In vitro liver clearance of tritiated estradiol-17β in the female rat after retrochiasmatic transection and ovariectomy

It was reported in a previous study that serum estradiol-17β (E₂) was elevated in rats after retrochiasmatic transection (FC). Serum E₂ was also higher in estradiol cypionate treated, ovariectomized (ovx) rats that had been subjected to FC than in those that had not. This suggested that increased secretion of E₂ was not the only factor responsible for elevated serum E₂ after FC. To ascertain the contribution of decreased metabolism of E₂ to this response, liver tissue slices were incubated with 3H-estradiol-17β, and the rates of ³h uptake and conversion to water-soluble conjugates were measured.The rate of uptake of ³H by the tissue was not indicative of the rate of conjugate formation. Livers of rats with high serum E₂ exhibited lower rates of ³h uptake than those of rats with low serum E₂. Even so, the formation of ³H conjugates was greater in liver of rats with high serum E₂. As hypothesized, livers of rats with FC formed conjugates at a significantly lower rate than those of similarly treated rats without FC. Thus, FC of an intact rat leads to an increase in serum E₂ by increasing the secretion of E₂, and apparently also by decreasing the rate of E₂ metabolism by the liver.     

Effect of prostaglandins E1, E2 and F2α on neutrophil aggregation

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E₁, E₂ and F_2α alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formly-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E₁, E₂ and F_2α were 10⁻⁷, 10⁻⁶ and 10⁻⁵M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Effects of prostaglandin E1 on the metabolism in rat parathyroid gland in vitro

We investigated some effects of prostaglandin E₁ on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5–10% heat-inactivated rat serum. Rat parathyroid glands incorporate [³H]fucose and ¹⁴C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E₁ (1 μg/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [³H]-fucose, ¹⁴C-labeled amino acids, or [³H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incroporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [³H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E₁. It is concluded that, in organ culture, prostaglandin E₁ inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone.     

Estriol metabolism in the baboon: analysis of urinary and biliary metabolites

The metabolism of i.v. estriol was investigated in two intact baboons and four with biliary fistulas. Urine and bile samples were collected periodically and the radioactivity extracted by Amberlite-XAD resin. Metabolites were separated and purified by a combination of DEAE-Sephadex chromatography, celite partition, specific enzyme hydrolysis of the conjugates and identification of the aglycones. The excretion and metabolism of estriol in the animals closely resembled those of the human. Intact animals excreted an average of 50% of the radioactivity in the urine during 12 hours and two animals with biliary drainage excreted an average of 40% in the urine and 49% in the bile. When the steroid was injected into the portal vein an average of 11.5% and 84% were excreted in the urine and bile, respectively.In the urine of intact animals, approximately 65.8% of the radioactivity was in the form of E₃-16G; 14.2% as E₃-3G; 13.4% as E₃-3S and 5.1% as E₃-3S-16G. Over 73% of biliary radioactivity from the peripheral injections was made up of E₃-3S-16G and 3.6% as E₃-16G and 8.3% as 3-sulfate. In the urine,however, 57% of the label was made up of E₃-16G. No radioactive E₃-3G was detected in the bile of any of the animals. Following simultaneous injection of ³H-E₃ peripherally and ¹⁴C-E₃ intraportally, the 3-glucosiduronate excreted in the urine was derived exclusively from the ³H-label. Based on the results obtained, the baboon has been shown to metabolize estriol in the same fashion as the human, with E³-3S-16G as the predominant biliary metabolite and E³-16G as the major urinary metabolite. As in the human, evidence was also found for an enterohepatic circulation of e³ in the baboon, 16-glucuronidation in the kidney, and extrahepatic (enteric?) formation of E³-3G. $\text{In vitro}$ incubation of the baboon liver yielded 94% of the total conjugate as E³-16G without any trace of E³-3G.     

Effects of various prostanoids on th in vitro metabolism of bovine articular chondrocytes

The dose-dependent effects of 9 prostanoids (PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂, PGI₂, 6 keto- PGF_1α) on metabolism of cultured bovine articular chrondrocytes were investigated. Most prostanoids dose-dependently inhibited ³⁵SO₄⁼ and ³H-glycine incorporation. At 25 μg/ml, the inhibitory sequence was A₂D₂>E₂ = E₁ = A₁>6 keto-F_1α>F₁>F₂, but sensivity (lowest dose eliciting inhibition) followed the sequence E₂ > 6 keto-F_1α = F₁ > A₂ = D₂>E₁>A₁. At 25 μg/ml PGA₂ also inhibited incorporation of ³H-cytidine and ^#H-thymidine, but had no significant effect on ³H-glucose or ¹⁴C-xylose incorporation. The inhibitory effect of PGA₂ was apparent after 30 minutes exposure for ³⁵SO₄= and after 60 minutesd for ³H-cytidine, and was still present up to 72 hours following incubation in fresh non-PG-containing medium. PGI₂ had no significant effect of ³⁵SO₄⁼ incorporation but at concentrations below 10 μg/ml enhanced uptake of ³H-glycine.The PG-induced inhibitory effect was apparently not due to cell damage as indicated by measurement of ³H-glucose metabolism and lactate production.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Metabolism of estrone sulfate in the pregnant sheep

The metabolism of ³H-estrone sulfate (³H-E₁S) in 4 pregnant sheep, two injected i.v. and two i.m., has been studied. Intravenously injected ³H-E₁S had a plasma half-life of approximately 8 min, and metabolic clearance rate of approximately 800 ml/min. Using this clearance rate and the previously published mean plasma concentration of E₁S, the estimated production rate of E₁S is between 8.8 nmol (3.3 μg) and 78.2 nmol (29.1 μg) per min from 2-day to 0-day before parturition.Intramuscularly injected ³H-E₁S disappeared from plasma linearly and was completely cleared well within 3 hours. In all cases, whether i.v. or i.m. injected, the main metabolite isolated was ³H-estradiol-17β-3-sulfate, with only a trace amount as ³H-estradiol-17β-3-sulfate.     

Mechanism of increased renal prostaglandin E2 in uranyl nitrate-induced acute renal failure

We have previously demonstrated that decreased cortical prostaglandin metabolism can contribute significantly to an increase in renal tissue levels and activity of prostaglandin E₂ in bilateral ureteral obstruction, a model of acute renal failure. In the present study, we have further investigated whether alterations in prostaglandin metabolism can occur in a nephrotoxic model of acute renal failure. Prostaglandin synthesis, prostaglandin E₂ metabolism (measured as both prostaglandin E₂-9-ketoreductase and prostaglandin E₂-15-hydroxydehydrogenase activity), and tissue concentration of prostaglandin E₂ were determined in rabbit kidneys following an intravenous administration of uranyl nitrate (5 mg/kg). No changes in the rates of cortical microsomal prostaglandin E₂ and prostaglandin F_2α synthesis were noted at the end of 1 and 3 days, while medullary synthesis of prostaglandin E₂ fell by 47% after 1 day and 43% after 3 days. Cortical cytosolic prostaglandin E₂-9-ketoreductase activity was found to be decreased by 36% and 76% after 1 and 3 days respectively. No significant changes were noted in cortical cytosolic prostaglandin E₂-15-hydroxydehydrogenase activity after 3 days. Cortical tissue levels of prostaglandin E₂ increased by 500% at the end of 3 days. These data demonstrate that in nephrotoxic acute renal failure, decreased prostaglandin metabolism (i.e., prostaglandin E₂-9-ketoreductase activity) can result in increased tissue levels of prostaglandin E₂ in the absence of increased prostaglandin synthesis and suggest that alterations in prostaglandin metabolism may be an important regulator of prostaglandin activity in acute renal failure.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.     

Effect of thyroid state on estradiol-17β metabolism in the rabbit

It is well known that the metabolic clearance rate (MCR) of a hormone is Influenced highly by the level of its specific binding protein. It was interesting, therefore, to study the metabolism of estradiol-17β (E₂) in an animal model such as the rabbit where there is a lack for a highly specific binding protein for the steroid. The kinetics of the hormone was studied in relation to the thyroid state, namely in rabbits receiving thyroxin or propylthiouracil.In the absence of any significant decrease of the level of the rabbit androgen binding protein (R-ABP), the accelerated MCR^E2 and the elevated conversion ratio of estradiol to estrone (CR ^E₂→E₁) observed in hyperthyroid rabbits were attributed to the important role of metabolizing enzymes in the liver and/or extrahepatic tissues. In hypothyroid rabbits, while the CR ^E₂→E₁ decreased significantly the MCR^E2 was not altered.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.     

The metabolism of synthetic estrogens in non-users and users of oral contraceptives

Using both pulse injections and constant infusions of ³H-mestranol (³H-ME) (1) and ³H-ethinyl estradiol (³H-EE) we have studied the metabolism of these compounds in non-users and users of oral contraceptives. Following pulse injection of ³H-ME the disappearance of radioactivity could be described as a function which was the sum of two exponentials. Studied by both types of administration there was no difference in the metabolism of ³H-ME in the two groups; the overall mean ± SE metabolic clearance rate (MCR) was 690 ± 45 1/day/m², the mean ratio of the concentrations of radioactivity as EE following administration of ME (CR_BB^{M, E}) was 0. 23 ± 0. 02 and the mean [ρ]_BB^{M, E} (fraction of administered ME measured in blood as EE) was 0. 19 (95% confidence limits = 0.15 – 0. 23).Following pulse injection of ³H-EE the disappearance of radioactivity was best described as a function which is the sum of three exponentials. Results from both types of administration revealed no difference in the metabolism of ³H-EE between non-users. The overall mean ± SE MCR^EE was 630 ± 30 I/day/m². The MCR^EE is significantly (0. 02 > P > 0. 01) less than the mean MCR for estradiol reported previously, in both non-users and users of oral contraceptives. The use of oral contraceptives containing estrogens and progestins does not appear to influence the metabolism of the estrogen used. Approximately 20% of mestranol is converted to and appears in the blood as ethinyl estradiol.     

Isolation,structural and toxicological characterization of three new mycotoxins produced by the fungusAureobasidium pullulans

3 substances, B₁, B₂, and E₁ were isolated from culture medium extracts ofAureobasidium pullulans by reversed phase liquid chromatography and subsequent liquid chromatographic purification steps on silica gel. The 3 compounds inhibited the metabolism ofSaccharomyces cerevisiae and showed toxic effects in the growth inhibition test toEscherichia coli andBacillus subtilis. Elementary analysis and mass spectroscopical methods revealed sum formulas of C₂₃H₂₂O₆, C₂₂H₂₀O₆ and C₂₄H₂₈O₃ for B₁ B₂, and E₁ and molecular weights of 394, 380, and 364, respectively. Mass spectroscopical, UV-, IR-,¹³C-NMR, and¹H-NMR-spectroscopical investigations revealed polycyclic, non-aromatic compounds containing several carbonyl functions and double bonds and, most notably, spiroepoxy-functions, in the case of B₁ and B₂.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E₁, E₂, F_2α and I₂ in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E₁, E₂ and F_2α at low concentrations (10⁻¹⁰ to 10⁻⁷ M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesentric artery. PGI₂ (10⁻⁹ to 10⁻⁶ M) produced marked relaxation in both arteries. The EC₂₅ for PGI₂ in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10⁻⁷ M) of PGs E₁, E₂ and F_2α. In mesentric arteries, a large contraction was induced by PGs F_2α and E₂ but not by PGE₁. Arachidonic acid (10⁻⁶ M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI₂ for the relaxant effect than was the cerebral artery, while PGs F_2α, E₁ and E₂ caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Basal level of human platelet prostaglandins: PGE1 is more elevated than PGE2

Radioimmunoassays of platelet prostaglandins E₁ and F_1α in platelet rich plasma or platelet suspension, demonstrate that both PGE₁ and PGF_1α are present at higher concentrations than prostaglandins E₂ and F_2α. Gas chromatography — mass spectrometry determinations of prostaglandins E₁ and E₂ in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1-¹⁴C] acetate into prostaglandins E₁ and F_1α compared to that into prostaglandins E₂ and F_2α.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

Uptake and metabolism of oestriol in human target tissues

The uptake, metabolism and subcellular distribution of oestradiol and oestriol in endometrial, myometrial and vaginal tissue of postmenopausal women under physiological conditions were studied by giving ³H-labelled oestradiol or oestriol in subphysiological doses by continuous infusion lasting 12 h before hysterectomy. The three tissues obtained from each woman were separated into three fractions: two cytosol fractions (free oestrogens and specifically bound) and one nuclear fraction. The results show an accumulation of both oestrogens in the target tissues, we found an approximately 33 times higher [³H]E₂ concentration in endometrium (dpm per g) than in plasma (dpm/ml), 20 times in myometrium and 10 times in vaginal tissue. After the E₃ infusions the tissue/plasma gradient was 37 for endometrium, 19 for myometrium and 11 for vagina. In plasma and tissues a metabolite of E₃ could tentatively be identified as 16α-hydroxyoestrone. The subcellular distribution showed that 60–80% of E₂ and E₃ is accumulated in the nuclear fraction of all tissues studied, no nuclear bound oestrone could be detected. From these results the conclusion was drawn that oestradiol still is the major tissue oestrogen in postmenopausal women and that it is mainly nuclear bound. Endometrium of postmenopausal women accumulates higher concentrations of E₂ and E₃ than vaginal tissue from the same individual, no preferential uptake of oestriol occurs under physiological conditions.