Metabolism of 17 beta-estradiol and the adrenal-derived estrogen 5-androstene-3 beta,17 beta-diol (hermaphrodiol) in human mammary cell lines.
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Acylation↗
Biomedical subjects
J B Adams
Publications and source records attributed to J B Adams.
User pays in the mental health service--256 discharges from Ashburn Hall.
A retrospective descriptive analysis was undertaken on all patients discharged from Ashburn Hall over a 14 month period. The sample of 256 patients was found to have a female to male ratio of 6:4 and contained 4.3% of individuals without school certificate (cf 48.3% national statistics) and 23% with a university degree or diploma (cf 13.2% in the general population). Seventy-four point six percent of the sample had some significant previous psychiatric history although for 68% of the sample this was their first admission to Ashburn Hall. A quarter of the sample came from the surrounding geographic area and patients were more likely to be referred by specialists rather than general practitioners as geographic distance increased from the hospital. Comparisons with the public system showed a predominance of patients with major affective disorder and less patients with a diagnosis of substance abuse and mental retardation.
Adult↗
Expression of hydroxysteroid sulphotransferase is related to estrogen receptor status in human mammary cancer.
A positive correlation between the expression of estrogen sulphotransferase (EC 2.8: 2.4) and the estrogen receptor (ER) in human breast cancer tissues was previously demonstrated. We have now established that a similar correlation exists between the expression of hydroxysteroid sulphotransferase (EC 2.8: 2.2) and ER in such tissues. Enzyme activity was present in 93% of the ER + tumor cytosols (mean 59 +/- 44 (SD) pmol dehydroepiandrosterone sulphate formed per mg protein per 2 h (n = 42). Activity was detected in 68% of ER - tumors and this was significantly lower (mean 21 +/- 26 (SD) (n = 19), P less than 0.001) than the former group. Metabolism of estradiol-17 beta (E2) and the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL), which is a substrate for hydroxysteroid sulphotransferase but not estrogen sulphotransferase, was studied in four ER + human mammary cancer cell lines (MCF-7, T47-D, MDA-MB-361 and ZR-75-1) and four ER-human mammary cell lines (BT-20, MDA-MB-231, MDA-MB-330 and HBL-100), employing steroid concentrations of 1 nM. At this concentration, formation of ester sulphates was a major route of metabolism in the ER + cell lines; E2 yielding a mean of 6.5 pmol estrogen monosulphates/mg DNA in 16 h and ADIOL yielding a mean of 9.4 pmol C19-5-ene steroid monosulphates/mg DNA in 16 h. In three of the four ER - cell lines, formation of sulphates from E2 occurred at an eight-fold lower rate (mean 0.8 pmol estrogen sulphates/mg DNA in 16 h), whereas MDA-MB-330 cells did not form estrogen sulphates. Only one of the four ER- cell lines (BT-20) sulphurylated ADIOL and this was at a 12-fold lower rate compared to the mean value for the ER + cel lines. Oxidation of E2 and ADIOL occurred in all cell lines and was generally the major route of metabolism in the ER - cells. A significant correlation between formation of estrone and dehydroepiandrosterone occurred for all cell lines (r = 0.98, P less than 0.001) indicating that the same 17 beta-hydroxysteroid dehydrogenase was probably involved. Since ADIOL is estrogenic in a number of systems at the concentration found in the blood of Western women (approximately 2 nM), the coordinated expression of hydroxysteroid sulphotransferase, estrogen sulphotransferase, and ER, supports the concept of a functional relationship between estrogen action via ER and sulphurylation reactions.
17-Hydroxysteroid Dehydrogenases↗
Formation of glucuronides of estradiol-17 beta by human mammary cancer cells.
The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.
Breast Neoplasms↗
Long-chain fatty acid esters of 5-androstene-3 beta, 17 beta-diol: composition and turnover in human mammary cancer cells in culture.
Long-chain fatty acid esters of the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL) were found to accumulate in four human mammary cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231 and MDA-MB-330) when explosed to 10-30 nM ADIOL for variable time periods. At each time point examined, the monoester fraction, which represented the major component of the total lipoidal fraction, contained fatty acids linked to either the 3 beta- or 17 beta-positions. However, there was considerable variation in the ratio of 3 beta- to 17 beta-monoesters in the four cell lines. By means of reverse phase HPLC and referral to authentic synthesized compounds, each monoester fraction was found to contain a number of long-chain fatty acid components whose composition resembled that previously determined for the fatty acid esters formed from 17 beta-estradiol. A specific and measurable turnover of a subfraction of ADIOL-17 beta-monoesters composed of essential fatty acids (22:6, 20:4, 18:3) occurred in MCF-7 cells, and to a lesser extent in ZR-75-1 cells. No changes were observed with time in any of the components of the 3 beta- or 17 beta-monoester fractions in MDA-MB-231 and MDA-MB-330 cells. These results, coupled with other studies, now suggest that a very rapid turnover of some components of these lipoidal derivatives may be occurring. If so, it is possible that the system of acylation-deacylation may be involved in a transport mechanism for estrogens and perhaps other steroid hormones.
Acylation↗
Hormone-sensitive lipase is involved in the hydrolysis of lipoidal derivatives of estrogens and other steroid hormones.
Long-chain fatty acid esters of 17 beta-estradiol and other steroid hormones, which are formed in hormone-sensitive tissues, can be regenerated to the free hormone by the action of an esterase present in the cytosol. This esterase has now been examined in bovine placenta cotyledons. Activity towards steroid fatty acid esters was accompanied by activity towards a diacylglycerol analogue and cholesteryl oleate. During purification procedures, the ratio of activities towards the diacylglycerol analogue and estradiol 17 beta-oleate remained approximately constant. Activity towards these two substrates was inhibited by increasing concentrations of HgCl2 and phenylmethanesulfonyl fluoride in a parallel manner. Upon treatment with [3H]diisopropyl fluorophosphate, a major labelled species of Mr approx. 84,000 was formed. Activation by ATP and the catalytic subunit of cAMP-dependent protein kinase occurred. These properties were very similar to those of the hormone-sensitive lipase of bovine adipose tissue previously reported and run in parallel in this study. A highly purified preparation of this latter enzyme was found to hydrolyse steroid fatty acid esters and relative activities towards such substrates, diacylglycerol analogue and cholesteryl oleate, were similar to the placenta esterase. When the two esterases were phosphorylated with [gamma-32P]ATP, a labelled species of Mr 84,000 was isolated in both cases by use of an antibody raised against purified hormone-sensitive lipase of bovine adipose tissue. It is concluded that hormone-sensitive lipase is very likely the enzyme responsible for hydrolysis of steroid fatty acid esters in bovine placenta and possibly steroid hormone target tissues in general.
Androgens↗
Properties of fatty acyl-coenzyme A: estradiol-17 beta acyltransferase in bovine placenta microsomes.
The properties of the enzyme catalyzing the formation of non-polar derivatives of estradiol-17 beta (E2) esterified to long-chain fatty acids have been investigated in microsomal preparations from bovine placenta cotyledons. A rapid enzyme assay has been developed which involves simple solvent partitioning. The membrane-bound enzyme showed a pH optimum of 5.0 and addition of fatty acyl-coenzymes A (CoAs), such as oleoyl-CoA, palmitoyl-CoA and palmitoleoyl-CoA, increased [3H]E2-fatty acyl ester formation from [3H]E2 by some 7-fold. Linoleoyl-CoA, linolenoyl-CoA and arachidonoyl-CoA were much less effective as acyl donors. Only 17 beta-fatty acyl monoesters were synthesized in each instance. Similar results were obtained with microsomes or mitochondria from bovine endometrium. The apparent Km for E2 employing placenta microsomes was 8.0 +/- 2.2 (SD) microM. Steroids such as testosterone, dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol acted as competitive inhibitors (Ki values 79, 46 and 39 microM, respectively). These, and other data to be reported separately, which showed that these steroids were substrates for the enzyme, demonstrate that the latter is not specific for E2. The [3H]E2-fatty acyl ester fractions biosynthesized from [3H]E2 and bovine placental or endometrial tissue were analyzed by high pressure liquid chromatography (HPLC) and were found to have similar compositions characterized by a high percentage of unsaturated fatty acids.
Acyl Coenzyme A↗
Metabolic fate of estradiol in human mammary cancer cells in culture: estrogen sulfate formation and cooperativity exhibited by estrogen sulfotransferase.
The metabolism of 17 beta-estradiol in both estrogen receptor positive and negative human breast cancer cell lines has been compared. Initial experiments in which confluent cells were exposed to 1 nM [3H]17 beta-estradiol for 24 h, revealed that the main metabolites formed by estrogen receptor positive MCF-7 and ZR-75-1 cells were 17 beta-estradiol-3-sulfate (together with lesser amounts of estrone sulfate) and estrone. In estrogen receptor negative cell lines, production of estrogen sulfates was either significantly lower (MDA-MB-231 cells) than receptor positive cells, or failed to be produced at all (MDA-MB-330 cells). In both these receptor negative cell lines, production of estrone was significantly higher than in receptor positive cells. Accumulation of estrogen sulfates resulted from attainment of a steady state between synthesis catalysed by estrogen sulfotransferase and degradation catalysed by estrogen sulfatase. The former was present in the cytosol and showed a very high affinity for 17 beta-estradiol and estrone (low nM range). Complex initial velocity versus estrogen substrate curves were obtained with enzyme purified 106-fold by affinity chromatography. Such curves were consistent with a rate equation of degree 3 or 4 and suggest the presence of cooperatively linked dependent binding sites.
Breast Neoplasms↗
Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase.
Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).
Acyltransferases↗
Assessing the preventability of emergency hospital admissions. A method for evaluating the quality of medical care in a primary care facility.
The quality of primary medical care was assessed by studying the events leading to 686 emergency admissions of patients from our hospital-based primary care practice. Independent physician reviewers determined that 59 (9 percent) of the admissions were potentially preventable; 40 were due to iatrogenic factors including inadequate follow-up and adverse drug reactions, 12 were due to lack of patient compliance, and seven were due to both iatrogenesis and noncompliance. Adverse drug reactions were the most common cause of iatrogenesis, and warfarin was the drug that most commonly caused an adverse reaction. Inadequate follow-up of abnormal physical findings, symptoms, and laboratory test results was also important. Patients with preventable admissions had more medical diagnoses (4.9 versus 4.1, p less than 0.01), were prescribed more medications (4.5 versus 3.7, p less than 0.01), and were older (66.5 years versus 60.2 years, p less than 0.01) than patients whose admissions were not preventable. It is concluded that a small percentage of emergency hospitalizations may be preventable and that systematic review of emergency hospitalizations may provide a means of measuring the quality of primary medical care.
Adult↗
Selective turnover of the essential fatty acid ester components of estradiol-17 beta lipoidal derivatives formed by human mammary cancer cells in culture.
The properties of fatty acyl coenzyme A: estradiol-17 beta acyl transferase in microsomes derived from pooled human mammary cancer tissue have been examined. A pH optimum of 5.5 was found and addition of long-chained fatty acyl CoAs increased estradiol-17 beta (E2) 17-monoacyl ester synthesis; the apparent Km for E2 being 8 microM when oleoyl CoA, linolenoyl CoA or palmitoyl CoA were employed. Testosterone, dehydroepiandrosterone, and 5-androsterone-3 beta, 17 beta-diol acted as competitive inhibitors with Ki values of 36, 36 and 46 microM, respectively. The composition of E2 fatty acyl esters (E2-L) formed by incubation of [3H]E2 with human mammary cancer tissue and human mammary cancer cell lines has been determined by HPLC. Although the composition of E2-L in estrogen receptor negative cell lines (MDA-MB-231 and MDA-MB-330) was generally similar to that found for MCF-7 cells (estrogen receptor positive) and pooled human mammary cancer tissue, the former cell lines contained a 3-fold higher relative concentration of E2-17 beta stearate. MCF-7 cells were exposed to 30 nM [3H]E2 and the composition of the isolated [3H]E2-L fraction studied at various time intervals. At 0.5 h, the intracellular concentration of E2-L was 1.8 +/- 0.4 (SEM) pmol/mg DNA which increased to values of 3.6 +/- 0.6 and 4.3 +/- 0.5 at 4 h and 16 h, respectively. In the subsequent 3 h following transfer to medium lacking [3H]E2, the concentration of E2-L declined to 3.7 +/- 0.3 pmol/mg DNA. The subfraction of E2-L composed of E2-17 beta arachidonate, linolenate and docosahexaenoate, was seen to decline in relative abundance after 0.5 h and to reach significantly lower relative levels at 16 h, and again in the 3 h period following estrogen withdrawal. The data suggests that these components, derived from essential fatty acids, are more metabolically active. This may then provide a new lead to link these novel estrogen derivatives with the established relationship between unsaturated fatty acids and an increased mammary cancer incidence.
Acyl Coenzyme A↗
Production and characterization of a monoclonal antibody to bovine estrogen sulfotransferase.
A murine IgGl (k) monoclonal antibody, termed 33-11, was produced against purified bovine estrogen sulfotransferase. After the enzyme was separated into its four isoenzyme forms by native polyacrylamide gel electrophoresis, a Western blotting analysis indicated that the antibody reacted to a similar extent with each of the isoenzymes. An immunoprecipitation and SDS-poly acrylamide gel electrophoresis analysis was performed with an I-125-labeled enzyme preparation. Antibody 33-11 precipitated protein in the molecular weight range 70,000-74,000, the size of native estrogen sulfotransferase, plus additional bands with molecular weights of 29,000, 54,000, and 61,000. The lower molecular weight polypeptides may represent fragments, bearing a common epitope, produced by limited endogenous proteolysis of the intact enzyme. Solid-phase radioimmunoassay testing demonstrated strong and specific binding of the antibody to the bovine enzyme. No cross-reactivity was observed with a panel of nine other purified bovine proteins. An immunohistochemical analysis with the antibody was performed on two bovine tissues with high levels of enzyme activity. Placenta showed strong, specific staining of fetal giant cells of chorionic villi, while adrenal had weak but widespread staining which tended to be more concentrated in the inner cortex. Monoclonal antibody 33-11 has potential utility in the purification, detection, and quantitation of bovine estrogen sulfotransferase.
Animals↗
Molecular endocrinology of breast cancer.
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Animals↗
Activation of soluble guanylate cyclase by NO-hemoproteins involves NO-heme exchange. Comparison of heme-containing and heme-deficient enzyme forms.
The mechanism of activation of soluble guanylate cyclase purified from bovine lung by high molecular weight, nitrosyl-hemoprotein complexes is reported. Heme-containing, heme-deficient, and heme-reconstituted forms of guanylate cyclase were studied. Nitric oxide (NO) and nitroso compounds activated heme-containing and heme-reconstituted enzymes (over 50-fold), with an accompanying shift in the Soret absorption peak from 431 to 398 nm, but failed to activate or alter the spectral characteristics of heme-deficient enzyme. In contrast, preformed NO-hemoprotein complexes as well as low molecular weight NO-heme activated all forms of guanylate cyclase. Heme-deficient guanylate cyclase was first reacted with excess amounts of NO-hemoglobin, NO-myoglobin, or NO-catalase and then rapidly separated from the NO-hemoprotein by column chromatography. Spectrophotometric analysis indicated that the NO-heme moiety was transferred from each of the NO-hemoproteins to heme-deficient guanylate cyclase. Approximately 1 mol of NO-heme was bound per mol of holoenzyme and the specific activity of this enzyme form was over 50-fold greater than that of unreacted, heme-deficient enzyme. NO-heme was tightly bound to guanylate cyclase as no transfer of enzyme-bound NO-heme to apohemoglobin was evident. Enzyme activated by NO-hemoproteins closely resembled, kinetically, that activated by NO or NO-heme. In contrast, reactions between heme-deficient guanylate cyclase and hemoproteins did not result in heme transfer, whereas heme alone rapidly reconstituted the enzyme. These observations indicate that soluble guanylate cyclase can be readily reconstituted with, and thereby activated by, NO-heme through an exchange reaction with NO-hemoproteins.
Animals↗
Esterification-deesterification of estradiol by human mammary cancer cells in culture.
Formation of lipoidal derivatives of estradiol-17 beta (E2) esterified to long-chained fatty acids has been reported to occur in estrogen target tissues. Employing human breast cancer cells in culture, we have detected the rapid synthesis of such compounds upon exposure of the cells to concentrations of [3H]E2 as low as 1 nM. When exposed to 10 nM [3H]E2 in the culture medium, synthesis of E2-lipoidal derivative (E2-L) reached 270 fmol/mg DNA in 2 h in the estrogen receptor positive MCF-7 human mammary cancer cell line. Higher rates (approximately 900 fmol/mg DNA in 2 h) were reached in 2 estrogen receptor negative human mammary cancer cell lines; MDA-MB-231 and MDA-MB-330. E2-L was the major form of estrogen in the latter cells at this time interval (E2-L/E2 approximately 3.0). Far higher concentrations of E2 were found in MCF-7 cells compared to 231 and 330 cells, and, in contrast to the latter, this was mostly specifically bound. Upon subsequent withdrawal of E2 from the medium, intracellular concentrations of E2-L decreased very rapidly in the first 5 h period, then declined more slowly to approximately 50 fmol/mg DNA at 24 h. Intracellular concentrations of E2 were maintained over this time period. E2-L was not present in the medium. Thus, accumulation of E2-L in cells upon continuous exposure to E2 represents the net result of esterification and deesterification reactions. These hydrophobic E2-derivatives may then be involved in the "capture" of E2 for transport through membranes and subsequent regeneration of E2 to maintain occupancy of the nuclear receptor.
Binding, Competitive↗
3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney: possible involvement in 11-deoxycorticosterone formation in situ.
3 beta-Hydroxysteroid isomerase dehydrogenase, capable of acting on C21- and C19-3 beta-hydroxy-5-ene-steroids has been found in guinea-pig kidney at equivalent levels to those in guinea pig testes. Of the 3 beta-hydroxy-5-ene-steroids present in guinea pig serum, 21-hydroxypregnenolone occurs in highest concentration (17 nM) followed by pregnenolone (10 nM), whereas 17 alpha-hydroxy-pregnenolone and dehydroepiandrosterone occur in very low concentrations (less than 0.5 nM). Furthermore, the concentration of 21-hydroxypregnenolone relative to 11-deoxycorticosterone (the mineralocorticoid of the guinea pig), is 10:1 (Nishikawa and Strott, Steroids 41 (1983) 105-120). The apparent Km value for 21-hydroxypregnenolone, for the reaction yielding 11-deoxycorticosterone as catalysed by guinea pig kidney microsomes, was 85 nM and the Vmax 33 pmol/min per mg protein. Pregnenolone was a competitive inhibitor (apparent Ki = 5 microM) in the above reaction. A sex difference in the level of the enzyme in the kidney was found (activity in the female was one-third of that in the male) which may indicate that the enzyme is under partial androgen control. 3 beta-Hydroxysteroid isomerase dehydrogenase activity was also detected in guinea pig liver and again it was lower in the female. Whilst the exact role of 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney remains uncertain, the data suggest that it may utilise blood-borne 21-hydroxypregnenolone, the later then playing the role of a prohormone.
17-Hydroxysteroid Dehydrogenases↗
Control of secretion and the function of C19-delta 5-steroids of the human adrenal gland.
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17-Ketosteroids↗
Sun Company's involvement in health cost management.
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Consultants↗