Biomedical subjects
Publications and source records attributed to J Fishman.
Acute peripheral arterial occlusion may lead to gangrene, with loss of parts of arms and legs. Three infants with disseminated intravascular clotting developed dark red discoloration of the tips of fingers and toes which progressed proximally. Repeated hyperbaric oxygenation treatments caused regression of the demarcation line and further progression of necrosis stopped.
We previously reported that saturable stereospecific binding of [3H]-naltrexone in rat brain homogenates prepared from castrated male rats was greater than the corresponding binding in intact animals. We now report that we have replicated these results and that the difficulty of other investigators in observing these differences is due to methodological factors. Specifically, when samples were filtered individually and rapidly, differences between castrated and intact rats were maintained. The increase in binding was also observed when tissues were washed to remove endogenous opioids prior to incubation, when [3H]-naloxone was used as the ligand, and when various antagonists were used as displacers in the radioreceptor assay.
The ketolic estrogen 16 alpha-hydroxyestrone (16 alpha OHE) reacts with lysine residues, forming stable covalent adducts with proteins. To determine the extent of protein modification by 16 alpha OHE in vivo, we measured the level of 16 alpha OHE-lysine present within proteins of varying half-lives obtained from normal subjects, patients with systemic lupus erythematosus (SLE), and pregnant women. The latter groups have higher than normal levels of plasma 16 alpha OHE. The proteins analyzed were membrane proteins of the red cell and the lymphocyte and basement membrane proteins of the glomerulus. We report that elevated levels of plasma 16 alpha OHE led to increased formation of 16 alpha OHE-protein adducts and that the level of these adducts increases with the half-life of the protein. In the case of erythrocyte membrane proteins, pregnant women and women with SLE had significantly higher mean levels of 16 alpha OHE-lysine than normal women (normal, 5.2 pmol 16 alpha OHE-lysine/mmol leucine; SLE, 15.7; pregnant, 24.9). A similar elevation in the modification of lymphocyte proteins in women was found (normal, 15.6; SLE, 40.5). Since the degree of protein modification also was dependent on the ambient level of free 16 alpha OHE, these measurements provide a useful indicator of the long term 16 alpha OHE status of an individual. The modification of proteins by 16 alpha OHE may be a link in the relationship between female hormones, pregnancy, and systemic lupus erythematosus.
Fatty acid esters of estradiol coupled at the C-17 position are naturally occurring metabolites of estradiol (E2). They have been recovered after incubation of E2 with human tissue and identified in human plasma. We investigated the primary metabolic transformation of E2, namely C-17 oxidation, of two representative fatty acid esters, in five normal subjects (four men and one woman), aged 25-55 yr, using a radiometric method. The transfer of tritium from the C-17 alpha position to tritium water after iv injection of free E2 was compared to that of E2-17 beta-stearate and E2-17 beta-arachidonate. Both esters were oxidized at the C-17 position to a greater extent than was free E2. In addition, the oxidation of the E2 fatty acid esters proceeded more slowly. Thus, the time necessary to reach half the maximal extent of reaction ranged from 30-45 min for the three E2-17 beta-arachidonate studies and from 2.5-4 h for the five E2-17 beta-stearate studies, while that for free E2 was less than 15 min in each instance. The disappearance of intact E2-17 beta-stearate from plasma after bolus injection was studied in two subjects (one of the five above and one additional subject). The t1/2 values calculated for the 0-60 min period were 24 and 16 min. The rate of disappearance E2-17 beta-stearate was slower than that of E2. The biological activity of E2 esters is thought to reside in their ability to be converted, by hydrolysis, to free E2. The E2 esters must undergo hydrolysis before the oxidation of free E2 to estrone can proceed. Since the oxidation reaction is extremely rapid, studying the rate of oxidation after injection of the E2 fatty acid esters provides an index of their in vivo hydrolysis and, thus, a measure of their subsequent biological activity as the free hormone. The unsaturated arachidonate ester of E2 was oxidized at a faster rate than the saturated stearate ester; this implies that the hydrolysis of the arachidonate ester was faster. Evidence of continuing oxidation of E2-17 beta-stearate at times well after its level in plasma markedly decreased indicates that the ester is removed from the circulation before its subsequent hydrolysis and oxidation. We conclude that in man, the C-17 oxidation of both E2-17 beta-arachidonate and E2-17 beta-stearate proceeds more slowly but to a greater extent than that of the free steroid.(ABSTRACT TRUNCATED AT 400 WORDS)
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16 alpha-Hydroxyestrone (16 alpha OHE ) has been shown previously to react with albumin, forming stable covalent adducts with lysine residues. The incubation of [3H]16 alpha OHE with whole blood also results in the incorporation of tritium into cells, with the rate of incorporation paralleling that of 16 alpha OHE -protein adduct formation. 32% of erythrocyte acid-precipitable radioactivity was found to be present within membrane proteins and electrophoresis demonstrated that several of these proteins are modified by [3H]16 alpha OHE . Membrane proteins from the red cells of ten individuals were reduced with sodium borohydride, acid hydrolyzed, and the 16 alpha OHE -lysine adducts purified by hydrophobic chromatography and reverse-phase high-pressure liquid chromatography. The amount of these adducts was quantitated by radioimmunoassay and found to be five times higher than the plasma levels of free 16 alpha OHE . This reflects most likely the accumulation of 16 alpha OHE -protein adducts during the course of the red cell life. Quantitation of 16 alpha OHE -protein adducts with other cellular proteins may contribute to our understanding of systemic lupus erythematosus, an autoimmune disease in which elevated levels of 16 alpha OHE occur.
The estrogen responsive human breast cancer MCF-7 cell culture was examined for its response to 2-hydroxyestrone a principal metabolite of estradiol. Addition of 2-hydroxyestrone to the cell cultures in concentration of 10(-9) - 10(-6) M had no effect on cell growth and proliferation because of rapid O-methylation of the catechol estrogen by catechol O-methyltransferase which is highly active in these cells. In the presence of quinalizarin, a potent catechol O-methyltransferase inhibitor which reduces the O-methylation of the steroid, 10(-7) M and 10(-8) M 2-hydroxyestrone markedly suppresses the growth and proliferation of the cells. The tumor cell growth-inhibitory action of the catechol estrogen was neutralized by the presence of 10(-9) M estradiol. The catechol estrogen inhibition of cell growth is not observed in the estrogen receptor-negative human breast cancer cell lines MDA-MB-231 and MDA-MB-330 providing evidence that the inhibition is specific and is estrogen receptor-mediated. In contrast, the 16 alpha-hydroxylated metabolites of estradiol, estriol and 16 alpha-hydroxyestrone, are effective stimulators of MCF-7 cell proliferation with the latter exhibiting potency in excess of that expected from its estrogen receptor affinity. The present results represent the first observation of a specific receptor-mediated antiestrogenic action of 2-hydroxyestrone and suggest that the physiological regulation of the agonist activity of the primary estrogen may involve in situ generation of catechol estrogen.
The terminal hydroxylation in placental estrogen biosynthesis from androgens is at the 2 beta position. The 2 beta-hydroxy-19-oxoandrogen derivative collapses nonenzymatically to estrogen and is therefore the proximate precursor of the female hormone. To establish the role of this pathway in biological aromatization, an immunological approach was employed in which an antibody was obtained which recognizes 2 beta-hydroxy-19-oxygenated androgens but not intermediates oxygenated at C-19 only. Binding of the 2 beta-hydroxy-19-oxo intermediate by the antibody stabilizes it so that its nonenzymatic transformation to estrogen is delayed and results in slower estrogen formation. When placental microsomes were incubated with [1,2-3H]androstenedione in the presence of the antibody antiserum, a 50% decrease in [3H]estradiol formation and 3H2O release was observed when compared with identical incubations containing normal rabbit serum alone. This inhibition is blocked when the antibody is inactivated by presaturation with 2 beta, 19-dihydroxyandrostenedione. Precipitation of immunoglobulins from the incubations followed by heating liberated the 2 beta-hydroxy-19-oxo intermediate (30%) from the antibody, and resulted in its nonenzymatic collapse to estrogen with concomitant release of 3H2O. Control normal rabbit serum or blocked antibody incubations did not show a similar increase in [3H]estradiol or 3H2O yields in the precipitate. Heat treatment (90 degrees C) of the antibody but not normal rabbit serum incubations resulted in a similar increase in [3H]estradiol and 3H2O yields. These results are consistent with the hypothesis that the final and rate-determining hydroxylation in aromatization of androgens is at the 2 beta position and that this pathway is the dominant, if not the sole, route of estrogen biosynthesis by placental aromatase. The antibody probe also permits the characterization of aromatization mechanisms in tissues other than the placenta.
The determination of steroid receptors in human breast cancers has assumed increasing importance over the past several decades. Improper handling of the specimens could affect results obtained. This study details the effects excessive levels of heat that occur with the use of electrocautery can have on steroid receptor quantities and localization. Twelve resected primary and metastatic human breast cancers were analyzed for cytoplasmic and nuclear receptors by biochemical analysis. In addition, steroid binding was determined by direct fluorescent histochemical techniques. To a portion of each resected specimen a Boviec was applied to simulate electrocautery resection. Analysis of the different portions of the same tumor revealed that there was a decrease in measurable cytoplasmic receptor in all cauterized specimens and a concomitant increase in the nuclear receptor. A similar shift in steroid binding was noted in all the specimens analyzed by fluorescent histochemical techniques. The results of this study show that the application of excessive heat to human breast cancers will lead to false negative biochemical steroid receptor determination by shifting the receptors intranuclear.
A case of a 12-year-old girl with Ewing sarcoma of the third metacarpal bone is described. Following an incisional biopsy, radiation therapy and adjuvant chemotherapy were given. The girl is now 34 months postdiagnosis and 25 months off therapy in sustained complete remission and functionally normal right hand. Of the seven previously published cases only two are long-term survivors (one had disarticulation, and the other resection of the fourth and fifth rays).
Peritalar dislocation is an uncommon injury and was first described after flying accidents. Ten patients are reported who sustained this injury after minor trauma. They were treated by closed reduction and immobilisation of the ankle in a plaster cast for between 10 and 12 weeks, the first 2 weeks being non-weightbearing. No degenerative changes were detectable in the subtalar joints one year after injury. Early reduction and immobilisation may be significant in reducing the incidence of degenerative arthritis in the subtalar joint.
We have measured the three principal oxidative transformations of estradiol by means of a radiometric procedure in women with breast or endometrial cancer and in age matched controls. No difference between the 17 beta-ol oxidation or 2-hydroxylation of the hormone was observed between the study groups. In contrast, 16 alpha-hydroxylation was strikingly elevated in the women with breast and endometrial cancer relative to the age matched controls. Evidence is presented that this increased activity precedes the clinical evidence of the disease and that it represents a significant risk factor for these estrogen dependent tumors. This risk may be mediated by one of the products of 16 alpha-hydroxylation, 16 alpha-hydroxyestrone, which exhibits unique biological properties.
The specificity of mushroom tyrosinase in displacing 3H from estradiol and catechol estrogens labeled at C-1, C-2, C-4 or C-6,7 was investigated under various conditions. [2-3H]E2 Yielded significant amounts of 3H2O, in the presence of NADH, and the rate of 3H loss from the steroid paralleled that of the radioactivity remaining in the aqueous fraction after extraction with organic solvents. NADH had little effect on the release of 3H from [1-3H]E2 or [4-3H]E2 but glutathione was highly active in this respect, with considerable differences being observed between lyophilizable 3H2O and yields of water-soluble products. It is proposed that 3H losses from C-2 of estradiol reflects oxidative displacement of this isotope by tyrosinase while the loss observed from C-1 and C-4 is the result of non-enzymatic conjugation with glutathione after the formation of the catechol estrogen. The difference between lyophilizable 3H2O and the yield of water-soluble products obtained with [1-3H]E2 and [4-3H]E2 provided a measure of the relative amount of conjugation occurring at C-1 and C-4. These findings were confirmed by double label experiments with 3H- and 14C-labeled estrogens and the isolation of the glutathionyl derivatives. The catechol estrogens did not serve as substrates for further hydroxylation by the enzyme even when C-2 was available for this reaction. These experiments give further information about the specificity of tyrosinase in its reaction with aromatic steroids and provide a simple and rapid method for confirming the distribution of 3H at C-2 or C-4 of estradiol.
Charcoal was found to catalyze the release of 3H2O from [1-3H]2-hydroxyestradiol-17 beta ([1-3H]2-OHE2) or [4-3H]2-hydroxyestradiol-17 beta ([4-3H]2-OHE2) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of 3H2O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of 3H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of 3H2O from [4-3H]2-OHE2 in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4 degrees C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.
Injection of 100 micrograms 2-hydroxyestrone (2OHE1) at various times on the morning of proestrus into normal 4-day-cycling rats results in abolition of the preovulatory LH surge in a number of animals tested. The greatest response was observed when the administration of 2OHE1 coincided with endogenous estradiol (E2) levels that were close to but not at their maximal proestrous levels. The catechol estrogen failed to abolish the LH surge if given much earlier or after the E2 maximum had already been reached. The effectiveness of 2OHE1 inhibition of the LH surge was greatly increased by the administration of 1 microgram E2 1 h before the catechol estrogen. 2OHE1 did not interfere with LH secretion in response to LHRH administration, indicating that the inhibitory action of the catechol estrogen is exercised at the hypothalamic level. In contrast to its inhibition of the positive feedback, 2OHE1 administered either before or after the injection of E2 to ovariectomized rats had no effect on the negative feedback of the hormone on pituitary LH secretion. The narrow and specific "time window" on proestrus when an injection of 2OHE1 results in the abolition of the preovulatory LH surge and its relation to the endogenous E2 preovulatory secretion suggest that the catechol estrogen interferes with a brief neuronal triggering event obligatory for LHRH release. The evidence also indicates that this action does not involve conventional competition for the E2 receptor.
Four-day cycling rats equipped with intracardiac catheters were injected with 2-hydroxyestradiol-17 beta (2OHE2-17 beta) at 0800, 0900, 1000, and 1200 h on the morning of proestrus. The administration at 0800 and 0900 h resulted in abolition of the afternoon preovulatory LH surge in virtually all animals. Injections given at 1000 and 1200 h were ineffective in this respect. The isomeric catechol estrogen 4-hydroxyestrone effectively inhibited the LH surge when given at 0900 h, but not if injected at 1000 or 1200 h. In contrast, the nonestrogenic 2-hydroxyestradiol-17 alpha was effective in blocking the LH surge when given at 0900 or 1000 h. Rats treated with 2OHE2-17 beta at 1000 h responded normally to exogenous LHRH administration in the afternoon, indicating that the action of 2OHE2-17 beta is at the hypothalamic level. 2OHE2-17 beta and 4-hydroxyestrone, which are potent estrogens, may act in this instance first as estrogen agonists, advancing the "time window" when their catechol antagonist properties in blocking the LH surge can be functional. The nonuterotropic 2-hydroxyestradiol-17 alpha and 2-hydroxyestrone act solely as catechol estrogens and inhibit the preovulatory LH surge if administered at a time when they be present during this physiological window, which is thought to involve events at the estrogen-catecholaminergic interphase.
Catechol estrogens labeled with 3H at different positions in rings A and B of the steroid were synthesized by chemical or enzymatic methods, and their oxidative transformation by male rat brain microsomes was followed by the transfer of 3H into 3H2O. This reaction was shown to occur more readily with the catechol estrogens than with the parent steroid and was also influenced by the position of the radiolabel. Tritium was displaced less readily from C-1 than from C-2 or C-4 of the aromatic ring. Spermine, which is known to increase cytochrome P-450-mediated hydroxylation reactions, had no effect on the release of 3H from ring A of either estradiol or 2-hydroxyestradiol with rat brain microsomes in contrast to liver. Glutathione and other thiols were able to cause a rapid loss of 3H from labeled catechol estrogens, even in the absence of tissue, but in double label experiments with [4-3H]- and [4-14C]2-hydroxyestradiol, the isotope ratio in the recovered catechol estrogen was unchanged. The results illustrate some of the problems in determining accurately the metabolism of estrogens by measuring 3H2O formation when aromatic hydroxylation is involved and also highlight the possible interaction of the catechol estrogens with cellular nucleophiles such as glutathione.
Dietary protein, when substituted for carbohydrate or fat, can increase cytochrome P-450-dependent drug oxidation rates in humans. Endogenous estrogens, as well as drugs, are also metabolized by cytochrome P-450 and other enzymes in the hepatic endoplasmic reticulum. Therefore, it was of interest to determine whether variations in diet can alter the major metabolic pathways for estrogens, as assessed by radiometric methods. Eight normal men were fed a high protein diet (44% of calories as protein, and 35% as carbohydrate for 2 weeks), followed by a high carbohydrate diet (70% of calories as carbohydrate and 10% as protein) for an additional 2 weeks. The fat and total energy contents of the two diets were equal. The percent oxidation of [2-3H]estradiol, measured as 3H2O released, which is an in vivo measure of 2-hydroxylation of endogenous estrogen, was greater in all eight men during the high protein dietary period than during the high carbohydrate dietary period (44 +/- 3% and 33 +/- 3%, respectively, means +/- SE, P less than 0.005). In contrast, 16 alpha-hydroxylation of estrogen, as measured using [16 alpha-3H]estradiol, did not change significantly. Our findings demonstrate that dietary components can alter estradiol oxidation in humans and that the 2- and 16 alpha-hydroxylases for estrogen are under separate regulatory control. The influences of specific nutrients on estrogen metabolism may have potential significance for diseases in which these hormones may play a role in clinical expression.