Comparison of aromatase activity in human prostatic, testicular and placental tissues.

The aromatase enzyme was quantified by the release of tritiated water from [1 beta-3H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0 microM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC50 values of 6.4, 0.17 and 0.0017 microM, respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075 microM, respectively).     

A site-directed mutagenesis study of human placental aromatase.

Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-450cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128----Gln, His-128----Ala, Cys-299----Ala, Glu-302----Leu, Asp-309----Asn, Asp-309----Ala, and Ser-312----Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] androstenedione (or [1 beta-3H]androstenedione), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19-hydroxyandrostenedione and 19-oxoandrostenedione by reverse phase high performance liquid chromatography using [7-3H]androstenedione as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis.     

Pseudoaromatase in circulating lymphocytes

A variety of data suggesting a relationship between estrogens and the immune system prompted a study of aromatase activity in blood lymphocytes. A tritiated water aromatase assay detected activity of 1.9 to 25.1 pmol/g protein/h in 14 samples of human lymphocytes. To confirm these results, additional tritiated water and product isolation assays were performed on a large pool of lymphocytes obtained from 4 U of blood. An assay using [1β-³H]androstenedione generated high apparent aromatase activity, 943 pmol/g protein/h, but this activity could not be blocked by the aromatase inhibitor, CGS 16949A. More direct methods of evaluation yielded the following results: (1) PCR demonstrated no aromatase mRNA production in lymphocytes; (2) direct product isolation using [1,2,6,7-³H]androstenedione yielded insignificant production of estrone and estradiol; (3) immunostaining of fixed lymphocyte smears with a polyclonal antibody to aromatase yielded equivocal results. These data suggest the presence of pseudoaromatase in blood lymphocytes. Since circulating lymphocyte pseudoaromatase levels can be correlated with various factors in patients, such as age, menopausal status, and glucose ingestion, further studies of this activity are warranted.     

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.     

Estrogen synthesis and metabolism in the hamster blastocyst, uterus and liver near the time of implantation

The steroidogenic potential of hamster tissues, just prior to implantation of the blastocyst in the uterus, was characterized by incubating blastocysts (14) and pieces of endometrium with [1, 2-3H]-androstenedione for 24 h. [3H]-2-Methoxyestradiol was synthesized, but intermediate estrogens were not found. To obtain a more quantitative assessment and comparison of steroidogenic activity, especially aromatase activity, in these tissues as well as in the uterine myometrium and liver and to increase the possibility of recovering estradiol, microsomes were isolated from 244 blastocysts and portions of the other tissues. Microsomes were incubated with [1 alpha, 2 alpha-3H]-testosterone plus [1 beta,2 beta-3H]-testosterone for 6 h. During this time [3H]-metabolites were synthesized by all tissues as indicated by HPLC. [3H]-Androstenedione was noted and values were higher than control levels (medium alone or microsomes from uterine flush fluid) in all samples but liver. [3H]-Estradiol was detected at an elevated level only in the blastocyst sample; however, addition of unlabeled estradiol during the subsequent incubation of endometrial, myometrial and liver microsomes increased the recovery of [3H]-estradiol. Identities of [3H]-2-methoxyestradiol from the first experiment and [3H]-androstenedione and [3H]-estradiol from the second experiment were confirmed by recrystallization. The formation of 3H2O from [beta-3H]-testosterone was used as an index of aromatase activity. After subtracting control medium values, blastocysts were 24-fold more active (dpm/microgram protein) than the endometrium and myometrium in synthesizing 3H2O. While there was no difference in synthetic potential between endometrium and myometrium, aromatase activity in these tissues was greater than that of the liver. Microsomes from uterine flush fluid displayed no capacity for synthesizing 3H2O indicating that the elevated blastocyst levels were not caused by contaminating endometrial cells. These results indicate that all of the tissues examined have the capacity to metabolize C19-steroids to a variety of hormones, including estrogens, and further, that estrogen metabolism occurs rapidly in these tissues. This capacity may be important for providing a suitable hormonal milieu at the time of implantation.     

Characterization of aromatase activity in the sea bass: effects of temperature and different catalytic properties of brain and ovarian homogenates and microsomes

Two aromatase genes have been discovered in the brain and ovary of some teleosts. However, data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, the tritiated water assay was optimized and validated to measure AA in the sea bass using 1 beta-[3H]-androstenedione as a substrate in crude homogenates and microsomes. Optimized assay variables included pH, temperature, buffer strength, incubation time, amount of fresh tissue, substrate, and cofactor concentration. Specificity of the assay was verified by using known inhibitors, inappropriate substrates, and heat-inactivation. Subcellular fractionation revealed ten-fold more activity in the microsomal over the cytosolic fraction. The assay was also validated by comparing results from the direct product isolation method. The validated assay described allows measurement of AA to levels as low as < 10 fmol/mg protein/hr. Sex differentiation is temperature-dependent in the sea bass. It was found that in the physiological range of temperatures where the sea bass can live, 10-30 degrees C, AA is highly dependent on temperature in a linear fashion (brain: r2 = 0.92; P < 0.001; ovary: r2 = 0.94; P < 0.001). When AA levels from brain and ovarian homogenates obtained from the same fish during the spawning season were compared, the respective Michaelis-Menten constant (Km) values were 7.3 nM vs. 4.6 nM, with no significant differences detected between the two tissues. Thus, sea bass aromatase has a very high affinity for androstenedione, similar to what has been found in goldfish, but much higher than other piscine or mammalian aromatases (30-435 nM). In contrast, the brain maximum reaction rate (Vmax 7.8 pmol/mg protein/hr) was four-fold higher (P < 0.001) than the ovarian Vmax (2.1 pmol/mg protein/hr). Consistent results were found using purified microsomes. Although this is the first time that the kinetic parameters are reported for a native piscine aromatase in two different tissues within the same fish, it remains to be determined whether this is a reflection of two distinct isoforms in this particular species.     

In vitro potency and selectivity of the non-steroidal androgen aromatase inhibitor CGS 16949A compared to steroidal inhibitors in the brain.

A sensitive in vitro 3H2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,4,6-androstatrien-3,17-dione (ATD) and 4-hydroxy-androstenedione (4-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 16949A [4-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [Vmax = 5.3 pmol testosterone (T) converted/h/mg protein], has high substrate binding affinity (Km = 4.0 nM), and direct involvement in control of sexual behaviour. With [1 beta-3H]T or [1 beta-3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 16949A. Thus, Kis and IC50s for aromatization were approximately 50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5 alpha/beta-reductase) occurred at concentrations at least one order of magnitude greater than for ATD and 4-OH-A. Neonatal male gerbil hypothalamic aromatase activity (Vmax = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 16949A is more potent in the neonatal gerbil than in the dove (Kis of 0.03 and 0.60 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.     

Comparison of Aromatase Activity in Human Prostatic,Testicular and Placental Tissues

Abstract

The aromatase enzyme was quantified by the release of tritiated water from [1β-³H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0μM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC₅₀ values of 6.4, 0.17 and 0.0017 μM. respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075μM, respectively).     

1-[(Benzofuran-2-yl)phenylmethyl]-triazoles and -tetrazoles - potent competitive inhibitors of aromatase.

The synthesis of a series of novel 1-[(benzofuran-2-yl)phenylmethyl]-triazoles and -tetrazoles is described. The compounds were tested for human placental aromatase inhibition in vitro, using [1beta-3H]-androstenedione as the substrate for the aromatase enzyme, the percentage inhibition and IC50 data is included.     

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-alpha-phosphatidylcholine with [1 beta-3H,4-14C]-androstenedione as substrate. The specific activity of purified aromatase was 65.0 (50.6-74.3) nmol.min-1.(mg of protein)-1 or a turnover rate of 5.0 (4.3-5.9) min-1. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The Km of immunoaffinity-purified aromatase was 12, 210, 41, and 2830 nM for androstenedione, 16 alpha-hydroxyandrostenedione, testosterone, and 16 alpha-hydroxytestosterone, respectively. The very high Km value for 16 alpha-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80 degrees C.     

Bile acids. XLVII. 12alpha-Hydroxylation of precursors of allo bile acids by rabbit liver microsomes.

Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3beta-3H]- or [24-14C]allochenodeoxycholic acid or [5alpha,6alpha-3H2]5alpha-cholestane-3alpha,7alpha-diol to their respective 12alpha-hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal 12alpha-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the 14C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.     

Measurement of aromatisation by a urine technique suitable for the evaluation of aromatase inhibitors in vivo.

By modification of a recently developed method for separation of radio-labelled urinary oestrogens we were able to separate oestrogen metabolites and measure their isotope ratios in urine following injections of [3H]delta 4-androstenedione and [14C]oestrone. This method provides a useful tool for studying in vivo aromatisation of delta 4-androstenedione into oestrone in breast cancer patients before and during treatment with aromatase inhibitors.     

In vitro and in vivo studies demonstrating potent and selective estrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A

CGS 16949A inhibited the conversion of [4-14C]androstenedione (A) to [4-14C]estrone by human placental microsomes in a competitive manner (Ki = 1.6 nM). Aminoglutethimide, also a competitive inhibitor, had a Ki = 0.7 microM in this assay system. The Km for the aromatization of A was 0.11 microM. Using ovarian microsomes from immature rats primed with pregnant mare's serum gonadotrophin and using [4-14C]testosterone conversion to [4-14C]estradiol as a measure of aromatase activity, the Km was 42 nM. At a substrate concentration 3-fold the Km, CGS 16949A was 180 times more potent as an inhibitor than aminoglutethimide, exhibiting half-maximal inhibition at 1.7 nM as compared to 0.3 microM. In vivo CGS 16949A lowered ovarian estrogen synthesis by gonadotropin-primed, androstenedione treated, immature rats by 90% at a dose of 260 micrograms/kg (PO). A dose of 100 mg/kg of aminoglutethimide was needed to produce this same effect. CGS 16949A at a dose of 4 mg/kg (PO) induced uterine atrophy (aromatase inhibition) without inducing adrenal hypertrophy - indicating a lack of inhibition of corticosterone secretion, while aminoglutethimide at 40 mg/kg (PO) induced adrenal hypertrophy without inducing uterine atrophy. CGS 16949A was neither androgenic nor estrogenic in rats using standard bioassays. The data suggest that CGS 16949A may serve as a potent and selective agent for modulating estrogen-dependent functions.     

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.     

Aromatase inhibition by 4-thiosubstituted-4-androstene-3,17-dione derivatives

The synthesis and evaluation of 4-thiosubstituted-4-androstenedione analogs as inhibitors of estrogen synthetase (aromatase) is described. All compounds were prepared by the addition of various thiol reagents to 4 beta,5 beta-epoxyandrostanedione. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]4-androstene-3,17-dione as substrate. Synthesized compounds exhibiting high inhibitory activity were further evaluated under initial velocity conditions to determine apparent Ki values. Several compounds were effective competitive inhibitors, and have apparent Ki values ranging from 34 to 52 nM, with the apparent Km for androstenedione being 54 nM. The results of these studies demonstrate a tightly fitted enzyme pocket that can accommodate bulky substituents at the C-4 position of androstenedione not to exceed 4.3 A in width and 5.5 A in length.     

Aromatase activity in microsomes from rat ventral prostate and Dunning R3327H rat prostatic adenocarcinoma

We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H2O release method as described by Weisz. Production of 3H2O from 1 beta-[3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein X h in 3-month old rats to 68 pmol/mg protein X h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.     

Measurement of Aromatisation by a Urine Technique Suitable for the Evaluation of Aromatase Inhibitors in Vivo

Abstract

By modification of a recently developed method for separation of radio-labelled urinary oestrogens we were able to separate oestrogen metabolites and measure their isotope ratios in urine following injections of [³H]Δ⁴-androstenedione and [¹⁴C]oestrone. This method provides a useful tool for studying in vivo aromatisation of Δ⁴-androstenedione into oestrone in breast cancer patients before and during treatment with aromatase inhibitors.     

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.