer Cell Line MCF‐7

Abstract: Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen‐like potency using several end‐points in MCF‐7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro‐ and tetrabromo‐bisphenol A, 4‐hydroxybiphenyl and 4,4′‐dihydroxybiphenyl all showed oestrogen‐like properties in MCF‐7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF‐7 cells, although their EC₅₀s were 1,000 to 80,000 times higher than the EC₅₀ of 17β‐oestradiol. Bisphenol A and 4‐hydroxybiphenyl induced cell growth in MCF‐7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17β‐oestradiol. The other chemicals tested induced less than 50% of the maximum 17β‐oestradiol‐stimulated cell growth. Bisphenol A, 4‐hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen‐regulated proteins, progesterone receptor and pS2, whereas 4,4′‐dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17β‐oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF‐7 cells. By measuring several oestrogen‐dependent endpoints it seems that some xeno‐oestrogens cause an imbalanced oestrogen‐response. Their ability and potency in mimicking 17β‐oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen‐linked parameter.     

Aromatase inhibitors and other novel agents in breast cancer treatment

Thirty years after the introduction of tamoxifen, which was expanded from palliation of metastatic cancer to recent application for chemoprevention, the primacy of this drug as the mainline pharmacological intervention is currently being challenged by the third generation aromatase inhibitors and inactivators. In contrast to the oestrogen receptor blockade provided by tamoxifen, aromatase inhibitors result in deprivation of oestrogens in postmenopausal women both through paracrine/intracrine and endocrine modulation. Experimental evidence has shown a significant (97-99%) reduction of in vivo aromatase activity and an equal or sometimes better antitumour activity compared with megestrol acetate when these drugs are used as second-line treatment for metastatic breast cancer. Recent pivotal studies in first-line settings comparing tamoxifen for metastatic breast cancer and preliminary results from the neoadjuvant trials demonstrate that third generation aromatase inhibitors are superior to tamoxifen. With a better understanding of local tissue production of oestrogen through oestrone sulfatase, which hydrolyses oestrone sulfate to oestrone, and 17-beta-hydroxysteroid dehydrogenase Type 1, which in turn catalyses the reduction of oestrone to oestradiol, more powerful tactics for oestrogen starvation of cancer may be realised in future.     

Metabolism of tamoxifen by isolated rat hepatocytes. Identification of the glucuronide of 4-hydroxytamoxifen

Metabolism of 4-hydroxytamoxifen by hepatocytes isolated from rats administered with phenobarbital and examination by TLC of the components not extractable into ethyl acetate revealed 4-hydroxytamoxifen beta-glucuronide; its identity was confirmed by comparison of its 1H NMR spectrum with that of synthetic material. This conjugate was also formed on metabolism of tamoxifen. It bound to cytosolic oestrogen receptors with only one thousandth the affinity of 4-hydroxytamoxifen and gave a correspondingly very weak inhibition of growth of the MCF-7 human breast cancer cell line. Therefore, in contrast to reported observations on the 3-glucuronide of oestradiol, the MCF-7 cells were unable to hydrolyse 4-hydroxytamoxifen glucuronide and on this evidence, formation of this metabolite is solely a deactivation pathway.     

Subcellular Distribution and Protein Binding of Perfluorooctanoic Acid in Rat Liver and Kidney

Perfluorooctanoic acid (PFOA) is an organic fluorochemical, and its elimination in rats is markedly sex-dependent. Liver and kidney are two primary tissues of distribution of PFOA in rats. In this study, the subcellular distribution of PFOA in male and female rat liver and kidney was examined. The results demonstrated that PFOA content in the liver cytosol of the female rat was significantly higher (49 ± 6% of total radioactive residues, TRR) than in the male liver (26 ± 5% TRR), whereas PFOA distribution in the heavier subcellular fractions, especially the nuclei and cell debris fraction, was marginally higher in male rat liver. In rat kidney, more than 70% of PFOA was distributed in the cytosolic fraction, with no significant difference between sexes. The degree of protein binding of PFOA in rat liver and kidney cytosol was analyzed by two different chromatographic methods. The percentage of protein-bound PFOA in the liver cytosol was found to be approximately 55% in both male and female rats. In contrast, significantly more PFOA was bound to cytosolic proteins in the kidney of male rats (42 ± 6% TRR) than in females (17 ± 5% TRR). Ligand blotting analysis revealed that multiple proteins from the liver cytosol, nuclei, and mitochondria fractions were capable of specific binding to PFOA.     

The citrus-derived flavonoid naringenin exerts uterotrophic effects in female mice at human relevant doses

Gavage administration of the citrus flavonoid naringenin, 3',4,5,7-tetrahydroxyflavanon for 4 consecutive days, to immature female mice (postnatal day 17-20) at 4 or 100 mg/kg b.wt. significantly increased uterine weights 3 and 4 times, respectively. Analysis of uterine oestrogen receptor alpha revealed that naringenin significantly increased the cytosolic concentration of oestrogen receptor alpha, whereas in nuclei the oestrogen receptor alpha concentration was significantly decreased as compared to the solvent control. This was in contrast to the positive control 17 beta-oestradiolacetate which acted as a true oestrogen by increasing the concentration of both total and nuclear oestrogen receptor alpha. Both naringenin and 17 beta-oestradiolacetate, however, significantly, induced nuclear oestrogen receptor alpha in the liver, suggesting a tissue specific effect of naringenin on oestrogen receptor alpha distribution. In order to investigate the tissue levels at which the uterotrophic effect was observed, the distribution of an oral dose of tritiated naringenin (4 mg/kg) was investigated in 3-week-old female mice. The radioactivity content (ng naringenin equivalents/g tissue) was found to be highest in the gastrointestinal-tract, followed by the kidneys and liver. Uterus and ovaries were also found to contain relatively high and approximately equal amounts of naringenin. The concentration of naringenin in uterus and ovaries was found to be ten times higher as compared to the mammary tissue. The urinary excretion of more than 25% of the administered dose, within 8 hr after dosing indicated that naringenin is absorbed extensively in mice. The plasma concentration of 0.5 microM found in the present study is similar to the peak plasma concentration of naringenin (0.6 microM) observed in man following ingestion of 400-760 ml of orange juice (Erlund et al. 2001). This could be taken to suggests that ingestion of orange juice and other citrus fruits and juices may give rise to sufficiently high tissue levels of naringenin in man to exert a biological effect.     

Clinical and biochemical investigations of a variable-dose combined type oral contraceptive

Summary

A 21-day treatment cycle oral contraceptive of variable composition was administered to 68 healthy young women, each of whom completed 6 cycles of treatment. For the first 11 days, the product contained 50 fig levonorgestrel plus 50 fig ethinyl oestradiol, then for treatment days 12 to 21, 125 fig levonorgestrel plus 50 fig ethinyl oestradiol. Cycle control was good with a mean length of 27.9±1.1 days. Breakthrough bleeding occurred in 1.2% of cycles, spotting in 2.8%, and amenorrhoea in 0.5%. There were no serious side-effects. A series of biochemical tests conducted on fasting blood specimens indicated metabolic changes similar to those seen with other 50 fig oestrogen oral contraceptives.     

改变奥扎格雷钠碱基对大鼠肝微粒体蛋白浓度和CYP450酶含量的影响

目的 改变奥扎格雷钠碱基为氨丁三醇后,研究药物对大鼠肝药酶的影响.方法 将健康SD大鼠分为7组(n=6),分别静脉注射给予高、中、低剂量的奥扎格雷氨丁三醇和奥扎格雷钠,并设定空白对照组,连续给药7d,于末次给药24 h后处死大鼠,取肝脏,制备肝微粒体.采用紫外-可见分光光度法测定大鼠肝微粒体蛋白的浓度和CYP 450酶的含量.结果 高、中、低剂量奥扎格雷氨丁三醇组大鼠的肝微粒体蛋白浓度均值分别为10.14±1.44、10.28±1.01、8.10±1.85 mg·mL-1,高、中、低剂量奥扎格雷钠组的肝微粒体蛋白浓度均值分别8.26--±1.55、8.19±0.49、8.72±1.76 mg· mL-1,与空白对照组比较,差异均无统计学意义.高、中、低剂量奥扎格雷氨丁三醇组大鼠的CYP 450酶含量分别为0.537±0.075、0.454±0.106、0.498±0.071 μmol·g-1,高、中、低剂量奥扎格雷钠组的CYP 450酶含量分别为0.504±0.092、0.450±0.086、0.476±0.028μmol·g-1,与空白对照组比较,差异均无统计学意义.结论 改变奥扎格雷钠碱基为氨丁三醇后,多剂量静脉给予大鼠奥扎格雷氨丁三醇后,其肝微粒体蛋白浓度和CYP 450酶的含量与奥扎格雷钠组及空白对照组相比,差异均无统计学意义.预测奥扎格雷改变碱基为氨丁三醇后对大鼠肝药酶无明显影响.     

The subcellular distribution of (plus or minus)-2,3-dehydroemetine and (-)-emetine in rat liver and changes in hepatic lipid content after treatment of rats with (plus or minus)-2,3-dehydroemetine

Treatment of female rats with (±)-2,3-dehydroemetine leads to increases in the total lipid content of their livers, without affecting phospholipid content appreciably. The subcellular distribution of (±)-2,3-dehydroemetine or (?)-emetine in the livers of rats pretreated with these drugs, shows that each compound is associated with the mitochondria rather than the microsomal fraction or the cell sap.     

ZD9583, an Orally Effective Thromboxane A2 Synthase Inhibitor and Receptor Antagonist with a Sustained Duration of Action in Rat and Dog

The thromboxane A₂ (TXA₂) synthase inhibitory activity and the TXA₂ receptor (TP-receptor) blocking action of ZD9583 ((4Z)-6-[(2S,4S,5R)-2-(1-[2-cyano-4-methylphenoxy]-1-methylethyl)-4-(3-pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in-vitro by use of whole blood and platelets from man, and ex-vivo by use of platelets and whole blood from rats and dogs. ZD9583 caused concentration–dependent inhibition of human platelet microsomal TXA₂ production with an IC50 of 0.017 ± 0.003 μm; this inhibition was associated with an increase in prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) formation. ZD9583 also inhibited collagen-stimulated TXA₂ synthesis in whole blood from man, rat and dog giving IC50 values of 0.027 ± 0.005, 0002 ± 0.006 and 0.013 ± 0.01 μm, respectively. The drug did not modify platelet cyclooxygenase activity as inhibition of thromboxane B₂ (TXB₂) formation was associated with a concomitant increased synthesis of prostaglandin D₂ (PGD₂), PGE₂ and PGF_2α. ZD9583 had little effect on cultured human umbilical vein endothelial cell prostacyclin synthase giving an IC50 of 24.2 ± 4.9 μm. In-vitro ZD9583 caused concentration-dependent inhibition of U46619-induced aggregation responses of platelets from man, rat and dog, yielding apparent log A₂ values of 8.7 ± 0.12, 8.8 ± 0.2 and 9.3 ± 0.2, respectively. The drug was selective; at concentrations up to 100 μm it did not affect 5-hydroxytryptamine or the primary phases of adenosine diphosphate and adrenaline-induced aggregation. ZD9583 (100 μm) did not, furthermore, modify the platelet inhibitory effects of PGD₂, prostaglandin E₁ (PGE₁) and prostacyclin. Oral administration of ZD9583 (3–10 mg kg^?1) to both rats and dogs caused dose-dependant inhibition of collagen-stimulated TXA₂ production ex-vivo which persisted for up to 12 h. The drug also caused profound TXA₂ receptor blockade in both species for in excess of 12-h after an oral dose of 3 mg kg^?1. ZD9583 (3 mg kg^?1, p.o.), when administered to dogs over a five-day period at 12 h intervals, did not cause either tachyphylaxis or an accumulation of effect. We conclude that ZD9583 is a potent, selective, orally active thromboxane synthase inhibitor and TXA₂ receptor antagonist.     

The effects of thromboxane receptor antagonists on oestrogen-induced uterotrophic responses in the spontaneously hypertensive rat.

1. The possible role of thromboxane in the uterotrophic response to oestrogen, in the spontaneously hypertensive rat was investigated by use of the thromboxane receptor antagonists EP092, AH23848 and BM 13.505. 2. The parameters studied were uterine blood flow (measured by the microsphere technique), uterine wet and dry weights and the concentrations of cytosolic and nuclear oestrogen receptors. 3. The antagonists attenuated oestradiol-induced uterine blood flow and significantly reduced both wet and dry uterine weight. These changes were accompanied by decreases in nuclear oestrogen receptor levels. 4. The results suggest a supportive role for thromboxane in oestradiol-induced uterine growth.     

Oestrogenic activity of benzylparaben

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10,000-fold molar excess, by 57% at 100 000-fold molar excess and by 100% at 1,000,000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive MDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping.     

Subcellular and extracellular localization of specific binding sites for triphenylethylene antiestrogens in human breast cancer

MCF-7 human breast cancer cell homogenates and subcellular organelles were submitted to isopycnic centrifugation on Percoll gradients to investigate the subcellular localization of triphenylethylene antiestrogen specific binding sites (AEBS). Electron microscopy revealed that gradient fractions coincident with the migration of [3H]tamoxifen-AEBS complexes were homogeneously represented by rough and smooth endoplasmic reticulum. Eighty percent of AEBS were localized in the endoplasmic reticulum [45,000 +/- 4,000 sites/cell, mean +/- S.D.), while 20% of these sites were also found in the nuclear fraction (12,000 +/- 1,000 sites/cell, mean +/- S.D.). A similar subcellular distribution of AEBS was observed in human breast cancer bioptic specimens. No differences in [3H]tamoxifen binding affinity between microsomal and nuclear AEBS were observed in MCF-7 and bioptic breast cancer. No major differences in microsomal AEBS levels were observed in the limited number of estrogen receptor-positive or -negative breast cancer specimens we have studied, whereas estrogen receptor-negative samples had higher levels of nuclear AEBS with respect to estrogen receptor-positive tumors. The presence of AEBS was also detected in the human serum of healthy and tumor-bearing subjects. The affinity and the binding specificity of serum AEBS were similar to those of intracellular AEBS. No differences in the levels of serum AEBS were observed between healthy and tumor-bearing subjects [19 +/- 4 and 22 +/- 4 pmoles/ml (mean +/- S.D.) respectively. Human serum AEBS did not appear to be associated to lipoproteins, whereas it migrated as a 5.5 S sedimenting molecule.     

Drug Evaluation: Oncologic,Endocrine & Metabolic: Toremifene

Toremifene is a triphenylethylene antioestrogenic derivative, chemically and pharmacologically related to tamoxifen. In animals, toremifene blocks the uterotrophic effect of oestradiol and, in intact animals, uterine weights are reduced, demonstrating the antioestrogenic activity of the compound. Such antioestrogenic effects are believed to underlie the antitumour actions of toremifene in breast cancer, i.e., it competes with oestrogen for binding sites in the cancer, thus preventing the growth-stimulating effects of oestrogen. The drug is well absorbed following oral administration and the mean elimination half-life is 5 days. Toremifene is extensively metabolised and the main metabolite in human serum is N-demethyltoremifene. This metabolite has a longer half-life (10 days) than toremifene and its steady state serum concentration is about 2 times higher than that of the parent compound.

Unlike tamoxifen, toremifene is not a genotoxic carcinogen in rodent studies and, similarly, is not associated with endometrial cancers following administration to patients with breast cancer. Phase I studies indicated that toremifene is well-tolerated by post-menopausal volunteers or women with various cancers in doses up to 400 mg/day. Phase II-III studies in patients with locally advanced and advanced breast cancer confirmed that toremifene is well-tolerated and, furthermore, demonstrated similar response rates to tamoxifen. Patients with oestrogen receptor (ER)-positive tumours fared better than those patients with unknown tumour composition. Toremifene's efficacy, excellent tolerability and low toxicity profile render this agent useful for the treatment of patients with locally advanced and advanced breast cancer.     

The effect of mepyramine and ranitidine on the oestrogen and anti-oestrogen stimulated rat uterus.

The aims of this study were to elucidate further the role of histamine in the rat uterotrophic response and to investigate the differences between the oestrogen and the anti-oestrogen induced uterine responses. The parameters examined were uterine blood flow (measured by the microsphere technique), uterine wet and dry weights. 17 beta-Oestradiol and the anti-oestrogen, tamoxifen, were used to stimulate the ovariectomised rat uterus and the antihistamines mepyramine (H1) and ranitidine (H2) were employed to modify these responses. The uterine changes evoked by oestradiol proved to be more susceptible to modification by the antihistamines than the tamoxifen-stimulated responses. The results suggest that histamine is involved in the early uterine response to oestradiol but histamine does not appear to play a major role in the uterine response to tamoxifen.