Urinary phytoestrogen excretion of rats bearing methylnitrosourea-induced mammary carcinoma in response to treatment with 2-methoxyestradiol.

The effect of treating mammary tumor-bearing rats with 2-methoxyestradiol (2-MeE2) on the urinary excretion of 12 phytoestrogens was investigated and compared with the changes in urinary excretion of estradiol metabolites. Alterations of excretion were registered for isoflavonoids, lignans and coumestans. However, due to large variations statistical significant differences were found only for two lignans, i.e. significant increases of enterodiol and matairesinol. Since the single components of phytoestrogens showed diverse alterations, excretions were expressed also by the ratio of total isoflavonoids to total lignans and compared with the estrogen ratios 2-hydroxyestrone to 16alpha-hydroxyestrone and A-ring to D-ring metabolites. The ratio of isoflavonoids to lignans was consistently decreased, whereas both ratios of estradiol metabolites were highly increased. The latter effect is probably due to demethylation of 2-methoxyestrone resulting in high catechol estrogen levels in urine. These results suggest that the high levels of catechol estrogens, produced by 2-MeE2 treatment, may have influenced the urinary excretion pattern of phytoestrogens.

Effect of 2-methoxyestradiol on the growth of methyl-nitroso-urea (MNU)-induced rat mammary carcinoma.

The present study investigated the influence of the endogenous estradiol metabolite 2-methoxyestradiol (2ME) on the growth of methyl-nitroso-urea (MNU)-induced mammary carcinoma in the rat. 2ME was administered by means of subcutaneously implanted osmotic pumps for a period of 4 weeks. The dosages of 2ME were 1 and 5mg/kg per day, the control animals received saline. At the low dosage of 2ME a stimulation of tumor growth was observed, whereas at the high dosage an inhibition was found. The urinary excretion of 15 estradiol metabolites revealed that 2ME triggered strong changes in estrogen metabolism in the organism. Our data show that 2ME may elicit both stimulation and inhibition of tumor growth depending on the dosage used, a fact which should be considered in case of therapeutic use.

The effect of zearalenone and 17 beta-estradiol on prostacyclin and thromboxane production in cell cultures from human umbilical cord veins.

The effect of zearalenone, a nonsteroidal mycotoxin with estrogenic activity, and of 17 beta-estradiol on prostacyclin and thromboxane production in human endothelial cells was investigated. Zearalenone stimulated prostacyclin production in low concentrations (10(-7) and 10(-8)M) and inhibited it at a higher concentration (10(-5)M). Estradiol alone in the concentration range 10(-5)-10(-8)M had no clear-cut effect on the prostacyclin production. The combination of both substances effected changes in prostacyclin production similar to that from zearalenone alone; with the exception of estradiol at a concentration of 10(-6)M which enhanced the effect of zearalenone. No distinct changes in the thromboxane production from the two substances could be found, either alone or in combination.

Immunoreactive ubiquitin in human seminal plasma.

The polypeptide ubiquitin, up to now almost exclusively discovered in intracellular spaces, was measured immunologically in a total of 187 samples of human seminal plasma. The values were between 1.83 and 19.11 micrograms/ml. In spermatozoa ubiquitin was detected too; the values, however, were significantly lower than in the seminal plasma. The origin and function of ubiquitin in human seminal plasma is still unclear. The possible role of ubiquitin in reproduction is discussed.

Effect of estradiol metabolites on prostacyclin synthesis in human endothelial cell cultures.

Estradiol can stimulate prostacyclin production in the vessel wall, thereby eliciting vasodilatation. In the present work the effect of the estradiol metabolites estrone, 2-methoxyestrone, 2-methoxyestradiol, and 16alpha-hydroxyestrone were investigated to find out if they are also able to stimulate prostacyclin synthesis. All metabolites triggered an increase of prostacyclin synthesis in human endothelial cells starting at a concentration of 10(-9) M. The parent substance, 17beta-estradiol, accomplished this effect only starting at a concentration of 10(-8) M. These results indicate that estradiol metabolites may take part in the estradiol-induced vasodilatation in vivo.

Effect of estradiol metabolites on the susceptibility of low density lipoprotein to oxidation.

The main estradiol metabolites 2-hydroxyestrone, 2-methoxyestrone and 16alpha-hydroxyestrone were investigated in vitro for the susceptibility of low density lipoprotein to oxidation and the effects compared with those of estradiol and vitamin E. 2-hydroxyestrone and 2-methoxyestrone had a greater inhibitory effect than estradiol and vitamin E whereas 16alpha-hydroxyestrone approximates the inhibition of estradiol. These results indicate that 2-hydroxyestrone and 2-methoxyestrone possess non-genomic actions which may play a role in the lipid metabolism.

The effects of A-ring and D-ring metabolites of estradiol on the proliferation of vascular endothelial cells.

The effects of 14 estradiol metabolites on the proliferation of cultured endothelial cells of human umbilical cord veins were examined and compared with that of their parent substance estradiol. The relationship between dosage and effect was tested over the pharmacological concentration range of 10(-8) to 10(-5) M. Estradiol showed a biphasic behaviour, in the form of stimulation at low concentrations and inhibition at the highest concentration. All 10 A-ring metabolites tested stimulated the growth of the endothelial cells at the lower concentrations. At the highest concentration, the 5 A-ring metabolites: 2-hydroxyestrone, 2-hydroxyestradiol, 2-hydroxyestriol, 4-hydroxyestrone and 4-hydroxyestradiol caused significant inhibitions. Except for the 2-hydroxyestradiol, methylation of these metabolites resulted in the loss of the proliferation inhibiting effect. The D-ring metabolites showed no marked effects compared to the A-ring metabolites except for 16alpha-hydroxyestrone which had an inhibiting effect from 10(-7) to 10(-5) M. Our results show that estradiol metabolites can influence the growth of vascular endothelial cells in the concentration range tested. While the antiproliferative action of 2-methoxyestradiol has been known for some time this study is the first to show the potential capacity of non-methylated metabolites of the A-ring metabolism in inhibiting endothelial proliferation. This may open up new clinical pharmacological aspects in the anti-angiogenetic treatment of tumors.

The impact of endogenous estradiol metabolites on carcinogenesis.

The available literature on estrogen metabolism and estrogen metabolites involved in carcinogenesis is reviewed. Endogenous estradiol metabolism leads to metabolic products that can have various, and, to some extent, contrary, biologic effects. Thus, there are numerous research findings on the stimulation and inhibition of cancer growth by estrogen metabolites. Furthermore, there are indications that, in certain types of cancer, the production of growth-stimulating estradiol metabolites is increased. There are also reports on substances that can influence estradiol metabolism. So far, only a few estradiol metabolites have been examined with respect to their influence on the development and growth of cancer. It is presumed that other metabolites can also intervene directly or indirectly in the cancer process, but there is a great lack of research in this area. An understanding of the actions of estradiol metabolites may open up new avenues for the therapy of malignant diseases. Although little is known about the biologic effects of most of the estradiol metabolites, the reported actions of certain estradiol metabolites already justify clinical investigations on their possible beneficial uses in tumor therapy.

Urinary cGMP excretion after hormone replacement therapy in postmenopausal women.

It is well established that estrogens and progestogens are able to influence the vasotonus in postmenopausal women. The present study was undertaken to find out if the NO/cGMP-system is involved in this hormone action. Urinary cGMP excretion which can reflect intracellular cGMP production elicited by NO (EDRF) was investigated in 20 postmenopausal women. In an open cross-over study design norethisterone acetate was administered orally for 8 days, estradiol valerate orally for 9 days and a combination of both substances for 12 days. After all three treatment phases urinary cGMP expressed as percentage of the pretreatment value was increased at a statistically significant level. Due to high individual variations no significant differences could be found among the values after the three treatment phases. It was concluded that the NO/cGMP-system may play a role in maintaining vasotonus in postmenopausal women under hormone replacement therapy.

Effects of valsartan and 17 beta-estradiol on the oxidation of low-density lipoprotein in vitro.

BACKGROUND: The 'sartans' are antagonists of the angiotensin type 1 (AT1) receptor that are mainly used for treatment of hypertension. Little is known about AT1-independent effects of these substances and interactions with other drugs used for prevention of cardiovascular diseases. Postmenopausal estradiol-replacement therapy has been shown to exert beneficial antiathero-sclerotic properties by inhibiting oxidation of low-density lipoprotein (LDL). OBJECTIVE: In the present study, the effects of valsartan alone and in combination with 17 beta-estradiol on the oxidation of isolated human LDL were investigated. METHODS: Oxidation of LDL, which was triggered by copper (II) chloride, was monitored spectrometrically at 234 nm. The test substances were added in vitro. RESULTS: Valsartan alone increased the duration of resistance of LDL to oxidation by 75.3 +/- 5.7 min at 5 mumol/l and by 138.2 +/- 8.1 min at 10 mumol/l. 17 beta-estradiol alone delayed the onset of oxidation of LDL by 75.7 +/- 5.1 min at 1 mumol/l. With the combination of 5 and 10 mumol/l valsartan with 1 mumol/l estradiol the time to onset of oxidation of LDL was increased by 142.8 +/- 4.9 and 215.3 +/- 6.9 min, respectively. CONCLUSIONS: There has been demonstrated an antioxidative effect of valsartan that was additive to that of 17 beta-estradiol. Thus this combination has the potential to be useful in the treatment of postmenopausal women with hypertension.

Serotonin metabolite excretion after postmenopausal estradiol therapy.

Serotonin, known for its beneficial action on mood and well-being, is also involved in cardiovascular functions. Thus the current work was undertaken to study the effect of hormone replacement therapy on serotonin turnover in postmenopausal women. Eighteen women received estradiol transdermally and 17 women estradiol valerate orally for 4 weeks. The serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) was determined in the urine before, and after 2 and 4 weeks' estradiol treatment. With both administration routes estradiol produced a significant increase in urinary 5-HIAA excretion, greatest with transdermal estradiol after 28 days of treatment. The enhancement of serotonin turnover may contribute not only to an improvement of mood and well-being but also to a cardioprotective effect of estradiol observed after hormone substitution in postmenopausal women.

Continuous-combined treatment of the menopause with combinations of oestradiol valerate and dienogest - a dose-ranging study.

OBJECTIVES: To determine the progestational efficacy of continuous treatment with various doses of dienogest, combined with oestradiol valerate, on the basis of endometrial histology, effect on climacteric symptoms and bleeding profile in postmenopausal women. METHODS: Patients were randomised to one of five fixed-combination treatments, oestradiol valerate 2.0 mg plus dienogest 0.5, 1.0, 2.0, 3.0 or 4.0 mg. Efficacy was assessed by endometrial biopsy, menstrual charts and change in climacteric symptoms. RESULTS: The endometrium was classified as atrophic in 20.0, 31.3, 25.0, 55.6 and 57.1% of patients in the 0.5, 1.0, 2.0, 3.0 and 4.0 mg dienogest groups, respectively. The frequency of uterine bleeding was dose-dependent. The most favourable bleeding profile was seen in the 3.0 mg dienogest group, whereas the lower doses of dienogest had advantages with respect to the efficacy of the combined preparation. CONCLUSIONS: Dienogest 2.0 and 3.0 mg are the optimal doses for combination with 2.0 mg oestradiol valerate for continuous-combined hormone replacement therapy.

Effect of 17 alpha-ethinylestradiol, levonorgestrel, 3-keto-desogestrel, and gestodene on calcium influx via voltage-gated calcium channels in human aortic smooth muscles.

The effect of the synthetic estradiol, 17 alpha-ethinylestradiol, and three progestogens on calcium influx was investigated in cell cultures of human aortic smooth muscle. Neither the synthetic estrogen nor the progestogens levonorgestrel, 3-keto-desogestrel, and gestodene showed, in the concentration range of 10(-9) to 10(-6) M, a significant effect on calcium influx both alone or in equimolar estrogen-gestagen combinations. The results indicate that these substances, commonly used in contraceptive pills, do not change vasotonus interfering with calcium homeostasis.

Seasonality of pineal melatonin production in the rat: possible synchronization by the geomagnetic field.

Pineal melatonin production was estimated by means of urinary 6-sulfatoxymelatonin (aMT6s) determination in two groups of female rats for 1 year each. Seasonal changes of nocturnal aMT6s excretion were found with peak levels in summer despite constant photoperiods. We hypothesize that the horizontal component H of the geomagnetic field may act as a seasonal zeitgeber because H shows a similar seasonal rhythm, and changes in the direction and intensity of H can affect pineal activity. The observed seasonal changes of pineal melatonin production stress that despite constant environmental conditions, endocrine experiments require consideration of season, neglect of which may lead to contradictory results.

Valsartan inhibits angiotensin II-stimulated proliferation of smooth muscle cells from human coronary artery.

OBJECTIVES: Angiotensin II is involved in the pathogenesis of atherosclerosis by inducing hyperproliferation of vascular smooth muscle cells. Little is known whether the sartans can inhibit the angiotensin-stimulated proliferation of smooth muscle cells. METHOD: The effect of valsartan on the angiotensin II-stimulated proliferation of smooth muscle cells from human coronary artery was investigated. RESULTS: Angiotensin II significantly increased cell proliferation by about 30% at a concentration of 10(-6) M without significant changes at the lower concentrations 10(-7) and 10(-8) M. Valsartan at the dosages 10(-8) to 10(-6) M had no effect on serum-stimulated proliferation. Valsartan at the dosages 10(-6) and 10(-7) M inhibited the cell proliferation induced by 10(-6) M angiotensin. CONCLUSION: Valsartan may prevent atherosclerosis by inhibiting angiotensin-induced vascular smooth muscle cell proliferation.

The inhibitory effect of endogenous estrogen metabolites on copper-mediated in vitro oxidation of LDL.

The antioxidant effects of 17beta-estradiol, its main A- and D-ring metabolites, and of vitamin E were compared in vitro. Low density lipoprotein (LDL) was isolated from fresh human blood, and LDL oxidation was evaluated spectrometrically by monitoring diene formation of fatty acids. All substances tested exhibited antioxidant potential. The A-ring metabolites (catecholestrogens) emerged as more potent inhibitors of LDL oxidation than the parent substance estradiol, its D-ring metabolites, and vitamin E. Since oxidized LDL seems to play a crucial role in the development of atherosclerosis, its inhibition may be of preventive value. In summary, A-ring metabolites of estradiol (catecholestrogens), substances that occur naturally in the body, may be involved in the physiologic inhibition of LDL oxidation.

Calciumantagonistic effect of natural and synthetic estrogens--investigations on a nongenomic mechanism of direct vascular action.

Cardiovascular effects of estrogens have been shown to be of great clinical importance treating patients with hormonal replacement therapy or using oral contraceptives. To test one nongenomic mechanism of direct vascular action, the calcium-antagonistic effect of natural and synthetic estrogens was investigated in cell cultures of human vascular muscle cells. 17 beta-estradiol significantly inhibited calcium influx at the concentrations of 10(-6) and 10(-7) M in resting and activated cells. Neither the natural estrogens, estrone, and estriol nor the synthetic estrogens, 17 alpha-estradiol and 17 alpha-ethinylestradiol, significantly changed calcium homeostasis in these cells. The results suggest that the vasodilatory effect of 17 beta-estradiol, seen with hormone substitution in postmenopausal women, could be mediated at least partly via influence on calcium homeostasis.

Fluvastatin increases prostacyclin and decreases endothelin production by human umbilical vein endothelial cells.

Fluvastatin, an agent of a class of lipid-lowering drugs, the "statins", significantly enhanced prostacyclin synthesis at the concentrations of 0.1 microM and 1 microM and significantly reduced endothelin production at the concentrations 0.01, 0.1 and 1 microM in cell cultures of human umbilical endothelial veins. Since prostacyclin is a vasodilator and endothelin a vasoconstrictor, fluvastatin may have a significant effect on hemodynamics by favoring the balance towards vasodilation. This mechanism may contribute to the prevention of cardiovascular diseases.

The effect of estradiol on the production of melatonin in postmenopausal women.

The influence of two routes of estradiol administration on pineal melatonin production in postmenopausal women was investigated. Both transdermal and oral estradiol treatment led to an increase as well as decrease of melatonin production in different patients. The reason why individuals respond either in a stimulatory or inhibitory manner is unknown and requires to be evaluated in further more extensive studies.

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.