Displacement by tamoxifen of the estradiol-estrogen receptor binding: a functional assay for breast cancer studies

Displacement curves with estradiol (E2) and Tamoxifen (Tam) of the [3H]E2-ER binding in 49 ER+ mammary neoplasia showed a great heterogeneity suggesting the existence of more than one population of ER+ tumors when the relative binding affinity of both ligands for the ER was considered. The (D50E2/D50Tam) x 100 ratio was called Displacement Index (DI) with values asymmetrically distributed from 0.05 to 2.90. The range from 0.18 to 0.54 was adopted as central interval given by the median +/- SE (median: 0.36; SE: 0.09). DI values below 0.18 (24% of the tumors in our series) were considered as "lower", indicating that higher Tam doses would be necessary to displace the E2-ER binding. The potency of Tam as displacer is dependent not only of its own affinity for the ER, but also of that of E2 for the same receptor. The DI expresses their relative binding "strength". DI values were not correlated with ER and progesterone receptor content nor with the D50 Tam and D50E2 taken separately. Antiestrogen binding sites (AEBS) were determined in the cytosol (AEBSc) and in the microsomal fraction of 10 ER+ tumors from our series. The AEBSc/ER ratio was inversely correlated with the DI, that is, displacement of 3HE2 from the E2-ER complex by Tam would be lower in tumors with higher AEBSc/ER ratio. The DI is another parameter to be considered in the study of the sensitivity of breast neoplasias to antiestrogen treatments.     

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.     

Characterization of an antiestrogen-binding protein in high salt extracts of human breast cancer tissue

An antiestrogen binding protein which binds [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)-phenyl]1,2-diphenylbut-1(Z)-ene) with high affinity (Kd = 1.1 X 10(-9) M) is present in high salt (0.6 M KCl) extracts of washed breast cancer tissue pellets. Its concentration in high salt extract is higher than its concentration in cytosol. The characteristics of the antiestrogen binding protein from cytosol and salt extract of breast cancer tissue are indistinguishable. It specifically binds triphenylethylene and other nonsteroidal antiestrogens and displays little or no binding affinity for estrogens, progesterone, dihydrotestosterone and cortisol. The antiestrogen binding protein is of unusually large size as judged by gel filtration on agarose 0.5 m and sedimentation analysis on 5-20% sucrose density gradients. Differential centrifugation studies indicate that it is not principally microsomal in origin. This protein is more thermostable than the estrogen receptor from which it can also be distinguished by ion exchange chromatography. The antiestrogen binding protein was eluted from DEAE-Sephacel by 0.05 M KCl indicating that it is less negatively charged than the estrogen receptor which was eluted by 0.1 M KCl. Lipoprotein fractionation of breast cancer cytosol using potassium bromide density gradients did not reveal specific antiestrogen binding activity associated with any recognized class of lipoprotein. Specific [3H]tamoxifen binding sites were pelleted in potassium bromide gradients consistent with the apparent large size of this protein. The physical characteristics of the antiestrogen binding protein in normal human tissue (myometrium) and neoplastic tissue (breast cancer) are remarkably similar, possibly reflecting a highly conserved structure.     

Prevention of quinone-mediated DNA arylation by antioxidants.

High performance liquid chromatographic (HPLC) analysis showed that the prototype antioxidant ascorbate (vitamin C) inhibits the DNA adducts induced by synthetic estrogen diethylstilbestrol (DES) and the antiestrogen metabolite 4-hydroxytamoxifen (4-OHTam). Treatment of salmon testes DNA with 4-OHTam quinone or 4-OHTam in the presence of horseradish peroxidase and hydrogen peroxide (H(2)O(2)) generated the same DNA adduct profile. Vitamin C and N-acetylcysteine (NAC) inhibited the formation of 4-OHTam-dG adducts in a dose-dependent manner. To determine whether the same antioxidants also protect cellular DNA, HL-60 cells were used as cell culture model. Cells treated with 10 microM 4-OHTam in the presence of 1 microM H(2)O(2 )for 24 h gave 4-OHTam-dG adducts approximately 4 x 10(-7), n = 3. Treatment of the cells with 100 microM 4-OHTam, without H(2)O(2), produced the same level of adducts. Supplementation of the incubation media with vitamin C (2.5 mM) or NAC (5 mM) inhibited the formation of DNA adducts. Thus, antioxidants may protect susceptible cells from genotoxicity associated with 4-OHTam activation.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.     

Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.     

Further studies on the antigonadotropic mechanism of action of norethisterone.

To examine the molecular mechanisms involved in the antigonadotropic effects of norethisterone (NET) and two of its A-ring reduced metabolites the 5 alpha-norethisterone (5 alpha-NET) and the 3 beta, 5 alpha-norethisterone (3 beta, 5 alpha-NET) at the neuroendocrine level, a series of experiments were undertaken in adult castrated rats. Animals were primed either with 0.2 mg of tamoxifen (Tam) for 4 consecutive days or 1.0 mg of cyproterone acetate (CPA) for 7 days followed by a single subcutaneous injection of 0.5 mg of NET, 5 alpha-NET or 3 beta, 5 alpha-NET. Four hours later, they were sacrificed and blood obtained for the measurement of immunoreactive serum LH and FSH. The results indicated that antiestrogen (Tam) pretreatment precluded the inhibitory effects of NET and the 3 beta, 5 alpha-NET but not those of the 5 alpha-NET derivative. Pretreatment with CPA did not modified the antigonadotropic action of the 3 beta, 5 alpha-NET metabolite but it markedly reduced the inhibitory action of the 5 alpha-NET, thus indicating that in the experimental model used, the antigonadotropic effects of NET, are in part the result of its metabolic conversion to its A-ring reduced metabolites. While the 5 alpha-NET displayed an androgenic effect, the 3 beta, 5 alpha-NET exhibited estrogen-like effect at the neuroendocrine level.     

Inhibitors of sterol synthesis. The effects of dietary 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one after intragastric administration to rats

The effect of dietary administration (0.1% in a rat chow diet) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol biosynthesis with marked hypocholesterolemic activity, on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one has been studied after intragastric administration of the labeled sterols to rats. In general, the distribution of 3H in major tissues paralleled that of 14C with no unusual concentration of 3H in any of the organs. Only trace amounts of 3H and 14C were recovered in urine. Administration of the 15-ketosterol was associated with decreased absorption of the labeled cholesterol as indicated by decreased levels of 14C in the various tissues and organs of the 15-ketosterol-treated rats (relative to ad libitum and pair-fed control animals) and increased levels of 14C in feces and intestinal contents at 12 and 48 h after the administration of the labeled cholesterol. Studies of the distribution of 3H in liver indicated rapid conversion of the 15-ketosterol to cholesterol and cholesteryl esters. The amounts of 3H recovered in the various tissues and organs at both 12 and 48 h after the administration of the labeled sterols were considerably less than the corresponding values for 14C, a finding which suggests a lower absorption of the 15-ketosterol (relative to cholesterol) and/or a more rapid clearance and biliary excretion of the 15-ketosterol and its metabolites.     

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

Direct evidence that an arene oxide is a metabolic intermediate of 2,2',5,5'-tetrachlorobiphenyl.

Radiolabeled arene oxide was recovered from incubations containing [³H]-2,2′,5,5′-tetrachlorobiphenyl (³H-TCB), unlabeled 2,2′,5,5′-tetrachlorobiphenyl-3,4-oxide (TCBAO), 3,3,3-trichloropropene-1,2-oxide (TCPO), NADPH, and liver microsomes from phenobarbital-induced rats. No labeled arene oxide was generated in the absence of NADPH, nor during the metabolism of unlabeled TCB in the presence of [³H]-H₂O. The recovered oxide (radiolabeled and carrier) was characterized by mobility on silica gel and by conversion to 3- and 4-hydroxy-TCB. Formation of a dihydrodiol metabolite was apparently blocked by inhibition of epoxide hydrase. These data provide the first direct evidence that arene oxides are intermediates of halogenated biphenyl metabolism.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.     

Maize and soybean response to phosphorus fertilization with blends of struvite and monoammonium phosphate

Plant and Soil - Struvite (MgNH4PO4·6H2O), a low water solubility (&lt;3%) mineral that is increasingly recovered from wastewater treatment plants, has potential to be used as a slow...     

Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways.

We hypothesized that hydrogen peroxide (H2O2) has a role in the local regulation of skeletal muscle blood flow, thus significantly affecting the myogenic tone of arterioles. In our study, we investigated the effects of exogenous H2O2 on the diameter of isolated, pressurized (at 80 mmHg) rat gracilis skeletal muscle arterioles (diameter of approximately 150 microm). Lower concentrations of H2O2 (10(-6)-3 x 10(-5) M) elicited constrictions, whereas higher concentrations of H2O2 (6 x 10(-5)-3 x 10(-4) M), after initial constrictions, caused dilations of arterioles (at 10(-4) M H2O2, -19 +/- 1% constriction and 66 +/- 4% dilation). Endothelium removal reduced both constrictions (to -10 +/- 1%) and dilations (to 33 +/- 3%) due to H2O2. Constrictions due to H2O2 were completely abolished by indomethacin and the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ-29548. Dilations due to H2O2 were significantly reduced by inhibition of nitric oxide synthase (to 38 +/- 7%) but were unaffected by clotrimazole or sulfaphenazole (inhibitors of cytochrome P-450 enzymes), indomethacin, or SQ-29548. In endothelium-denuded arterioles, clotrimazole had no effect, whereas H2O2-induced dilations were significantly reduced by charybdotoxin plus apamin, inhibitors of Ca(2+)-activated K+ channels (to 24 +/- 3%), the selective blocker of ATP-sensitive K+ channels glybenclamide (to 14 +/- 2%), and the nonselective K(+)-channel inhibitor tetrabutylammonium (to -1 +/- 1%). Thus exogenous administration of H2O2 elicits 1) release of PGH2/TxA2 from both endothelium and smooth muscle, 2) release of nitric oxide from the endothelium, and 3) activation of K+ channels, such as Ca(2+)-activated and ATP-sensitive K+ channels in the smooth muscle resulting in biphasic changes of arteriolar diameter. Because H2O2 at low micromolar concentrations activates several intrinsic mechanisms, we suggest that H2O2 contributes to the local regulation of skeletal muscle blood flow in various physiological and pathophysiological conditions.     

Measurement of gluconeogenesis by deuterated water: the effect of equilibration time and fasting period

Fasting gluconeogenesis (GNG) is often quantified using the 2H2O technique, which is based on plasma 2H2O enrichment and ensuing enrichment of plasma glucose at the C5 and C2 positions. Fractional (fr)GNG can be calculated using the ratio of C5 to C2 enrichment or the ratio of C5 to plasma 2H2O enrichment. For the latter, equilibration of 2H2O and C2 is required. The optimal equilibration period of 2H2O and C2 remains to be elucidated. In six healthy male subjects fasted for 18 h, we studied the effects of 3-, 5-, and 15-h 2H2O incubation periods on 1) the equilibration of plasma 2H2O and C2 glucose enrichment, 2) the measurement of frGNG, and 3) C5 labeling of hepatic glycogen after 1 mg of glucagon administration. After 3-h 2H2O incubation, plasma 2H2O and C2 were not equilibrated, frGNG C5/2H2O and C5/C2 were also different as was gluconeogenesis calculated with C5/2H2O and C5/C2. After 5- and 15-h 2H2O incubation, plasma 2H2O and C2 were equilibrated, and frGNG C5/2H2O and C5/C2 were similar, as was GNG calculated with C5/2H2O and C5/C2. After glucagon administration, no difference of C5 enrichment was found between 3, 5, and 15 h of 2H2O incubation. In conclusion, for reliable measurement of GNG in healthy subjects with C5/2H2O incubation periods longer than 3 h are required. After 5- and 15-h 2H2O incubation, GNG can be reliably measured with C5/2H2O. Gluconeogenetic labeling of glycogen did not affect the results after 3, 5, or 15 h of 2H2O incubation.     

高温胁迫对两种常绿杜鹃亚属植物幼苗生理生化特性的影响

为探明常绿杜鹃亚属植物高温致伤的生理机制,以猴头杜鹃（Rhododendron simiarum Hance）、井冈山杜鹃（R.jinggangshanicumTam）四年生实生苗为材料,通过人工气候箱中的盆栽实验,研究了不同程度的高温胁迫对两种常绿亚属杜鹃幼苗叶片生理生化特性的影响。结果表明：（1）两种杜鹃叶片的丙二醛（MDA）、过氧化氢（H2O2）及脯氨酸（Pro）、可溶性蛋白质含量均随胁迫温度的升高而增大;猴头杜鹃的H2O2、可溶性蛋白含量及增幅均明显小于井冈山杜鹃,而MDA、Pro的增幅则因胁迫温度而异。（2）两种杜鹃叶片抗坏血酸（AsA）含量均随胁迫温度的升高而降低,但猴头杜鹃的降幅小于井冈山杜鹃。（3）高温胁迫下,猴头杜鹃除过氧化氢酶（CAT）活性增幅略小外,超氧化物歧化酶（SOD）、过氧化物酶（POD）及抗坏血酸过氧化物酶（APX）增幅均大于井冈山杜鹃。可见,随着胁迫温度的升高两种杜鹃叶片膜脂过氧化作用加重,而猴头杜鹃较井冈山杜鹃具有更强的酶促和非酶促清除活性氧能力。     

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.     

Synergistic induction of ovulation and prostaglandin synthesis in goldfish (Carassius auratus) follicles by sodium orthovanadate and hydrogen peroxide.

The effects of hydrogen peroxide (H2O2) and sodium orthovanadate (Na3VO4) on ovulation and prostaglandin (PG) production were investigated in goldfish (Carassius auratus) follicles. H2O2, at levels that did not stimulate ovulation, significantly increased the ability of Na3VO4 to induce ovulation. The enhancing effect of H2O2 on Na3VO4-induced (10 microM) ovulation was observed over a wide range of concentrations (0.3-19.2 ppm) but was maximal at 1.2-4.8 ppm. The H2O2 effect on ovulation diminished at concentrations greater than 4.8 ppm. Na3VO4 and H2O2 also stimulated prostaglandin E (PGE) and prostaglandin F (PGF) levels in incubates. An interactive effect of the two agents was significant only on PGE production. However, optimal H2O2/Na3VO4 concentrations for the stimulation of PG production were much higher than those for stimulating ovulation. In most incubations, Na3VO4-induced or Na3VO4/H2O2-induced ovulation was not inhibited by the cyclooxygenase inhibitor indomethacin (IM), but was blocked by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Treatment of an Na3VO4/H2O2 mixture with catalase before the start of incubation totally abolished the enhancing effect of H2O2 on ovulation. This suggests that the enhancing effect of H2O2 on ovulation may not be a result of a chemical metabolite(s) produced by the two agents in mixture but rather is due to some direct effect of H2O2. This may have physiological significance in light of the published effects of H2O2 on various processes known to be involved in ovulation.